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Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model

  • Marcus Y. Chin
    Marcus Y. Chin
    Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States
    Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
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    Orcidhttps://orcid.org/0000-0002-7037-1603
  • , 
  • Kean-Hooi Ang
    Kean-Hooi Ang
    Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
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  • , 
  • Julia Davies
    Julia Davies
    Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
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  • , 
  • Carolina Alquezar
    Carolina Alquezar
    Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States
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  • , 
  • Virginia G. Garda
    Virginia G. Garda
    Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States
    Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
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  • , 
  • Brendan Rooney
    Brendan Rooney
    Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States
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  • , 
  • Kun Leng
    Kun Leng
    Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States
    Biomedical Sciences Graduate Program, University of California, San Francisco, California 94158, United States
    Medical Scientist Training Program, University of California, San Francisco, California 94158, United States
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  • , 
  • Martin Kampmann
    Martin Kampmann
    Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States
    More by Martin Kampmann
    Orcidhttps://orcid.org/0000-0002-3819-7019
  • , 
  • Michelle R. Arkin*
    Michelle R. Arkin
    Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-9366-6770
  • , and 
  • Aimee W. Kao*
    Aimee W. Kao
    Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States
    *Email: [email protected]
    More by Aimee W. Kao
    Orcidhttps://orcid.org/0000-0002-7686-7968
Cite this: ACS Chem. Neurosci. 2022, 13, 10, 1505–1516
Publication Date (Web):May 6, 2022
https://doi.org/10.1021/acschemneuro.1c00804

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

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1. Introduction

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Lysosomes are specialized membrane-bound organelles that participate in many crucial cellular functions such as macromolecular degradation, nutrient sensing, and secretion. (1−3) They are intimately involved in autophagy, which serves as a key pathway for maintaining protein homeostasis within the cell. Lysosomes derive their degradative functions by possessing a very acidic lumen (pH ∼ 4.5–4.7), (4,5) allowing the optimal activation of hydrolytic enzymes that are ultimately responsible for substrate breakdown. The lysosomal pH is tightly regulated through the vacuolar-type H+-ATPase (V-ATPase) proton pump and other counter-ion channels. (6)
Defective lysosomes are a common feature of age-related and neurodegenerative disorders. Numerous mutations have been found in genes directly involved in the endolysosomal pathway. (7,8) Pathological accumulation of proteins is also seen across various neurodegenerative diseases, (9,10) implicating a role in aberrant cellular clearance. While the exact mechanisms causing neurodegeneration remain elusive, these observations suggest an overall breakdown in protein homeostasis stemming from lysosomal dysfunction. Indeed, lysosomal acidity has been described to be impaired in studies of age-related neurodegenerative diseases. (11−16)
With increasing evidence underscoring its critical role in neurodegenerative diseases, correcting lysosomal function and pH regulation may be therapeutically tractable strategies for future drug development. However, relatively few phenotypic screens have been conducted with a specific focus on lysosomes. High-throughput screening (HTS) studies have explored lysosomal morphology, positioning, and calcium regulation, with regard to lysosomal storage disorders and cancer. (17) Importantly, to our knowledge, no group has conducted a phenotypic screen on lysosomal pH. Such is the focus in the current study.
Novel lysosomal pH probes that specifically target lysosomes and accurately measure intralumenal pH have been described by various groups. (17−20) Recently, we engineered FIRE-pHLy, a genetically encoded ratiometric lysosomal pH biosensor with a reported pKa of ∼4.4. (20,21) FIRE-pHLy presents advantages in automated, HTS, including stable expression in cells, accurate targeting to lysosomal compartments, and resistance to fluorescence quenching during fixation. Here, we utilized FIRE-pHLy to develop a cell-based phenotypic assay to identify small molecules that affect lysosomal pH. Ultimately, the modulation of lysosomal acidity may restore protein homeostasis defects and serve as a novel therapeutic strategy for neurodegenerative disease-related drug discovery.

2. Results and Discussion

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2.1. High-Content Imaging Screen to Identify Modulators of Lysosomal pH

To identify small molecules and biological pathways that regulate lysosomal pH, we developed a cell-based high-content imaging screen that measured relative changes in intralumenal pH of lysosomes through fluorescence detection (Figure 1). We utilized the previously validated genetically encoded pH biosensor, FIRE-pHLy or fluorescence indicator reporting pH of lysosomes. (21) FIRE-pHLy is composed of monomeric teal fluorescent protein 1 (mTFP1), mCherry, and lysosomal associated membrane protein 1 (LAMP1) that targets the fusion protein to lysosomal membranes. The fluorescence of mTFP1 is pH-dependent, while mCherry serves as an expression control and internal lysosome marker. Ratiometric imaging of mTFP1 and mCherry reports relative changes (herein referred to as the FIRE-pHLy ratio or mTFP1/mCherry) in the lysosomal pH environment.

Figure 1

Figure 1. HTS flowchart for identifying lysosomal pH modulators.

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FIRE-pHLy expressing SH-SY5Y cells were differentiated on 96-well microplates for 10 days and treated with bioactive compounds [10 μM/0.2% dimethyl sulfoxide (DMSO)] for their potential ability to decrease (acidify) or increase (alkalinize) lysosomal pH, as measured by the change in the FIRE-pHLy ratio. Compound ratios were calculated through the ratiometric quantification of mTFP1 and mCherry fluorescence intensities. Data was analyzed in parallel through two distinct pipelines (population-based and object-based quantification) and compared to select hits.
FIRE-pHLy can be stably expressed in a variety of cell models including human neuroblastoma SH-SY5Y cells, (21) which were selected for this HTS study due to their ability to be differentiated into neuron-like cells. (22) Because terminally differentiated SH-SY5Y cells have qualities appropriate to model aspects of neurodegenerative diseases, including endogenous expression of the aggregation-prone proteins such as tau, (23,24) they may provide enhanced therapeutic relevance over actively dividing cells in drug screening campaigns. Morphologically, undifferentiated cells are non-polarized with shortened processes, while their differentiated counterparts exhibit elongated and branching neurites extending from the cell body. Lysosomes are found both as perinuclear clusters within the cell body and as more individual entities along the processes of neurons, both of which we observed using FIRE-pHLY visualization. (21)
Cells were differentiated within 96-well microplates and then treated with a 1835-member library of bioactive compounds at the UCSF small-molecule discovery center (SMDC) for 6 h. This time point was rationalized to capture small molecules that changed lysosomal pH through fast-acting mechanisms (e.g., ion channels and transporters that generated the proton gradient) while avoiding longer-term global changes such as cellular proliferation and survival that could have non-specifically impacted lysosomal activity. The final screening concentration was 10 μM with a DMSO concentration of 0.2%, which did not artificially alter FIRE-pHLy ratio measurements (Figure S1A) and was non-toxic to cells (Figure S1B). Single plane images were acquired and analyzed through a custom segmentation protocol for mTFP1, mCherry and nuclei target sets (Figure 1). (21)
First, we designed a population-based analysis approach that quantified the FIRE-pHLy ratio averaged across the entire well. Given that there were no well-validated compounds that decreased lysosomal pH, we established variability in the screening data by the percent coefficient of variation (CV) of the FIRE-pHLy ratio. A CV of 5% in negative controls indicated that the assay was consistent across all assay plates (Figure S1C), with a mean FIRE-pHLy ratio of 0.36 ± 0.02. The CV for cell count was also acceptable (CV = 22%) with a mean of 1,163 ± 261 quantified cells per well (Figure S1D). Compounds that decreased the FIRE-pHLy ratio (i.e. decreased pH) were considered acidic hits, while any that increased the FIRE-pHLy ratio (i.e. increased pH) were labeled alkaline hits.
As a secondary method for hit selection, we developed an object-based analysis approach in order to focus on lysosomes, optimize sensitivity, and account for different populations of lysosomes based on coordinate location (25−27) in the differentiated SH-SY5Y cells. FIRE-pHLy ratios from individually segmented lysosomes in the DMSO controls were binned according to their ratio values, normalized from 0.0 to 1.0 with an increment of 0.05 per bin, and plotted as a histogram. FIRE-pHLy ratios from compound-treated wells were then normalized to the DMSO bins (see the Supporting Information). The data were normally distributed in the negative control DMSO-treated wells; assay means of “bin at max” and “median bin” were 9.88 ± 0.42 and 9.39 ± 0.24, respectively (Figure S1E). Shifts in the distribution caused by modulators of lysosomal pH would result in acidic (leftward curve shift) or alkaline (rightward curve shift) phenotypes; skew in the distribution could indicate that a subset of lysosomes were affected by compound treatment. The CVs of “bin at max” and “median bin” were 4 and 3%, supporting the consistency of our assay. Hits were selected from both population- and object-based approaches in tandem and compared to generate the primary hit list.

2.2. Primary Hit Selection, Filtering, and Comparison of Analysis Approaches

Thresholding for primary hits was performed using both parallel quantification pipelines, which will herein be referred to as “population-based analysis” (Figure 2A–C) and “object-based analysis” (Figure 2D–H). With population-based analysis, controls were visualized along a two-dimensional (2D) plot of FIRE-pHLy ratio fold change (FC) and nuclear count (“nucleus”) FC to define the boundaries for determining hits (Figure 2A). Compounds that exhibited a nucleus FC of less than 0.48 [−3 standard deviations; (SD)] were considered cytotoxic and excluded. Compounds with the FIRE-pHLy ratio FCs within ± 3SD of control were considered inactive (Figures 2B and S2A). Primary alkaline hits were identified based on the FIRE-pHLy ratio FC of at least 1.12 and nucleus FC of at least 0.48 (Figure S2B). Conversely, acidic hits were identified based on a FIRE-pHLy ratio FC of less than or equal to 0.88 and nucleus FC of at least 0.48 (Figure 2C). Importantly, we note that the FIRE-pHLy ratio could be artificially altered by changes in mCherry fluorescence. This may be caused by compound autofluorescence or off-target pH changes in the cytosolic environment where mCherry resides. To exclude these artifacts, a filter of mCherry fluorescence intensity FC was applied. Alkaline compounds with mCherry fluorescence FC > 0.7 and acidic compounds with FC < 1.5 were shortlisted. Overall, the population-based analysis approach identified 29 filtered alkaline hits (Figure S2B) and 13 filtered acidic hits (Figure 2C).

Figure 2

Figure 2. Hit selection for lysosomal acidifiers. (A–C) Population-based analysis. (A) 2D plot of FIRE-pHLy ratio FC versus nuclei FC (i.e., cell count) for all DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (B) 2D FIRE-pHLy ratio FC versus nuclei FC plot for all test compounds (n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds and yellow dots represent toxic or inactive compounds. (C) Expanded inset of acidic hits from Figure 2B. Acidic hits were selected using nucleus FC ≤ 3SD and FIRE-pHLy ratio FC ≤ 3SD compared to controls. Compounds that artificially altered the FIRE-pHLy ratio FC through mCherry fluorescence were excluded (green dots with black cross marks). (D–H) Lysosomal object-based analysis. (D) 2D plot of median bin versus bin at max for DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (E) 2D plot of median bin versus bin at max for all the test compounds (shown in yellow dots; n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds. (F) Lysosomal object-based acidic hits from Figure 2E. Acidic hits were selected using median bin ≤ 3SD and bin at max ≤ 3SD. (Inset) Frequency distribution for the hit compound highlighted with a red circle. Gray bars represent negative control distribution. Red bars represent hit compound distribution. (G) Filtering hits for cell toxicity. 2D plot of median bin versus nucleus FC for all DMSO negative controls. (H) 2D plot of median bin versus nucleus FC for test compounds. Compounds highlighted in the red box were excluded due to cell toxicity; alkaline hits are highlighted by the blue box; and acidic hits are highlighted in the orange box. Compounds that altered mCherry fluorescence were excluded (green dots with black cross marks). (I) Venn diagram showing the overlap of final filtered alkaline and acidic hits selected from population-based and lysosomal object-based analyses. Data in this figure was visualized in DataWarrior.

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A similar thresholding paradigm was used for object-based analysis. Compared to controls (Figure 2D), compounds that increased or decreased median bin and bin at max by 3SD were considered as hits (Figure 2E). Alkaline hits were selected based on a bin at the max and median of at least 13 and 11.5, respectively (Figure S2C). Acidic hits were identified based on a bin at the max and median of less than or equal to 8.7 and 8.6, respectively (Figure 2F). Subsequently, compounds that reduced nucleus FC compared to the control (Figure 2G,H) were eliminated. Finally, compounds that artificially altered mCherry fluorescence intensity were also removed (Figure 2H).
Hits were compiled from population-based and object-based analyses to generate the finalized filtered hit list. Thirteen acidic hits were identified from population-based analysis (Table 1), while four hits were identified from object-based analysis (Table 2). One compound, OSI-027, was found in both analysis pipelines. For alkaline hits, 29 compounds were identified from population-based methods (Table S1), 7 of which overlapped with object-based methods (Table S2). No additional alkaline hits were identified by object-based analysis. Overall, the population-based analysis method identified more hits than object-based analysis (Figure 2I) in both acidic and alkaline hit types. More alkalinizing hits were identified than acidifying hits.
Table 1. Primary Filtered Acidifying Compounds Identified from Population-Based Analysis
a

OSI-027 was identified as a hit in both population- and object-based analysis. Compounds highlighted in red passed dose-response retesting in differentiated SH-SH5Y cells.

Table 2. Primary Filtered Acidifying Compounds Identified from Object-Based Analysis
a

OSI-027 was identified as a hit in both population- and object-based analysis. Compounds highlighted in red passed dose-response retesting in differentiated SH-SH5Y cells.

2.3. Hit Confirmation with Dose-Response Retesting

Twenty-three compounds (i.e. all 16 acidic hits and the 7 alkaline hits identified in both population- and object-based analyses) were retested in a twofold dose response over a range of 80–0.156 μM in differentiated and undifferentiated SH-SY5Y cells. Undifferentiated cells were evaluated to support hit confirmation in differing cellular states. Among the 16 primary acidic hits, five compounds lowered lysosomal pH in a dose-dependent manner in differentiated cells (Figure 3A–E). Among these five primary hits, OSI-027 and PP242 reproduced in undifferentiated cells (Figure 3F,G). The estimated EC50 values for OSI-027 (Figure 3F) and PP242 (Figure 3G) in undifferentiated cells were 35 and 2 μM, respectively. The narrow range for differentiated SH-SY5Y cells makes it difficult to precisely measure EC50. The remaining three compounds─buparlisib, teniposide, and AZ960─exhibited marginal dose-response with a much narrower range in FIRE-pHLy ratio FC in undifferentiated cells (Figure 3H–J). We observed a FC reversal at higher doses for these compounds, which may be due to cell death. Finally, in the alkaline direction, reserpine robustly increased lysosomal pH, with a ∼8.3-fold decrease in EC50 post-differentiation (Figure S3).

Figure 3

Figure 3. Top acidic hits tested in differentiated and undifferentiated SH-SY5Y cells.

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Ten-point dose-response curves (2-fold serial dilution) from 0.15 to 80 μM. Cells were treated with compounds for 6 h before imaging. FIRE-pHLy ratios were taken from total mTFP1/mCherry fluorescence, displayed as a FC relative to control, and plotted according to dose. (A–E) Five primary hits tested in differentiated SH-SY5Y cells. (A) OSI-027. (B) PP242. (C) Buparlisib. (D) Teniposide. (E) AZ960. (F-J) Hits retested in undifferentiated cells. (F) OSI-027. (G) PP242. (H) Buparlisib. (I) Teniposide. (J) AZ960. OSI-027 and PP242, highlighted in red, yielded the strongest responses and were selected for further validation studies. Data points are presented as mean ± SD, from 3 biological replicates; n = ∼3000–5000 differentiated cells or ∼15,000–20,000 undifferentiated cells quantified per dose per time point. Dose-response curves were generated using a simple linear regression model from which EC50 values were estimated. Red asterisk: single data point not displayed within the Y-axis range.
Overall, we note the consistent expansion of the ratio FC range across the ten-point dose response in undifferentiated cells compared to their differentiated counterparts. In differentiated cells, the ratio FC range for OSI-027 and PP-242 was from ∼1.00 to ∼0.90 and ∼1.05 to ∼0.93, respectively. For the undifferentiated cells, the ranges for OSI-027 and PP242 were from ∼0.97 to ∼0.81 and ∼0.97 to ∼0.75, respectively. These data suggest that undifferentiated SH-SY5Y cells are more sensitive to pH lowering effects induced by OSI-027 and PP242 than differentiated cells. We elected to proceed with validating the top two acidic hits, OSI-027 and PP242, because of their chemical similarity and potential mechanistic interest in reacidifying lysosomes. The overall summary of the primary screen is detailed in Figure 4.

Figure 4

Figure 4. Hit selection summary. Summary of small molecule hits that modulate lysosomal pH in undifferentiated and differentiated SH-SY5Y cells.

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2.4. Functional Validation of Top Acidic Hits OSI-027 and PP242

To functionally validate acidification of lysosomal pH, we treated live undifferentiated SH-SY5Y cells (without FIRE-pHLy) with compounds and assessed cathepsin D activity (Figure 5A). Because cathepsin D auto-activates at acidic pH, (28) its activity can be used as a functional readout of lysosomal pH; BODIPY FL-Pepstatin A is a cathepsin D antagonist that binds to the active form of the enzyme. (29) Cells were treated with OSI-027 and PP-242 at 10 μM for various times before BODIPY FL Pepstatin A fluorescence was assayed (Figure 5B). Compared to DMSO control, cathepsin D activity was significantly increased with OSI-027 and PP242 treatment. As a negative control, we tested bafilomycin A1 (BafA1), which increased pH by inhibiting the V-ATPase proton pump. BafA1 slightly decreased cathepsin D activity, but not significantly, suggesting that the alkalinization of lysosomes did not further lower basal activated enzyme levels. Overall, the correlation between the FIRE-pHLy ratio FC decrease and cathepsin D level increase validated OSI-027 and PP242 as robust─and functionally relevant─lysosomal pH acidifiers.

Figure 5

Figure 5. OSI-027 and PP242 increases mature cathepsin D levels in SH-SY5Y cells and acidifies pH in human iAstrocytes. (A) Representative fluorescence images of undifferentiated SH-SY5Y cells treated with DMSO, 100 nM BafA1, 10 μM OSI-027, and 10 μM PP242 at t = 6 h and stained with BODIPY FL Pepstatin A probe. Scale bar = 10 μm. (B) Time course of cells treated with compounds for 0.5, 1, 1.5, 2, 4, and 6 h. Cells were incubated with BODIPY FL Pepstatin A for 30 min before live-imaging. BODIPY FL fluorescence was normalized to cell number, displayed as a FC relative to control, and plotted against time (hours). Data points are presented as mean ± SD, from 3 biological replicates; n = ∼15,000–20,000 cells quantified per condition group per time point. Statistical analysis was performed using two-way ANOVA for multiple comparisons. *p ≤ 0.05 and **p ≤ 0.01. (C) Bar graph quantification of FIREpHLy ratio fold-change (FC) in human iPSC-derived astrocytes (iAstrocytes) treated with OSI-027 and PP242 at 10 μM for 24 h. Data points are presented as median ± SD from three technical replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. ***p ≤ 0.001.

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After confirming that OSI-027 and PP-242 acidified lysosomal pH and increased active cathepsin D levels, we sought to validate these compounds in another native cell model, namely, human-induced pluripotent stem cell (iPSC)-derived astrocytes, or iAstrocytes. Quiescent (non-reactive) iAstrocytes stably expressing FIRE-pHLy were subsequently treated for 24 h with OSI-027 and PP242 (Figure 5C). Both compounds acidified pH in iAstrocytes compared to control treatment (∼50% reduction in FIRE-pHLy ratio FC), providing further support that these compounds acidified lysosomes across multiple cell types.
Reactive astrocytes secrete neurotoxic factors and have been implicated in the neuroinflammatory pathogenesis of neurodegenerative diseases. (30) Recently, Rooney et al. described an in vitro system to model inflammatory reactive astrocytes. (31,32) Reactive iAstrocytes activated by cytokines exhibited an alkaline lysosomal pH, which was accompanied by increased levels of lysosomal exocytosis, a contributor to neurotoxicity. (32) When reactive iAstrocytes were treated with 10 μM of PP242, elevated lysosomal pH was restored to control levels and was accompanied by a reduction in lysosomal exocytosis. (32) Ultimately, these data supported the notion that aberrant lysosomal pH was a contributing factor to neuroinflammation-induced functional changes in neurodegenerative disease and may be corrected with small molecules such as PP242 and OSI-027. Taken together, the acidifying effect of OSI-027 and PP242 in lysosomes was recapitulated in disease models. We posit that its effects may be therapeutic in other contexts of lysosomal dysfunction.

2.5. OSI-027 and PP242 Inhibit mTOR and Induce Autophagy in SH-SY5Y Cells

Both OSI-027 and PP242 are described as potent and selective ATP-competitive inhibitors of mammalian target of rapamycin (mTOR). (33−35) mTOR forms two protein complexes, mTORC1 and mTORC2; these signaling complexes (Figure 6A) form a major hub that regulates cellular processes such as metabolism, growth, and proliferation. mTOR inhibition is coupled with autophagy induction, which is associated with lysosomal activation and acidification. (36−38)

Figure 6

Figure 6. OSI-027 and PP242 inhibits mTORC1/2 and activates autophagy markers. (A) Simplified schematic of the proposed mechanism for OSI-027 and PP242-mediated lysosomal acidification through autophagy (highlighted in red arrows). Compounds are shown in orange. (B) Representative immunoblots for mTORC1 and mTORC2 phosphorylation substrates P70S6KThr389 and AktSer473, respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 and PP242 at 0.1, 1, and 10 μM. (C) Representative immunoblots for autophagy markers ULK1Ser757, LC3B-I/LC3B-II, and p62 respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 (OSI) and PP242 (PP) (same as above). GAPDH was used as the housekeeping protein. Note: WT SH-SY5Y cells were used to generate the p62 immunoblots. (D) Bar graphs showing quantification of P70S6KThr389/total (E) AktSer473/total, (F) ULK1Ser757/total, (G) LC3B-II/LC3B-I, and (H) p62/GAPDH. OSI-027 shown in the top row and PP242 shown in the bottom row. Data is normalized to DMSO controls. Bars are presented as mean ± SD from three independent replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

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To understand whether the lysosomal acidification promoted by OSI-027 and PP242 was related to their role as mTOR inhibitors, we assessed mTOR inhibition and autophagy activation (Figure 6). Both compounds dose dependently inhibited downstream targets of mTORC1 (Figure 6B,D) and mTORC2 (Figure 6B,E) in undifferentiated SH-SY5Y cells. mTORC1 activity was measured by the phosphorylation state of P70 S6 Kinase (P70S6K) at position Thr389 and mTORC2 activity was assessed by the phosphorylation of Akt at position Ser473.
After confirming that OSI-027 and PP242 inhibited mTOR, we measured their ability to activate autophagy. mTORC1 negatively regulates autophagy through the phosphorylation of Unc-51-like autophagy activating kinase (ULK1) at Ser757. (39) OSI-027 and PP242 reduced ULK1Ser757 levels dose dependently with near complete reduction at 10 μM (Figure 6C,F), suggesting that both drugs initiated mTORC1-dependent autophagy. We then measured the levels of microtubule-associated protein light chain 3B (LC3B). The conversion of LC3B-I to LC3B-II correlates with the number of formed autophagosomes and therefore reflects autophagy activation. (40,41) The ratio of LC3B-II to LC3B-I increased at higher OSI-027 and PP242 doses, achieving significance at 10 μM (Figure 6C,G). Finally, we tested for p62, which is an autophagic cargo adaptor that is shuttled into lysosomes during autophagy for degradation. (42) Corroborating our data above, PP242 treatment lowered p62 protein levels, while OSI-027 trended to decreased p62 levels, although this result was not statistically significant (Figure 6C,H). Together, these results demonstrated that OSI-027 and PP242 induced autophagy, indicating that their ability to acidify lysosomes may be secondary to the induction of autophagy rather than a direct action on the lysosome.

2.6. OSI-027 and PP242 Acidifies Lysosomes More Potently than Other mTOR Inhibitors

Interestingly, our compound screen included other mTOR inhibitors, such as rapamycin and torin1, that were not identified as primary hits in our screen. To assess the differential effectiveness of additional mTOR inhibitors in modulating lysosomal pH, we retested rapamycin and torin 1 in undifferentiated FIRE-pHLy expressing SH-SY5Y cells over 24 h (Figure 7). Confirming our screening results, OSI-027 and PP242 treatment induced a dose- and time-dependent decrease in lysosomal pH in cells (Figure 7A,B), but treatment with rapamycin and torin1 did not acidify lysosomes across the tested dose range up to 24 h (Figure 7C,D). These results suggested that at the dosage and timing used in these experiments, OSI-027 and PP242 were more effective in acidifying lysosomal pH in undifferentiated SH-SY5Y cells than the mTOR inhibitors torin1 and rapamycin.

Figure 7

Figure 7. Dose-response and time-course comparison of mTOR inhibitors on lysosomal acidification. Five-point dose response (10-fold serial dilution) from 0.0001 to 10 μM treatment of (A) OSI-027, (B) PP242, (C) Rapamycin, and (D) Torin1 measured after 2, 6, and 24 h in undifferentiated FIRE-pHLy expressing SH-SY5Y cells. FIRE-pHLy ratio measurements were normalized to dose- and time-matched controls. Data points are presented as mean ± SD, from 3 technical replicates; n = ∼15,000–20,000 cells quantified per condition group per time point.

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Because we proposed that OSI-027 and PP242 induced pH acidification by activating autophagy, we hypothesized that rapamycin and torin1 did not activate autophagy in SH-SY5Y cells, at the doses and timings used in this study. Indeed, though we confirmed that these compounds inhibited mTORC (Figure S4A), rapamycin did not significantly reduce ULK1Ser757 levels nor increase the ratio of LC3B-I and LC3B-II, indicating that autophagy was not activated under these conditions (Figure S4A). Although torin1 treatment did reduce ULK1Ser757 levels starting at 100 nM compound, it did not significantly increase the LC3B-II/LC3B-I ratio (Figure S4B), suggesting that torin1 did not induce autophagy in as strongly as OSI-027 and PP242.
In summary, we performed a high-content imaging-based phenotypic screen to identify small molecules that modulate lysosomal pH with a specific focus on acidifying compounds. We identified 16 acidic and twenty29 alkaline compounds using two distinct hit selection approaches. Population-based analysis is used in standard plate-based HTS studies, while object-based analysis provides a novel technique that could be used in future studies to dissect organelle subpopulation phenotypes. While the latter technique yielded an overall lower acidic hit rate, the validation rate was superior to population-based analysis. Out of the four acidic hits identified using the object-based approach, two were confirmed as top hits and validated in subsequent orthogonal assays. Indeed, we were able to visualize more nuanced changes in the distribution of lysosomes according to their pH, which improves upon existing analysis methods of image-based cellular assays.
Ultimately, we validated 2 out of the 16 primary acidic hits. This hit rate may be a product of both biological factors such as lysosomal pH dynamics and screening limitations such as the library size and protein druggability of the target(s). We speculate that because basal lumenal pH of lysosomes is already highly acidic (∼4.5) compared to other cellular compartments, acidification beyond this set-point may be tightly regulated or even perhaps detrimental to the cell in certain contexts. This may explain the reduced dynamic range of FIRE-pHLy signal exhibited by acidifiers compared to alkalinizing compounds. Indeed, only a few examples highlight specific roles of lysosomal hyper-acidification in melanosome trafficking (43) and phospholipid biosynthesis. (44)
Alkaline compartments, on the other hand, are more common in the cell. In fact, lysosomes mature from the endolysosomal network, which maintains more alkaline pH ranges. Exogenous agents, such as drugs, have also been known to accumulate in acidic vesicles, such as lysosomes, and affect the local pH. (45,46) It is conceivable that perturbations in the alkaline direction are generally better tolerated in the cell (at least over short time periods), supporting our observation that alkalinizing compounds exhibited a larger signal range in the primary screen.
OSI-027 and PP242 were identified as top acidic hits, demonstrating lysosomal pH lowering effects in undifferentiated and differentiated SH-SY5Y neuronal-like cells and in basal and activated iAstrocytes, possibly through activation of autophagy. Indeed, other groups have utilized OSI-027 and PP242 to study autophagy in the context of neurodegenerative disease models. For example, Silva et al. identified OSI-027 as a top hit in a mutant tau protein lowering screen performed in patient iPSC-derived neurons. (47) Consistent with our data, OSI-027 lowered total mutant tauA152T and hyperphosphorylated tauSer396 levels at 1 and 10 μM, suggesting the correlation between lysosomal acidification and enhanced tau clearance. Interestingly, the tau lowering effect for OSI-027 was much stronger than that of rapamycin. Moreover, one group showed in a Parkinson’s disease neuroinflammation model that impaired lysosomal acidification was accompanied by alpha-synuclein accumulation. (48) Treatment with 40 nM PP242 rescued lysosomal acidity as measured by LysoTracker dye and normalized alpha-synuclein protein levels in mouse PC12 cells and primary midbrain neurons. Together, these data support the supposition that OSI-027 and PP242 acidify lysosomes and thereby restore normal degradative function in neuronal cells.
The link between lysosomal acidification defects and clinical phenotypes, such as changes in the lysosomal morphology, remains an unstudied question in the field. To begin to understand this connection, high-resolution imaging techniques such as electron microscopy (EM) may be used. Past studies have already described EM as a robust method to visualize defective lysosomes in the context of lysosomal storage disorders and autophagy. (49,50) Conceivably compounds that rescue lysosomal functions through acidification such as OSI-027 and PP242 may also reverse abnormal morphologies.
It is still not entirely clear why OSI-027 and PP242 are more effective in activating autophagy and decreasing lysosomal pH than other mTOR inhibitors such as torin1 and rapamycin. It is plausible that OSI-027 and PP242 have undescribed targets independent of mTOR that may be contributing to autophagy and lysosomal acidity. In order to investigate additional targets, we measured ULK1Ser555 phosphorylation (Figure S5A). ULK1Ser555 is a mTOR-independent phosphosite controlled by AMP-activated protein kinase (AMPK) activity. (51) OSI-027 (Figure S5B) and rapamycin (Figure S5D) did not lower phosphorylated ULK1Ser555/total levels, while a significant reduction was observed for PP242 (Figure S5C) at 10 μM and torin1 (Figure S5E) at 1 and 10 μM. Because these two compounds had differential effects on lysosomal acidification, we concluded that this particular non-mTOR phosphosite was unlikely to affect pH.
Nevertheless, kinase inhibitors are likely to have additional targets. According to the KINOMEscan database, (52) an assay platform that annotates competitive binding between inhibitors and a panel of known kinases, PP242 binds to multiple other kinases. These include phosphoinositide 3-kinase and ABL proto-oncogene 1, which have reported roles in regulating autophagy from cancer studies. (53−56) Thus, by evaluating the other targets of OSI-027 and PP242, one may identify additional, mTOR-independent, mechanisms of lysosomal acidification. Importantly, OSI-027 and PP242 may serve as “tool” compounds for the further investigation of the mechanisms driving autophagy-mediated lysosomal activation in the context of neurodegenerative diseases.

3. Methods

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3.1. Compound Library and Repurchased Compounds

The library consisted of 1835 compounds assembled from the commercially available SelleckChem bioactive collection (SelleckChem, Houston, TX). For dose-response and further validations, compounds were repurchased from SelleckChem, unless otherwise indicated. Repurchased compounds were evaluated for purity by liquid chromatography/mass spectrometry.

3.2. Cell Line Maintenance and Differentiation in 96-Well Microplates

Human WT and stably expressing FIRE-pHLy SH-SY5Y neuroblastoma cells (21) were maintained in 1:1 Eagle’s minimum essential medium (ATCC, #30-003) and F12 medium (Life Technologies; Carlsbad, CA, #11765062) with 10% fetal bovine serum (FBS) and 1% pen/strep under standard humidified conditions of 37 °C and 5% CO2 atmosphere. (21) Cells were trypsinized with 0.25% trypsin/EDTA solution (Sigma, #T4049) and seeded into collagen type-I-coated μClear 96-well microplates (Greiner Bio-One, #655956) at a low density of 10,000 cells/cm2 with a total well volume of 100 μL using a WellMate microplate dispenser (Thermo Scientific, Waltham, MA) to allow for proliferation during the first phase of differentiation. Plates were incubated overnight at 37 °C/5% CO2 before the start of differentiation, as previously described. (21) Briefly, cells were maintained in FBS(+) media supplemented with 10 μM retinoic acid from days 0 to 6 and FBS-free media supplemented with a 50 ng/mL brain-derived growth factor until compound pinning on day 10.

3.3. HTS Ratiometric FIRE-pHLy Lysosomal pH Reporter Assay

The drug screen was performed at the UCSF SMDC. From the library plate (5 mM stock dissolved in DMSO), 200 nL of the compound was added in singlicate to 96-well assay plates (10 μM final screening concentration) using a fixed-volume pin tool (V&P Scientific, San Diego, CA) loaded onto the BioMek-FXP liquid handling automation workstation (Beckman Coulter, Brea, CA). DMSO was added to negative control wells on every assay plate. Assay plates were incubated for 6 h (37 °C and 5% CO2). 50 μL of 6% PFA (diluted in serum-free media) was dispensed directly into each 100 μL assay well (2% PFA final concentration) and shaken briefly using an EL406 Combination Washer Dispenser (BioTek, Winooski, VT). Plates were fixed at room temperature (RT) for 15 min and washed once with 100 μL 1× D-PBS (with MgCl2 and CaCl2). Cells were stained with 1:1000 (vol/well) Hoechst dye (10 mg/mL Hoechst 33342 solution, Thermo Fisher, #H3570) diluted in D-PBS for 20 min at RT protected from light and washed once with 1× D-PBS (with MgCl2 and CaCl2). Plates were wrapped and stored at 4 °C protected from light.

3.4. High-Content Confocal Imaging, Analysis Types, and Data Output

Following our previously described imaging methods and feature extraction protocols, (21) assay plates were imaged (at a single Z-plane) on the IN Cell 6500 HS Analyzer (General Electric Life Sciences/Cytiva, Marlborough, MA) and quantified on the IN Cell Developer Toolbox v1.9 (GE Life Sciences/Cytiva). Both population- and object-based analysis were used to quantify primary screening imaging data. Population-based analysis was used to validate hits in subsequent dose-response and validation assays. A detailed description of population- and object-based image analysis is available in the Supporting Information.

3.5. BODIPY FL Pepstatin A Live-Cell Time Course Assay

Mature cathepsin D levels were assessed via BODIPY FL Pepstatin A staining on live cells. The staining protocol was performed according to the manufacturer’s protocol. (29) Briefly, cells were seeded in 96-well plates, cultured overnight, and treated for 30, 60, 90, 120, 240, and 360 min with 100 nM bafilomycin A1, 10 μM OSI-027, 10 μM PP-242, and DMSO as the solvent control. Prior to imaging, cells were incubated for 30 min with a staining solution consisting of 1 μM BODIPY FL Pepstatin A and 1:1000 (vol/well) Hoescht nuclear dye. Cells were washed once with D-PBS and imaged live on an IN cell analyzer 6500 HS and processed according to an adapted protocol on IN Cell Developer Toolbox v1.9.

3.6. Immunoblotting

Western blots were performed as previously described. (21)

3.6.1. Primary Antibodies

Rabbit anti-ULK (1:1000, cell signaling, #8054).
Rabbit anti-ULKSer757 (1:1000, cell signaling, #14202).
Rabbit anti-ULKSer555 (1:1000, cell signaling, #5869).
Rabbit anti-p70S6K (1:1000, cell signaling, #2708).
Rabbit anti-P70S6KThr389 (1:1000, cell signaling, #9234).
Rabbit anti-Akt (1:1000, cell signaling, #9272).
Rabbit anti-AktSer437 (1:1000, cell signaling, #4060).
Rabbit anti-LC3B (1:1000, Sigma, #L7543).
Mouse anti-SQSTM1/p62 (1:1000, Santa Cruz, #48402).
Mouse anti-GAPDH (1:5000, Abcam, #8245).

3.6.2. Secondary Antibodies

Donkey anti-Mouse Green (1:10,000, LICOR, #926-32212).
Donkey anti-Rabbit Green (1:10,000, LICOR, #926-32213).
Donkey anti-Rabbit Red (1:10,000, LICOR, #926-68073).

3.7. iAstrocyte Experiments

iAstrocytes were generated as detailed in Leng et al. (31) Day 20 iAstrocytes were plated in ScienCell Astrocyte Media (ScienCell Research Laboratories cat. no. 1801) at 20,000 cells/cm2 on BioLite Cell Culture Treated 96-well plates (ThermoFisher Scientific cat. no. 12-556-008) coated with growth factor reduced, Phenol Red-Free, LDEV-Free Matrigel Basement Membrane Matrix (Corning cat. no. 356231) diluted 1:200 in DMEM/F12 (ThermoFisher Scientific cat. no. 11330032). iAstrocytes were transduced with FIRE-pHLy lentivirus at the time of plating. Full media changes with ScienCell Astrocyte Media were conducted on days 1, 3, and 5 after plating. On day 5, small-molecule compounds were diluted in media to 10 μM. After 24 h (i.e. on day 6 after plating), iAstrocytes were incubated with Accutase Cell Dissociation Reagent (ThermoFisher Scientific cat. no. A11105-01) for 10 min at 37 °C and diluted in Dulbecco’s phosphate buffered saline (Milipore Sigma cat. no. D8537) for flow cytometry analysis.
Data from flow cytometry experiments were analyzed using FlowJo (version 10.7.1). FIRE-pHLy-positive populations were determined through live cell (SSC-A vs FSC-A), single cell (FSC-H vs FSC-A), and transduced (mCherry+) gating strategies. FIRE-pHLy signal was quantified as the Median FITC-A/mCherry-A ratio for each well.

3.8. Data Presentation, Statistical Analysis, and Illustrations

Visualization of control and screening hit data was performed on the SMDC HiTS server and DataWarrior, an open-source cheminformatics tool. Pre-processing of data was organized in Microsoft Excel. Calculations of hit selection measurements was conducted on Pipeline Pilot (Biovia) (see the Supporting Information). Dose-response curves were generated using a simple linear regression model in GraphPad Prism 9. For validation experiments, all data were generated from randomly selected sample populations from at least three independent experiments represented unless otherwise mentioned in the corresponding figure legends. Statistical data were presented as mean ± S.D or S.E.M. Multiple comparisons between groups were analyzed by the one-way or two-way ANOVA test. All data plots and statistical analyses were performed using GraphPad Prism 9 with no samples excluded. Significant differences between experimental groups were indicated as *P < 0.05; **P < 0.01; and ***P < 0.001; only P < 0.05 was considered as statistically significant. NS = not significant. Immunoblot images and quantifications were acquired from Image Studio (LI-COR Biosciences). Cartoon schematics were created on Biorender.com. Figures were assembled on Adobe Illustrator and Adobe Photoshop.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.1c00804.

  • Assay performance for negative controls, hit selection for lysosomal alkalinizers, hit confirmation for top alkaline hit, mTORC1/2 immunoblots of cells treated with rapamycin and torin1, ULK1Ser555 immunoblots of cells treated with mTOR inhibitors, and methods describing the population- and object-based analysis pipelines (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
  • Authors
    • Marcus Y. Chin - Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States;  Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States;  Orcidhttps://orcid.org/0000-0002-7037-1603
    • Kean-Hooi Ang - Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
    • Julia Davies - Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
    • Carolina Alquezar - Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States
    • Virginia G. Garda - Memory and Aging Center, Department of Neurology, University of California, San Francisco, California, California 94158, United States;  Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, United States
    • Brendan Rooney - Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States
    • Kun Leng - Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States;  Biomedical Sciences Graduate Program, University of California, San Francisco, California 94158, United States;  Medical Scientist Training Program, University of California, San Francisco, California 94158, United States
    • Martin Kampmann - Institute for Neurodegenerative Diseases, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, United States;  Orcidhttps://orcid.org/0000-0002-3819-7019
  • Author Contributions

    M.Y.C., M.R.A., and A.W.K. for conceptualization and design; M.Y.C., K.A., J.D., C.A., and V.G.G. for experimental design and data acquisition; B.R., K.L, and M.K. for conceptualization, design, and data acquisition of iAstrocyte experiments; all authors analyzed and interpreted data; M.Y.C., M.R.A., and A.W.K. wrote the manuscript; and all authors edited the manuscript.

  • Notes
    The authors declare no competing financial interest.

    The authors declare that all relevant data supporting the findings of this study are available within the paper. Any data or reagents can be obtained from the corresponding authors (M.R.A. and A.W.K.) and on reasonable request.

Acknowledgments

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We thank the UCSF SMDC for their assistance on drug screening procedures, data management, and storage. We also thank Deanna Kroetz for insights on drug screening and validation. Additionally, we thank members of the Kao and Arkin labs for their thoughtful discussions. This work has received support from the NIH/NIA (R01 AG055342 to A.W.K.; R01 AG062359 to M.K.; and F30 AG066418 to K.L.). Finally, this work was included in the dissertation of M.Y.C for the Pharmaceutical Sciences and Pharmacogenomics doctoral program at UCSF (https://escholarship.org/uc/item/3xs387n6). (20)

Abbreviations

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FIRE-pHLy

Fluorescence Indicator REporting pH in Lysosomes
mTFP1

monomeric teal fluorescent protein
V-ATPase

vacuolar-type ATPase
LAMP1

lysosomal-associated membrane protein 1
BafA1

bafilomycin A1
iPSC

induced pluripotent stem cells
RA

retinoic acid
BDNF

brain-derived neurotrophic factor
HTS

high-throughput screening
FC

fold change
CV

coefficient of variation
EC50

half maximal effective concentration
DMSO

dimethyl sulfoxide
WT

wildtype
ATP

adenosine triphosphate
mTOR

mammalian target of rapamycin
mTORC

mTOR complex
ULK

unc-51-like autophagy activating kinase
LC3B

microtubule-associated protein light chain 3B
PI3K

phosphoinositide 3-kinase
ALB1

ABL proto-oncogene 1
AMP

activated protein kinase
AMPK

AMP-activated protein kinase

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    Monaco, A.; Fraldi, A. Protein Aggregation and Dysfunction of Autophagy-Lysosomal Pathway: A Vicious Cycle in Lysosomal Storage Diseases. Front. Mol. Neurosci. 2020, 13, 37,  DOI: 10.3389/fnmol.2020.00037
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    9
    Protein aggregation and dysfunction of autophagy-lysosomal pathway: a vicious cycle in lysosomal storage diseases
    Monaco, Antonio; Fraldi, Alessandro
    Frontiers in Molecular Neuroscience (2020), 13 (), 37CODEN: FMNRAJ; ISSN:1662-5099. (Frontiers Media S.A.)
    Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In postmitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often assocd. to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degrdn. function, thus generating a vicious cycle, which boost neurodegenerative cascades.
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  10. 10
    Ross, C. A.; Poirier, M. A. Protein Aggregation and Neurodegenerative Disease. Nat. Med. 2004, 10, S10– S17,  DOI: 10.1038/nm1066
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    10
    Protein aggregation and neurodegenerative disease
    Ross Christopher A; Poirier Michelle A
    Nature medicine (2004), 10 Suppl (), S10-7 ISSN:1078-8956.
    Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.
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  11. 11
    Baxi, K.; Ghavidel, A.; Waddell, B.; Harkness, T. A.; de Carvalho, C. E. Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis Elegans. Genetics 2017, 207, 83– 101,  DOI: 10.1534/genetics.117.204222
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    11
    Regulation of lysosomal function by the daf-16 forkhead transcription factor couples reproduction to aging in Caenorhabditis elegans
    Baxi, Kunal; Ghavidel, Ata; Waddell, Brandon; Harkness, Troy A.; de Carvalho, Carlos E.
    Genetics (2017), 207 (1), 83-101CODEN: GENTAE; ISSN:0016-6731. (Genetics Society of America)
    Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans. In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signaling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-assocd. phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny prodn. and somatic aging and identify the loss of lysosomal acidity-a crit. regulator of the proteolytic output of these organelles-as a novel biomarker of aging in C. elegans. The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reprodn., and correlates with the redn. in proteostasis in early adult life. Our results further implicate the steroid signaling pathway and DAF-16 in dynamically regulating lysosomal pH in the intestine of wildtype worms in response to the reproductive cycle. In the intestine of reproducing worms, DAF-16 promotes acidic lysosomes by upregulating the expression of v-ATPase genes. These findings support a model in which protein clearance in the soma is linked to reprodn. in the gonad via the active regulation of lysosomal acidification.
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  12. 12
    Colacurcio, D. J.; Nixon, R. A. Disorders of Lysosomal Acidification─The Emerging Role of v-ATPase in Aging and Neurodegenerative Disease. Ageing Res. Rev. 2016, 32, 75– 88,  DOI: 10.1016/j.arr.2016.05.004
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    12
    Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease
    Colacurcio, Daniel J.; Nixon, Ralph A.
    Ageing Research Reviews (2016), 32 (), 75-88CODEN: ARRGAK; ISSN:1568-1637. (Elsevier B.V.)
    Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degrdn. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metab. and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson disease and Alzheimer disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal hydrolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clin. and exptl. studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum.
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  13. 13
    Hughes, A. L.; Gottschling, D. E. An Early Age Increase in Vacuolar PH Limits Mitochondrial Function and Lifespan in Yeast. Nature 2012, 492, 261– 265,  DOI: 10.1038/nature11654
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    13
    An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast
    Hughes, Adam L.; Gottschling, Daniel E.
    Nature (London, United Kingdom) (2012), 492 (7428), 261-265CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asym. divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degrdn., but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a crit. regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.
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  14. 14
    Lee, J.-H.; McBrayer, M. K.; Wolfe, D. M.; Haslett, L. J.; Kumar, A.; Sato, Y.; Lie, P. P. Y.; Mohan, P.; Coffey, E. E.; Kompella, U.; Mitchell, C. H.; Lloyd-Evans, E.; Nixon, R. A. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating VATPase-Mediated Lysosome Acidification. Cell Rep. 2015, 12, 1430– 1444,  DOI: 10.1016/j.celrep.2015.07.050
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    14
    Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification
    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.
    Cell Reports (2015), 12 (9), 1430-1444CODEN: CREED8; ISSN:2211-1247. (Cell Press)
    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common mol. mechanism.
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    Sun, Y.; Li, M.; Zhao, D.; Li, X.; Yang, C.; Wang, X. Lysosome Activity Is Modulated by Multiple Longevity Pathways and Is Important for Lifespan Extension in C. Elegans. Elife 2020, 9, e55745  DOI: 10.7554/eLife.55745
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    15
    Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans
    Sun, Yanan; Li, Meijiao; Zhao, Dongfeng; Li, Xin; Yang, Chonglin; Wang, Xiaochen
    eLife (2020), 9 (), e55745/1-e55745/28CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Lysosomes play important roles in cellular degrdn. to maintain cell homeostasis. In order to understand whether and how lysosomes alter with age and contribute to lifespan regulation, we characterized multiple properties of lysosomes during the aging process in C. elegans. We uncovered age-dependent alterations in lysosomal morphol., motility, acidity and degrdn. activity, all of which indicate a decline in lysosome function with age. The ageassocd. lysosomal changes are suppressed in the long-lived mutants daf-2, eat-2 and isp-1, which extend lifespan by inhibiting insulin/IGF-1 signaling, reducing food intake and impairing mitochondrial function, resp. We found that 43 lysosome genes exhibit reduced expression with age, including genes encoding subunits of the proton pump V-ATPase and cathepsin proteases. The expression of lysosome genes is upregulated in the long-lived mutants, and this upregulation requires the functions of DAF-16/FOXO and SKN-1/NRF2 transcription factors. Impairing lysosome function affects clearance of aggregate-prone proteins and disrupts lifespan extension in daf-2, eat-2 and isp-1 worms. Our data indicate that lysosome function is modulated by multiple longevity pathways and is important for lifespan extension.
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  16. 16
    Tong, B. C.-K.; Wu, A. J.; Huang, A. S.; Dong, R.; Malampati, S.; Iyaswamy, A.; Krishnamoorthi, S.; Sreenivasmurthy, S. G.; Zhu, Z.; Su, C.; Liu, J.; Song, J.; Lu, J.-H.; Tan, J.; Pan, W.; Li, M.; Cheung, K.-H. Lysosomal TPCN (Two Pore Segment Channel) Inhibition Ameliorates Beta-Amyloid Pathology and Mitigates Memory Impairment in Alzheimer Disease. Autophagy 2021, 18, 624– 642,  DOI: 10.1080/15548627.2021.1945220
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    Chin, M. Y.; Espinosa, J. A.; Pohan, G.; Markossian, S.; Arkin, M. R. Reimagining Dots and Dashes: Visualizing Structure and Function of Organelles for High-Content Imaging Analysis. Cell Chem. Biol. 2021, 28, 320– 337,  DOI: 10.1016/j.chembiol.2021.01.016
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    17
    Reimagining dots and dashes: visualizing structure and function of organelles for high-content imaging analysis
    Chin, Marcus Y.; Espinosa, Jether Amos; Pohan, Grace; Markossian, Sarine; Arkin, Michelle R.
    Cell Chemical Biology (2021), 28 (3), 320-337CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    A review. Organelles are responsible for biochem. and cellular processes that sustain life and their dysfunction causes diseases from cancer to neurodegeneration. While researchers are continuing to appreciate new roles of organelles in disease, the rapid development of specifically targeted fluorescent probes that report on the structure and function of organelles will be crit. to accelerate drug discovery. Here, we highlight four organelles that collectively exemplify the progression of phenotypic discovery, starting with mitochondria, where many functional probes have been described, then continuing with lysosomes and Golgi and concluding with nascently described membraneless organelles. We introduce emerging probe designs to explore organelle-specific morphol. and dynamics and highlight recent case studies using high-content anal. to stimulate further development of probes and approaches for organellar high-throughput screening.
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  18. 18
    Ponsford, A. H.; Ryan, T. A.; Raimondi, A.; Cocucci, E.; Wycislo, S. A.; Fröhlich, F.; Swan, L. E.; Stagi, M. Live Imaging of Intra-Lysosome PH in Cell Lines and Primary Neuronal Culture Using a Novel Genetically Encoded Biosensor. Autophagy 2020, 17, 1500,  DOI: 10.1080/15548627.2020.1771858
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    Webb, B. A.; Aloisio, F. M.; Charafeddine, R. A.; Cook, J.; Wittmann, T.; Barber, D. L. PHLARE: A New Biosensor Reveals Decreased Lysosome PH in Cancer Cells. MBoC 2021, 32, 91,  DOI: 10.1091/mbc.E20-06-0383
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    Chin, M. Y.-Y. Exploring Lysosomal PH as a Therapeutic Strategy for Neurodegeneration; UCSF, 2021.
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    There is no corresponding record for this reference.
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    Chin, M. Y.; Patwardhan, A. R.; Ang, K.-H.; Wang, A. L.; Alquezar, C.; Welch, M.; Nguyen, P. T.; Grabe, M.; Molofsky, A. V.; Arkin, M. R.; Kao, A. W. Genetically Encoded, PH-Sensitive MTFP1 Biosensor for Probing Lysosomal PH. ACS Sens. 2021, 6, 2168,  DOI: 10.1021/acssensors.0c02318
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    21
    Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH
    Chin, Marcus Y.; Patwardhan, Anand R.; Ang, Kean-Hooi; Wang, Austin L.; Alquezar, Carolina; Welch, Mackenzie; Nguyen, Phi T.; Grabe, Michael; Molofsky, Anna V.; Arkin, Michelle R.; Kao, Aimee W.
    ACS Sensors (2021), 6 (6), 2168-2180CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    Lysosomes are important sites for macromol. degrdn., defined by an acidic lumenal pH of ~ 4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-assocd. membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quant. imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.
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    Encinas, M.; Iglesias, M.; Liu, Y.; Wang, H.; Muhaisen, A.; Ceña, V.; Gallego, C.; Comella, J. X. Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells. J. Neurochem. 2000, 75, 991– 1003,  DOI: 10.1046/j.1471-4159.2000.0750991.x
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    22
    Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells
    Encinas, Mario; Iglesias, Montse; Liu, Yuhui; Wang, Hongyin; Muhaisen, Ashraf; Cena, Valentin; Gallego, Carme; Comella, Joan X.
    Journal of Neurochemistry (2000), 75 (3), 991-1003CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
    A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphol., avoiding the presence of other neural crest derivs. that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-assocd. protein-43 as well as neuronal polarity markers such as tau and microtubule-assocd. protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the neg. staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochem. and mol. studies due to its susceptibility to genetic manipulation and the availability of an unlimited amt. of cells.
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    Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability. J. Biomol. Screen 2016, 21, 496– 509,  DOI: 10.1177/1087057115625190
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    23
    Characterization of differentiated SH-SY5Y as neuronal screening model reveals increased oxidative vulnerability
    Forster, J. I.; Koglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.
    Journal of Biomolecular Screening (2016), 21 (5), 496-509CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with -hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chem. high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.
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    Xicoy, H.; Wieringa, B.; Martens, G. J. M. The SH-SY5Y Cell Line in Parkinson’s Disease Research: A Systematic Review. Mol. Neurodegener. 2017, 12, 10,  DOI: 10.1186/s13024-017-0149-0
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    24
    The SH-SY5Y cell line in Parkinson's disease research: a systematic review
    Xicoy, Helena; Wieringa, Be; Martens, Gerard J. M.
    Molecular Neurodegeneration (2017), 12 (), 10/1-10/11CODEN: MNOEAZ; ISSN:1750-1326. (BioMed Central Ltd.)
    Parkinson's disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the mol. and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, are difficult to obtain and maintain as primary cells, current PD research is mostly performed with permanently established neuronal cell models, in particular the neuroblastoma SH-SY5Y lineage. This cell line is frequently chosen because of its human origin, catecholaminergic (though not strictly dopaminergic) neuronal properties, and ease of maintenance. However, there is no consensus on many fundamental aspects that are assocd. with its use, such as the effects of culture media compn. and of variations in differentiation protocols. Here we present the outcome of a systematic review of scientific articles that have used SH-SY5Y cells to explore PD. We describe the cell source, culture conditions, differentiation protocols, methods/approaches used to mimic PD and the preclin. validation of the SH-SY5Y findings by employing alternative cellular and animal models. Thus, this overview may help to standardize the use of the SH-SY5Y cell line in PD research and serve as a future user's guide.
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    Bright, N. A.; Davis, L. J.; Luzio, J. P. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity. Curr. Biol. 2016, 26, 2233– 2245,  DOI: 10.1016/j.cub.2016.06.046
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    25
    Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity
    Bright, Nicholas A.; Davis, Luther J.; Luzio, J. Paul
    Current Biology (2016), 26 (17), 2233-2245CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    The endocytic delivery of macromols. from the mammalian cell surface for degrdn. by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochem. to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake expts., we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equil. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.
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    Cabukusta, B.; Neefjes, J. Mechanisms of Lysosomal Positioning and Movement. Traffic 2018, 19, 761– 769,  DOI: 10.1111/tra.12587
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    Mechanisms of lysosomal positioning and movement
    Cabukusta, Birol; Neefjes, Jacques
    Traffic (Oxford, United Kingdom) (2018), 19 (10), 761-769CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
    A review. Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule-organizing center in a "cloud," and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biol. functions, such as cargo degrdn., antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biol. events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid-protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the mol. machineries that control this mobility.
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    Johnson, D. E.; Ostrowski, P.; Jaumouillé, V.; Grinstein, S. The Position of Lysosomes within the Cell Determines Their Luminal PH. J. Cell Biol. 2016, 212, 677– 692,  DOI: 10.1083/jcb.201507112
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    27
    The position of lysosomes within the cell determines their luminal pH
    Johnson, Danielle E.; Ostrowski, Philip; Jaumouille, Valentin; Grinstein, Sergio
    Journal of Cell Biology (2016), 212 (6), 677-692CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
    We examd. the luminal pH of individual lysosomes using quant. ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+-ATPase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered compn. of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b dets. the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 d. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is assocd. with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.
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  28. 28
    Gulnik, S.; Baldwin, E. T.; Tarasova, N.; Erickson, J. Human Liver Cathepsin D. J. Mol. Biol. 1992, 227, 265– 270,  DOI: 10.1016/0022-2836(92)90696-H
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    Human liver cathepsin D. Purification, crystallization and preliminary x-ray diffraction analysis of a lysosomal enzyme
    Gulnik, Sergei; Baldwin, Eric T.; Tarasova, Nadezhda; Erickson, John
    Journal of Molecular Biology (1992), 227 (1), 265-70CODEN: JMOBAK; ISSN:0022-2836.
    The 2-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, was isolated from human liver. Isoelec. focusing revealed 2 major species of enzyme that differed by approx. 0.2 pI unit. Crystals suitable for x-ray diffraction anal. were prepd. from acidic solns. using pptn. with ammonium sulfate. The hexagonal crystals diffracted x-rays to beyond 3.1 Å resoln. and belonged to space group P61 (or P65) with cell consts. a = b = 125.9 Å, c = 104.1 Å, γ = 120.0°. The crystals likely contain 2 mols. in the asym. unit, giving a solvent content of 56% (v/w). Biochem. anal. of crystals indicated that both isoforms were present in approx. equimolar proportions. Full structure detn. of the enzyme is underway.
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    Chen, C.-S.; Chen, W.-N. U.; Zhou, M.; Arttamangkul, S.; Haugland, R. P. Probing the Cathepsin D Using a BODIPY FL–Pepstatin A: Applications in Fluorescence Polarization and Microscopy. J. Biochem. Biophys. Methods 2000, 42, 137– 151,  DOI: 10.1016/S0165-022X(00)00048-8
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    29
    Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy
    Chen, C.-S.; Chen, W.-N. U.; Zhou, M.; Arttamangkul, S.; Haugland, R. P.
    Journal of Biochemical and Biophysical Methods (2000), 42 (3), 137-151CODEN: JBBMDG; ISSN:0165-022X. (Elsevier Science Ireland Ltd.)
    Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathol. progressions during tumor formation and oxidn. stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.
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    Phatnani, H.; Maniatis, T. Astrocytes in Neurodegenerative Disease. Cold Spring Harbor Perspect. Biol. 2015, 7, a020628,  DOI: 10.1101/cshperspect.a020628
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    Leng, K.; Rooney, B.; Kim, H.; Xia, W.; Koontz, M.; Krawczyk, M.; Zhang, Y.; Ullian, E. M.; Fancy, S. P. J.; Schrag, M. S.; Lippmann, E. S.; Kampmann, M. CRISPRi Screens in Human Astrocytes Elucidate Regulators of Distinct Inflammatory Reactive States; preprint. Neuroscience 2021,  DOI: 10.1101/2021.08.23.457400
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    Rooney, B.; Leng, K.; McCarthy, F.; Rose, I. V. L.; Herrington, K. A.; Bax, S.; Chin, M. Y.; Fathi, S.; Leonetti, M.; Kao, A. W.; Elias, J. E.; Kampmann, M. MTOR Controls Neurotoxic Lysosome Exocytosis in Inflammatory Reactive Astrocytes; preprint. Cell Biol. 2021,  DOI: 10.1101/2021.09.11.459904
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    Apsel, B.; Blair, J. A.; Gonzalez, B.; Nazif, T. M.; Feldman, M. E.; Aizenstein, B.; Hoffman, R.; Williams, R. L.; Shokat, K. M.; Knight, Z. A. Targeted Polypharmacology: Discovery of Dual Inhibitors of Tyrosine and Phosphoinositide Kinases. Nat. Chem. Biol. 2008, 4, 691– 699,  DOI: 10.1038/nchembio.117
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    Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases
    Apsel, Beth; Blair, Jimmy A.; Gonzalez, Beatriz; Nazif, Tamim M.; Feldman, Morri E.; Aizenstein, Brian; Hoffman, Randy; Williams, Roger L.; Shokat, Kevan M.; Knight, Zachary A.
    Nature Chemical Biology (2008), 4 (11), 691-699CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    The clin. success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of mols. that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chem. synthesis, X-ray crystallog. and kinome-level biochem. profiling, we identified compds. that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these mols. is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that compd. I blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These mols. demonstrate the feasibility of accessing a chem. space that intersects two families of oncogenes.
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  34. 34
    Bhagwat, S. V.; Gokhale, P. C.; Crew, A. P.; Cooke, A.; Yao, Y.; Mantis, C.; Kahler, J.; Workman, J.; Bittner, M.; Dudkin, L.; Epstein, D. M.; Gibson, N. W.; Wild, R.; Arnold, L. D.; Houghton, P. J.; Pachter, J. A. Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of MTORC1 and MTORC2: Distinct from Rapamycin. Mol. Cancer Ther. 2011, 10, 1394– 1406,  DOI: 10.1158/1535-7163.MCT-10-1099
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    34
    Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin
    Bhagwat, Shripad V.; Gokhale, Prafulla C.; Crew, Andrew P.; Cooke, Andy; Yao, Yan; Mantis, Christine; Kahler, Jennifer; Workman, Jennifer; Bittner, Mark; Dudkin, Lorina; Epstein, David M.; Gibson, Neil W.; Wild, Robert; Arnold, Lee D.; Houghton, Peter J.; Pachter, Jonathan A.
    Molecular Cancer Therapeutics (2011), 10 (8), 1394-1406CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
    The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clin. validated target. MTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metab., proliferation, and survival. Rapamycin and its analogs partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clin. utility in certain cancers. Here, we report the preclin. characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochem. IC50 values of 22 nmol/L and 65 nmol/L, resp. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analog of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concn.-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clin. trials in cancer patients.
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    Falcon, B. L.; Barr, S.; Gokhale, P. C.; Chou, J.; Fogarty, J.; Depeille, P.; Miglarese, M.; Epstein, D. M.; McDonald, D. M. Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual MTORC1/MTORC2 Inhibitors. Cancer Res. 2011, 71, 1573– 1583,  DOI: 10.1158/0008-5472.CAN-10-3126
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    35
    Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual mTORC1/mTORC2 Inhibitors
    Falcon, Beverly L.; Barr, Sharon; Gokhale, Prafulla C.; Chou, Jeyling; Fogarty, Jennifer; Depeille, Philippe; Miglarese, Mark; Epstein, David M.; McDonald, Donald M.
    Cancer Research (2011), 71 (5), 1573-1583CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
    The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiol. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examd. the effects of mTORC1/2 dual inhibition on VEGF prodn., tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF prodn. in tumors in a manner assocd. with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral prodn. of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist. Cancer Res; 71(5); 1573-83.
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    Kim, Y. C.; Guan, K.-L. MTOR: A Pharmacologic Target for Autophagy Regulation. J. Clin. Invest. 2015, 125, 25– 32,  DOI: 10.1172/JCI73939
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    mTOR: a pharmacologic target for autophagy regulation
    Kim Young Chul; Guan Kun-Liang
    The Journal of clinical investigation (2015), 125 (1), 25-32 ISSN:.
    mTOR, a serine/threonine kinase, is a master regulator of cellular metabolism. mTOR regulates cell growth and proliferation in response to a wide range of cues, and its signaling pathway is deregulated in many human diseases. mTOR also plays a crucial role in regulating autophagy. This Review provides an overview of the mTOR signaling pathway, the mechanisms of mTOR in autophagy regulation, and the clinical implications of mTOR inhibitors in disease treatment.
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    Yim, W. W.-Y.; Mizushima, N. Lysosome Biology in Autophagy. Cell Discov. 2020, 6, 1– 12,  DOI: 10.1038/s41421-020-0141-7
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    Zhou, J.; Tan, S.-H.; Nicolas, V.; Bauvy, C.; Yang, N.-D.; Zhang, J.; Xue, Y.; Codogno, P.; Shen, H.-M. Activation of Lysosomal Function in the Course of Autophagy via MTORC1 Suppression and Autophagosome-Lysosome Fusion. Cell Res. 2013, 23, 508– 523,  DOI: 10.1038/cr.2013.11
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    Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion
    Zhou, Jing; Tan, Shi-Hao; Nicolas, Valerie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming
    Cell Research (2013), 23 (4), 508-523CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is assocd. with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examd. the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.
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    Jung, C. H.; Jun, C. B.; Ro, S.-H.; Kim, Y.-M.; Otto, N. M.; Cao, J.; Kundu, M.; Kim, D.-H. ULK-Atg13-FIP200 Complexes Mediate MTOR Signaling to the Autophagy Machinery. Mol. Biol. Cell 2009, 20, 1992– 2003,  DOI: 10.1091/mbc.E08-12-1249
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    ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery
    Jung, Chang Hwa; Jun, Chang Bong; Ro, Seung-Hyun; Kim, Young-Mi; Otto, Neil Michael; Cao, Jing; Kundu, Mondira; Kim, Do-Hyung
    Molecular Biology of the Cell (2009), 20 (7), 1992-2003CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    Autophagy, the starvation-induced degrdn. of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homolog of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
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    Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Arozena, A. A.; Adachi, H.; Adams, C. M.; Adams, P. D. Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition). Autophagy 2016, 12, 1– 222,  DOI: 10.1080/15548627.2016.1139264
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    Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
    Autophagy (2016), 12 (1), 1-222 ISSN:.
    There is no expanded citation for this reference.
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    Menzies, F. M.; Moreau, K.; Puri, C.; Renna, M.; Rubinsztein, D. C. Measurement of Autophagic Activity in Mammalian Cells. Curr. Protoc. Cell Biol. 2012, 54, 15.16.1– 15.16.25,  DOI: 10.1002/0471143030.cb1516s54
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    Bjørkøy, G.; Lamark, T.; Pankiv, S.; Øvervatn, A.; Brech, A.; Johansen, T. Monitoring Autophagic Degradation of P62/SQSTM1. Methods Enzymol. 2009, 452, 181– 197,  DOI: 10.1016/S0076-6879(08)03612-4
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    Monitoring autophagic degradation of p62/SQSTM1
    Bjorkoy Geir; Lamark Trond; Pankiv Serhiy; Overvatn Aud; Brech Andreas; Johansen Terje
    Methods in enzymology (2009), 452 (), 181-97 ISSN:.
    The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative diseases and proteinopathies of the liver. The protein is able to polymerize via an N-terminal PB1 domain and can interact with ubiquitinated proteins via the C-terminal UBA domain. Also, p62/SQSTM1 binds directly to LC3 and GABARAP family proteins via a specific sequence motif. The protein is itself degraded by autophagy and may serve to link ubiquitinated proteins to the autophagic machinery to enable their degradation in the lysosome. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 may be used as a marker to study autophagic flux. Here, we present several protocols for monitoring autophagy-mediated degradation of p62 using Western blots, pulse-chase measurement of p62 half-life, immunofluorescence and immuno-electron microscopy, as well as live cell imaging with a pH-sensitive mCherry-GFP double tag. We also present data on species-specificity and map the epitopes recognized by several commercially available anti-p62 antibodies.
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    van der Poel, S.; Wolthoorn, J.; van den Heuvel, D.; Egmond, M.; Groux-Degroote, S.; Neumann, S.; Gerritsen, H.; van Meer, G.; Sprong, H. Hyperacidification of Trans-Golgi Network and Endo/Lysosomes in Melanocytes by Glucosylceramide-Dependent V-ATPase Activity. Traffic 2011, 12, 1634– 1647,  DOI: 10.1111/j.1600-0854.2011.01263.x
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    Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity
    van der Poel, Selene; Wolthoorn, Jasja; van den Heuvel, Dave; Egmond, Maarten; Groux-Degroote, Sophie; Neumann, Sylvia; Gerritsen, Hans; van Meer, Gerrit; Sprong, Hein
    Traffic (Oxford, United Kingdom) (2011), 12 (11), 1634-1647CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
    Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addn. to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H+-translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent Ki for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These expts. establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.
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    Lenk, G. M.; Park, Y. N.; Lemons, R.; Flynn, E.; Plank, M.; Frei, C. M.; Davis, M. J.; Gregorka, B.; Swanson, J. A.; Meisler, M. H.; Kitzman, J. O. CRISPR Knockout Screen Implicates Three Genes in Lysosome Function. Sci. Rep. 2019, 9, 9609,  DOI: 10.1038/s41598-019-45939-w
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    CRISPR knockout screen implicates three genes in lysosome function
    Lenk Guy M; Park Young N; Lemons Rosemary; Flynn Emma; Plank Margaret; Frei Christen M; Meisler Miriam H; Kitzman Jacob O; Davis Michael J; Gregorka Brian; Swanson Joel A
    Scientific reports (2019), 9 (1), 9609 ISSN:.
    Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.
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    De Duve, C.; De Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P.; Van Hoof, F. o. Lysosomotropic Agents. Biochem. Pharmacol. 1974, 23, 2495– 2531,  DOI: 10.1016/0006-2952(74)90174-9
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    Lysosomotropic agents
    De Duve, Christian; De Barsy, Thierry; Poole, Brian; Trouet, Andre; Tulkens, Paul; Van Hoof, Francois
    Biochemical Pharmacology (1974), 23 (18), 2495-531CODEN: BCPCA6; ISSN:0006-2952.
    A review of the nature, mechanisms of entry, and therapeutic possibilities of lysosomotropic agents, i.e., substances that are taken up selectively into lysosomes.
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    Effect of lysosomotropic molecules on cellular homeostasis
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    Pharmacological Research (2017), 117 (), 177-184CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
    Weak bases that readily penetrate through the lipid bilayer and accumulate inside the acidic organelles are known as lysosomotropic mols. Many lysosomotropic compds. exhibit therapeutic activity and are commonly used as antidepressant, antipsychotic, antihistamine, or antimalarial agents. Interestingly, studies also have shown increased sensitivity of cancer cells to certain lysosomotropic agents and suggested their mechanism of action as a promising approach for selective destruction of cancer cells. However, their chemotherapeutic utility may be limited due to various side effects. Hence, understanding the homeostatic alterations mediated by lysosomotropic compds. has significant importance for revealing their true therapeutic potential as well as toxicity. In this review, after briefly introducing the concept of lysosomotropism and classifying the lysosomotropic compds. into two major groups according to their cytotoxicity on cancer cells, we focused on the subcellular alterations mediated by class-II lysosomotropic compds. Briefly, their effect on intracellular cholesterol homeostasis, autophagy and lysosomal sphingolipid metab. was discussed. Accordingly, class-II lysosomotropic mols. inhibit intracellular cholesterol transport, leading to the accumulation of cholesterol inside the late endosomal-lysosomal cell compartments. However, the accumulated lysosomal cholesterol is invisible to the cellular homeostatic circuits, hence class-II lysosomotropic mols. also upregulate cholesterol synthesis pathway as a downstream event. Considering the fact that Niemann-Pick disease, a lysosomal cholesterol storage disorder, also triggers similar pathol. abnormalities, this review combines the knowledge obtained from the Niemann-Pick studies and lysosomotropic compds. Taken together, this review is aimed at allowing readers a better understanding of subcellular alterations mediated by lysosomotropic drugs, as well as their potential therapeutic and/or toxic activities.
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    Wang, M.-X.; Cheng, X.-Y.; Jin, M.; Cao, Y.-L.; Yang, Y.-P.; Wang, J.-D.; Li, Q.; Wang, F.; Hu, L.-F.; Liu, C.-F. TNF Compromises Lysosome Acidification and Reduces α-Synuclein Degradation via Autophagy in Dopaminergic Cells. Exp. Neurol. 2015, 271, 112– 121,  DOI: 10.1016/j.expneurol.2015.05.008
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    Experimental Neurology (2015), 271 (), 112-121CODEN: EXNEAC; ISSN:0014-4886. (Elsevier Inc.)
    Tumor necrosis factor-α (TNF) is increasingly implicated as a crit. pro-inflammatory cytokine involved in chronic inflammation and neurodegeneration of Parkinson's disease (PD). However, the cellular and mol. events that lead to dopaminergic neuron degeneration are not fully understood. In this study, we demonstrated that microglia-released and recombinant TNF disrupted α-synuclein (α-SYN) degrdn. and caused its accumulation in PC12 cells and midbrain neurons. At subtoxic doses, recombinant TNF was found to increase the no. of LC3 puncta dots and LC3II protein level, assocd. with the increases of P62 protein level. Inhibition of lysosomal degrdn. with Bafilomycin A1 pretreatment abrogated the TNF-induced elevation in LC3II protein level whereas autophagy inhibitor 3-methyladenine did not affect it. Moreover, TNF led to a marked increase in the no. of yellow LC3 dots with a marginal elevation in red-only dots in RFP-GFP-tandem fluorescent LC3 (tf-LC3) transfected PC12 cells, implying the impairment in autophagic flux. Furthermore, TNF treatment reduced lysosomal acidification, as LysoTracker Red fluorescence and LysoSensor fluorescence shift from blue to yellow was markedly decreased in TNF-treated PC12 cells. Co-treatment with mammalian target of rapamycin kinase complex 1 (mTORC1) inhibitor PP242, which activated transcription factor EB (TFEB) signaling and lysosome biogenesis, partially rescued the accumulation of α-SYN in PC12 cells and midbrain neurons. Taken together, our results demonstrated that at subtoxic levels, TNF was able to impair autophagic flux and result in α-SYN accumulation by compromising lysosomal acidification in dopaminergic cells. This may represent a novel mechanism for TNF-induced dopaminergic neuron degeneration in PD.
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    Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metab. to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a mol. mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homolog of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. This study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
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    Kinase inhibitors show great promise as a new class of therapeutics. Here the authors describe an efficient way to det. kinase inhibitor specificity by measuring binding of small mols. to the ATP site of kinases. The authors have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clin. development, against a panel of 119 protein kinases. The authors find that specificity varies widely and is not strongly correlated with chem. structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. The authors also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small mol.-protein interaction map for clin. compds. across a large no. of related proteins.
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    American Journal of Hematology (2013), 88 (6), 455-462CODEN: AJHEDD; ISSN:0361-8609. (Wiley-Liss, Inc.)
    Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are crit. for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been assocd. with the clearance of large macromol. signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This assocn. is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacol. inhibition of autophagy also reduced Bcr-Abl/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an addnl. and important feature of the activity of imatinib. 88:455-462, 2013. © 2013 Wiley Periodicals, Inc.
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    Nature communications (2019), 10 (1), 1566 ISSN:.
    The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.
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    Journal of Pharmacological Sciences (Amsterdam, Netherlands) (2017), 134 (4), 197-202CODEN: JPSTGJ; ISSN:1347-8613. (Elsevier B.V.)
    S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissocd. the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two crit. targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.
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    Abl Kinases Regulate Autophagy by Promoting the Trafficking and Function of Lysosomal Components
    Yogalingam, Gouri; Pendergast, Ann Marie
    Journal of Biological Chemistry (2008), 283 (51), 35941-35953CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Autophagy is a lysosome-dependent degradative pathway that regulates the turnover of intracellular organelles, parasites, and long-lived proteins. Deregulation of autophagy results in a variety of pathol. conditions, but little is known regarding the mechanisms that link normal cellular and pathol. signals to the regulation of distinct stages in the autophagy pathway. Here we uncover a novel role for the Abl family kinases in the regulation of the late stages of autophagy. Inhibition, depletion, or knockout of of the Abl family kinases, Abl and Arg, resulted in a dramatic redn. in the intracellular activities of the lysosomal glycosidases α-galactosidase, α-mannosidase and neuraminidase. Inhibition of Abl kinases also reduced the processing of the precursor forms of cathepsin D and cathepsin L to their mature, lysosomal forms, which coincided with the impaired turnover of long-lived cytosolic proteins and accumulation of autophagosomes. Furthermore, defective lysosomal degrdn. of long-lived proteins in the absence of Abl kinase signaling was accompanied by a perinuclear redistribution of lysosomes and increased glycosylation and stability of lysosome-assocd. membrane proteins, which are known to be substrates for lysosomal enzymes and play a role in regulating lysosome mobility. Our findings reveal a role for Abl kinases in the regulation of late-stage autophagy and have important implications for therapies that employ pharmacol. inhibitors of the Abl kinases.
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  • Abstract

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    Figure 1

    Figure 1. HTS flowchart for identifying lysosomal pH modulators.

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    Figure 2

    Figure 2. Hit selection for lysosomal acidifiers. (A–C) Population-based analysis. (A) 2D plot of FIRE-pHLy ratio FC versus nuclei FC (i.e., cell count) for all DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (B) 2D FIRE-pHLy ratio FC versus nuclei FC plot for all test compounds (n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds and yellow dots represent toxic or inactive compounds. (C) Expanded inset of acidic hits from Figure 2B. Acidic hits were selected using nucleus FC ≤ 3SD and FIRE-pHLy ratio FC ≤ 3SD compared to controls. Compounds that artificially altered the FIRE-pHLy ratio FC through mCherry fluorescence were excluded (green dots with black cross marks). (D–H) Lysosomal object-based analysis. (D) 2D plot of median bin versus bin at max for DMSO negative controls (shown in red dots; n = 384 wells across all assay plates). (E) 2D plot of median bin versus bin at max for all the test compounds (shown in yellow dots; n = 1 per compound; 1835 total compounds). Green dots represent primary hit compounds. (F) Lysosomal object-based acidic hits from Figure 2E. Acidic hits were selected using median bin ≤ 3SD and bin at max ≤ 3SD. (Inset) Frequency distribution for the hit compound highlighted with a red circle. Gray bars represent negative control distribution. Red bars represent hit compound distribution. (G) Filtering hits for cell toxicity. 2D plot of median bin versus nucleus FC for all DMSO negative controls. (H) 2D plot of median bin versus nucleus FC for test compounds. Compounds highlighted in the red box were excluded due to cell toxicity; alkaline hits are highlighted by the blue box; and acidic hits are highlighted in the orange box. Compounds that altered mCherry fluorescence were excluded (green dots with black cross marks). (I) Venn diagram showing the overlap of final filtered alkaline and acidic hits selected from population-based and lysosomal object-based analyses. Data in this figure was visualized in DataWarrior.

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    Figure 3

    Figure 3. Top acidic hits tested in differentiated and undifferentiated SH-SY5Y cells.

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    Figure 4

    Figure 4. Hit selection summary. Summary of small molecule hits that modulate lysosomal pH in undifferentiated and differentiated SH-SY5Y cells.

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    Figure 5

    Figure 5. OSI-027 and PP242 increases mature cathepsin D levels in SH-SY5Y cells and acidifies pH in human iAstrocytes. (A) Representative fluorescence images of undifferentiated SH-SY5Y cells treated with DMSO, 100 nM BafA1, 10 μM OSI-027, and 10 μM PP242 at t = 6 h and stained with BODIPY FL Pepstatin A probe. Scale bar = 10 μm. (B) Time course of cells treated with compounds for 0.5, 1, 1.5, 2, 4, and 6 h. Cells were incubated with BODIPY FL Pepstatin A for 30 min before live-imaging. BODIPY FL fluorescence was normalized to cell number, displayed as a FC relative to control, and plotted against time (hours). Data points are presented as mean ± SD, from 3 biological replicates; n = ∼15,000–20,000 cells quantified per condition group per time point. Statistical analysis was performed using two-way ANOVA for multiple comparisons. *p ≤ 0.05 and **p ≤ 0.01. (C) Bar graph quantification of FIREpHLy ratio fold-change (FC) in human iPSC-derived astrocytes (iAstrocytes) treated with OSI-027 and PP242 at 10 μM for 24 h. Data points are presented as median ± SD from three technical replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. ***p ≤ 0.001.

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    Figure 6

    Figure 6. OSI-027 and PP242 inhibits mTORC1/2 and activates autophagy markers. (A) Simplified schematic of the proposed mechanism for OSI-027 and PP242-mediated lysosomal acidification through autophagy (highlighted in red arrows). Compounds are shown in orange. (B) Representative immunoblots for mTORC1 and mTORC2 phosphorylation substrates P70S6KThr389 and AktSer473, respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 and PP242 at 0.1, 1, and 10 μM. (C) Representative immunoblots for autophagy markers ULK1Ser757, LC3B-I/LC3B-II, and p62 respectively, in FIRE-pHLy SH-SY5Y cells treated with OSI-027 (OSI) and PP242 (PP) (same as above). GAPDH was used as the housekeeping protein. Note: WT SH-SY5Y cells were used to generate the p62 immunoblots. (D) Bar graphs showing quantification of P70S6KThr389/total (E) AktSer473/total, (F) ULK1Ser757/total, (G) LC3B-II/LC3B-I, and (H) p62/GAPDH. OSI-027 shown in the top row and PP242 shown in the bottom row. Data is normalized to DMSO controls. Bars are presented as mean ± SD from three independent replicates. Statistical analysis was performed using one-way ANOVA for multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

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    Figure 7

    Figure 7. Dose-response and time-course comparison of mTOR inhibitors on lysosomal acidification. Five-point dose response (10-fold serial dilution) from 0.0001 to 10 μM treatment of (A) OSI-027, (B) PP242, (C) Rapamycin, and (D) Torin1 measured after 2, 6, and 24 h in undifferentiated FIRE-pHLy expressing SH-SY5Y cells. FIRE-pHLy ratio measurements were normalized to dose- and time-matched controls. Data points are presented as mean ± SD, from 3 technical replicates; n = ∼15,000–20,000 cells quantified per condition group per time point.

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    This article references 56 other publications.

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      Lysosomes as dynamic regulators of cell and organismal homeostasis
      Ballabio, Andrea; Bonifacino, Juan S.
      Nature Reviews Molecular Cell Biology (2020), 21 (2), 101-118CODEN: NRMCBP; ISSN:1471-0072. (Nature Research)
      Abstr.: Exciting new discoveries have transformed the view of the lysosome from a static organelle dedicated to the disposal and recycling of cellular waste to a highly dynamic structure that mediates the adaptation of cell metab. to environmental cues. Lysosome-mediated signalling pathways and transcription programs are able to sense the status of cellular metab. and control the switch between anabolism and catabolism by regulating lysosomal biogenesis and autophagy. The lysosome also extensively communicates with other cellular structures by exchanging content and information and by establishing membrane contact sites. It is now clear that lysosome positioning is a dynamically regulated process and a crucial determinant of lysosomal function. Finally, growing evidence indicates that the role of lysosomal dysfunction in human diseases goes beyond rare inherited diseases, such as lysosomal storage disorders, to include common neurodegenerative and metabolic diseases, as well as cancer. Together, these discoveries highlight the lysosome as a regulatory hub for cellular and organismal homeostasis, and an attractive therapeutic target for a broad variety of disease conditions.
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    2. 2
      Lawrence, R. E.; Zoncu, R. The Lysosome as a Cellular Centre for Signalling, Metabolism and Quality Control. Nat. Cell Biol. 2019, 21, 133– 142,  DOI: 10.1038/s41556-018-0244-7
      2
      The lysosome as a cellular centre for signalling, metabolism and quality control
      Lawrence, Rosalie E.; Zoncu, Roberto
      Nature Cell Biology (2019), 21 (2), 133-142CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
      A review. Long known as terminal degrdn. stations, lysosomes have emerged as sophisticated signalling centers that govern cell growth, division and differentiation. Lysosomes interface phys. and functionally with other organelles, and the master regulator mechanistic target of rapamycin complex 1 kinase is activated on lysosomes in response to nutrient and growth factor inputs. Lysosomes also enable autophagy, a 'self-eating' process essential for quality control and stress adaptation. Faulty execution of lysosomal growth and catabolic programs drives cancer, neurodegeneration and age-related diseases.
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    3. 3
      Mony, V. K.; Benjamin, S.; O’Rourke, E. J. A Lysosome-Centered View of Nutrient Homeostasis. Autophagy 2016, 12, 619– 631,  DOI: 10.1080/15548627.2016.1147671
      3
      A lysosome-centered view of nutrient homeostasis
      Mony, Vinod K.; Benjamin, Shawna; O'Rourke, Eyleen J.
      Autophagy (2016), 12 (4), 619-631CODEN: AUTOC9; ISSN:1554-8635. (Taylor & Francis Ltd.)
      Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromols. for the regeneration of basic building blocks, cellular housekeeping, or pathogen degrdn. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different sol. hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense mols. like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis.
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    4. 4
      Casey, J. R.; Grinstein, S.; Orlowski, J. Sensors and Regulators of Intracellular PH. Nat. Rev. Mol. Cell Biol. 2010, 11, 50– 61,  DOI: 10.1038/nrm2820
      4
      Sensors and regulators of intracellular pH
      Casey, Joseph R.; Grinstein, Sergio; Orlowski, John
      Nature Reviews Molecular Cell Biology (2010), 11 (1), 50-61CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. Protons dictate the charge and structure of macromols. and are used as energy currency by eukaryotic cells. The unique function of individual organelles therefore depends on the establishment and stringent maintenance of a distinct pH. This, in turn, requires a means to sense the prevailing pH and to respond to deviations from the norm with effective mechanisms to transport, produce, or consume proton equiv. A dynamic, finely tuned balance between proton-extruding and proton-importing processes underlies pH homeostasis not only in the cytosol, but in other cellular compartments as well.
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    5. 5
      Ohkuma, S. Use of Fluorescein Isothiocyanate-Dextran to Measure Proton Pumping in Lysosomes and Related Organelles. Methods Enzymol. 1989, 174, 131– 154,  DOI: 10.1016/0076-6879(89)74015-5
      5
      Use of fluorescein isothiocyanate-dextran to measure proton pumping in lysosomes and related organelles
      Ohkuma, Shoji
      Methods in Enzymology (1989), 174 (Biomembranes, Pt. U), 131-54CODEN: MENZAU; ISSN:0076-6879.
      The applications and methodol. of using fluorescein isothiocyanate-dextran for the measurement of proton transport by cells, lysosomes, and organelles are discussed.
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    6. 6
      Mindell, J. A. Lysosomal Acidification Mechanisms. Annu. Rev. Physiol. 2012, 74, 69– 86,  DOI: 10.1146/annurev-physiol-012110-142317
      6
      Lysosomal acidification mechanisms
      Mindell, Joseph A.
      Annual Review of Physiology (2012), 74 (), 69-86CODEN: ARPHAD; ISSN:0066-4278. (Annual Reviews Inc.)
      A review. Lysosomes, the terminal organelles on the endocytic pathway, digest macromols. and make their components available to the cell as nutrients. Hydrolytic enzymes specific to a wide range of targets reside within the lysosome; these enzymes are activated by the highly acidic pH (between 4.5 and 5.0) in the organelles' interior. Lysosomes generate and maintain their pH gradients by using the activity of a proton-pumping V-type ATPase, which uses metabolic energy in the form of ATP to pump protons into the lysosome lumen. Because this activity separates elec. charge and generates a transmembrane voltage, another ion must move to dissipate this voltage for net pumping to occur. This so-called counterion may be either a cation (moving out of the lysosome) or an anion (moving into the lysosome). Recent data support the involvement of ClC-7, a Cl-/H+ antiporter, in this process, although many open questions remain as to this transporter's involvement. Although functional results also point to a cation transporter, its mol. identity remains uncertain. Both the V-ATPase and the counterion transporter are likely to be important players in the mechanisms detg. the steady-state pH of the lysosome interior. Exciting new results suggest that lysosomal pH may be dynamically regulated in some cell types.
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    7. 7
      Koh, J.-Y.; Kim, H. N.; Hwang, J. J.; Kim, Y.-H.; Park, S. E. Lysosomal Dysfunction in Proteinopathic Neurodegenerative Disorders: Possible Therapeutic Roles of CAMP and Zinc. Mol. Brain 2019, 12, 18,  DOI: 10.1186/s13041-019-0439-2
      7
      Lysosomal dysfunction in proteinopathic neurodegenerative disorders: possible therapeutic roles of cAMP and zinc
      Koh Jae-Young; Kim Ha Na; Hwang Jung Jin; Kim Yang-Hee; Park Sang Eun
      Molecular brain (2019), 12 (1), 18 ISSN:.
      A number of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, share intra- and/or extracellular deposition of protein aggregates as a common core pathology. While the species of accumulating proteins are distinct in each disease, an increasing body of evidence indicates that defects in the protein clearance system play a crucial role in the gradual accumulation of protein aggregates. Among protein degradation systems, the endosome-autophagosome-lysosome pathway (EALP) is the main degradation machinery, especially for large protein aggregates. Lysosomal dysfunction or defects in fusion with vesicles containing cargo are commonly observed abnormalities in proteinopathic neurodegenerative diseases. In this review, we discuss the available evidence for a mechanistic connection between components of the EALP-especially lysosomes-and neurodegenerative diseases. We also focus on lysosomal pH regulation and its significance in maintaining flux through the EALP. Finally, we suggest that raising cAMP and free zinc levels in brain cells may be beneficial in normalizing lysosomal pH and EALP flux.
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    8. 8
      Settembre, C.; Fraldi, A.; Medina, D. L.; Ballabio, A. Signals for the Lysosome: A Control Center for Cellular Clearance and Energy Metabolism. Nat. Rev. Mol. Cell Biol. 2013, 14, 283– 296,  DOI: 10.1038/nrm3565
      8
      Signals from the lysosome: a control centre for cellular clearance and energy metabolism
      Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L.; Ballabio, Andrea
      Nature Reviews Molecular Cell Biology (2013), 14 (5), 283-296CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degrdn. and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metab. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.
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    9. 9
      Monaco, A.; Fraldi, A. Protein Aggregation and Dysfunction of Autophagy-Lysosomal Pathway: A Vicious Cycle in Lysosomal Storage Diseases. Front. Mol. Neurosci. 2020, 13, 37,  DOI: 10.3389/fnmol.2020.00037
      9
      Protein aggregation and dysfunction of autophagy-lysosomal pathway: a vicious cycle in lysosomal storage diseases
      Monaco, Antonio; Fraldi, Alessandro
      Frontiers in Molecular Neuroscience (2020), 13 (), 37CODEN: FMNRAJ; ISSN:1662-5099. (Frontiers Media S.A.)
      Many neurodegenerative conditions are characterized by the deposition of protein aggregates (mainly amyloid-like) in the central nervous system (CNS). In postmitotic CNS cells protein aggregation causes cytotoxicity by interfering with various cellular functions. Mutations in different genes may directly cause protein aggregation. However, genetic factors together with aging may contribute to the onset of protein aggregation also by affecting cellular degradative functions, in particular the autophagy-lysosomal pathway (ALP). Increasing body of evidence show that ALP dysfunction and protein aggregation are functionally interconnected and induce each other during neurodegenerative processes. We will summarize the findings supporting these concepts by focusing on lysosomal storage diseases (LSDs), a class of metabolic inherited conditions characterized by global lysosomal dysfunction and often assocd. to a severe neurodegenerative course. We propose a model by which the inherited lysosomal defects initiate aggregate-prone protein deposition, which, in turns, worsen ALP degrdn. function, thus generating a vicious cycle, which boost neurodegenerative cascades.
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    10. 10
      Ross, C. A.; Poirier, M. A. Protein Aggregation and Neurodegenerative Disease. Nat. Med. 2004, 10, S10– S17,  DOI: 10.1038/nm1066
      10
      Protein aggregation and neurodegenerative disease
      Ross Christopher A; Poirier Michelle A
      Nature medicine (2004), 10 Suppl (), S10-7 ISSN:1078-8956.
      Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.
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    11. 11
      Baxi, K.; Ghavidel, A.; Waddell, B.; Harkness, T. A.; de Carvalho, C. E. Regulation of Lysosomal Function by the DAF-16 Forkhead Transcription Factor Couples Reproduction to Aging in Caenorhabditis Elegans. Genetics 2017, 207, 83– 101,  DOI: 10.1534/genetics.117.204222
      11
      Regulation of lysosomal function by the daf-16 forkhead transcription factor couples reproduction to aging in Caenorhabditis elegans
      Baxi, Kunal; Ghavidel, Ata; Waddell, Brandon; Harkness, Troy A.; de Carvalho, Carlos E.
      Genetics (2017), 207 (1), 83-101CODEN: GENTAE; ISSN:0016-6731. (Genetics Society of America)
      Aging in eukaryotes is accompanied by widespread deterioration of the somatic tissue. Yet, abolishing germ cells delays the age-dependent somatic decline in Caenorhabditis elegans. In adult worms lacking germ cells, the activation of the DAF-9/DAF-12 steroid signaling pathway in the gonad recruits DAF-16 activity in the intestine to promote longevity-assocd. phenotypes. However, the impact of this pathway on the fitness of normally reproducing animals is less clear. Here, we explore the link between progeny prodn. and somatic aging and identify the loss of lysosomal acidity-a crit. regulator of the proteolytic output of these organelles-as a novel biomarker of aging in C. elegans. The increase in lysosomal pH in older worms is not a passive consequence of aging, but instead is timed with the cessation of reprodn., and correlates with the redn. in proteostasis in early adult life. Our results further implicate the steroid signaling pathway and DAF-16 in dynamically regulating lysosomal pH in the intestine of wildtype worms in response to the reproductive cycle. In the intestine of reproducing worms, DAF-16 promotes acidic lysosomes by upregulating the expression of v-ATPase genes. These findings support a model in which protein clearance in the soma is linked to reprodn. in the gonad via the active regulation of lysosomal acidification.
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    12. 12
      Colacurcio, D. J.; Nixon, R. A. Disorders of Lysosomal Acidification─The Emerging Role of v-ATPase in Aging and Neurodegenerative Disease. Ageing Res. Rev. 2016, 32, 75– 88,  DOI: 10.1016/j.arr.2016.05.004
      12
      Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease
      Colacurcio, Daniel J.; Nixon, Ralph A.
      Ageing Research Reviews (2016), 32 (), 75-88CODEN: ARRGAK; ISSN:1568-1637. (Elsevier B.V.)
      Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degrdn. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metab. and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson disease and Alzheimer disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal hydrolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clin. and exptl. studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum.
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    13. 13
      Hughes, A. L.; Gottschling, D. E. An Early Age Increase in Vacuolar PH Limits Mitochondrial Function and Lifespan in Yeast. Nature 2012, 492, 261– 265,  DOI: 10.1038/nature11654
      13
      An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast
      Hughes, Adam L.; Gottschling, Daniel E.
      Nature (London, United Kingdom) (2012), 492 (7428), 261-265CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asym. divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degrdn., but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a crit. regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.
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    14. 14
      Lee, J.-H.; McBrayer, M. K.; Wolfe, D. M.; Haslett, L. J.; Kumar, A.; Sato, Y.; Lie, P. P. Y.; Mohan, P.; Coffey, E. E.; Kompella, U.; Mitchell, C. H.; Lloyd-Evans, E.; Nixon, R. A. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating VATPase-Mediated Lysosome Acidification. Cell Rep. 2015, 12, 1430– 1444,  DOI: 10.1016/j.celrep.2015.07.050
      14
      Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification
      Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.
      Cell Reports (2015), 12 (9), 1430-1444CODEN: CREED8; ISSN:2211-1247. (Cell Press)
      Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common mol. mechanism.
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    15. 15
      Sun, Y.; Li, M.; Zhao, D.; Li, X.; Yang, C.; Wang, X. Lysosome Activity Is Modulated by Multiple Longevity Pathways and Is Important for Lifespan Extension in C. Elegans. Elife 2020, 9, e55745  DOI: 10.7554/eLife.55745
      15
      Lysosome activity is modulated by multiple longevity pathways and is important for lifespan extension in C. elegans
      Sun, Yanan; Li, Meijiao; Zhao, Dongfeng; Li, Xin; Yang, Chonglin; Wang, Xiaochen
      eLife (2020), 9 (), e55745/1-e55745/28CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Lysosomes play important roles in cellular degrdn. to maintain cell homeostasis. In order to understand whether and how lysosomes alter with age and contribute to lifespan regulation, we characterized multiple properties of lysosomes during the aging process in C. elegans. We uncovered age-dependent alterations in lysosomal morphol., motility, acidity and degrdn. activity, all of which indicate a decline in lysosome function with age. The ageassocd. lysosomal changes are suppressed in the long-lived mutants daf-2, eat-2 and isp-1, which extend lifespan by inhibiting insulin/IGF-1 signaling, reducing food intake and impairing mitochondrial function, resp. We found that 43 lysosome genes exhibit reduced expression with age, including genes encoding subunits of the proton pump V-ATPase and cathepsin proteases. The expression of lysosome genes is upregulated in the long-lived mutants, and this upregulation requires the functions of DAF-16/FOXO and SKN-1/NRF2 transcription factors. Impairing lysosome function affects clearance of aggregate-prone proteins and disrupts lifespan extension in daf-2, eat-2 and isp-1 worms. Our data indicate that lysosome function is modulated by multiple longevity pathways and is important for lifespan extension.
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    16. 16
      Tong, B. C.-K.; Wu, A. J.; Huang, A. S.; Dong, R.; Malampati, S.; Iyaswamy, A.; Krishnamoorthi, S.; Sreenivasmurthy, S. G.; Zhu, Z.; Su, C.; Liu, J.; Song, J.; Lu, J.-H.; Tan, J.; Pan, W.; Li, M.; Cheung, K.-H. Lysosomal TPCN (Two Pore Segment Channel) Inhibition Ameliorates Beta-Amyloid Pathology and Mitigates Memory Impairment in Alzheimer Disease. Autophagy 2021, 18, 624– 642,  DOI: 10.1080/15548627.2021.1945220
      There is no corresponding record for this reference.
    17. 17
      Chin, M. Y.; Espinosa, J. A.; Pohan, G.; Markossian, S.; Arkin, M. R. Reimagining Dots and Dashes: Visualizing Structure and Function of Organelles for High-Content Imaging Analysis. Cell Chem. Biol. 2021, 28, 320– 337,  DOI: 10.1016/j.chembiol.2021.01.016
      17
      Reimagining dots and dashes: visualizing structure and function of organelles for high-content imaging analysis
      Chin, Marcus Y.; Espinosa, Jether Amos; Pohan, Grace; Markossian, Sarine; Arkin, Michelle R.
      Cell Chemical Biology (2021), 28 (3), 320-337CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      A review. Organelles are responsible for biochem. and cellular processes that sustain life and their dysfunction causes diseases from cancer to neurodegeneration. While researchers are continuing to appreciate new roles of organelles in disease, the rapid development of specifically targeted fluorescent probes that report on the structure and function of organelles will be crit. to accelerate drug discovery. Here, we highlight four organelles that collectively exemplify the progression of phenotypic discovery, starting with mitochondria, where many functional probes have been described, then continuing with lysosomes and Golgi and concluding with nascently described membraneless organelles. We introduce emerging probe designs to explore organelle-specific morphol. and dynamics and highlight recent case studies using high-content anal. to stimulate further development of probes and approaches for organellar high-throughput screening.
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    18. 18
      Ponsford, A. H.; Ryan, T. A.; Raimondi, A.; Cocucci, E.; Wycislo, S. A.; Fröhlich, F.; Swan, L. E.; Stagi, M. Live Imaging of Intra-Lysosome PH in Cell Lines and Primary Neuronal Culture Using a Novel Genetically Encoded Biosensor. Autophagy 2020, 17, 1500,  DOI: 10.1080/15548627.2020.1771858
      There is no corresponding record for this reference.
    19. 19
      Webb, B. A.; Aloisio, F. M.; Charafeddine, R. A.; Cook, J.; Wittmann, T.; Barber, D. L. PHLARE: A New Biosensor Reveals Decreased Lysosome PH in Cancer Cells. MBoC 2021, 32, 91,  DOI: 10.1091/mbc.E20-06-0383
      There is no corresponding record for this reference.
    20. 20
      Chin, M. Y.-Y. Exploring Lysosomal PH as a Therapeutic Strategy for Neurodegeneration; UCSF, 2021.
      There is no corresponding record for this reference.
    21. 21
      Chin, M. Y.; Patwardhan, A. R.; Ang, K.-H.; Wang, A. L.; Alquezar, C.; Welch, M.; Nguyen, P. T.; Grabe, M.; Molofsky, A. V.; Arkin, M. R.; Kao, A. W. Genetically Encoded, PH-Sensitive MTFP1 Biosensor for Probing Lysosomal PH. ACS Sens. 2021, 6, 2168,  DOI: 10.1021/acssensors.0c02318
      21
      Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH
      Chin, Marcus Y.; Patwardhan, Anand R.; Ang, Kean-Hooi; Wang, Austin L.; Alquezar, Carolina; Welch, Mackenzie; Nguyen, Phi T.; Grabe, Michael; Molofsky, Anna V.; Arkin, Michelle R.; Kao, Aimee W.
      ACS Sensors (2021), 6 (6), 2168-2180CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
      Lysosomes are important sites for macromol. degrdn., defined by an acidic lumenal pH of ~ 4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-assocd. membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quant. imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.
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    22. 22
      Encinas, M.; Iglesias, M.; Liu, Y.; Wang, H.; Muhaisen, A.; Ceña, V.; Gallego, C.; Comella, J. X. Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differentiated, Neurotrophic Factor-Dependent, Human Neuron-Like Cells. J. Neurochem. 2000, 75, 991– 1003,  DOI: 10.1046/j.1471-4159.2000.0750991.x
      22
      Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells
      Encinas, Mario; Iglesias, Montse; Liu, Yuhui; Wang, Hongyin; Muhaisen, Ashraf; Cena, Valentin; Gallego, Carme; Comella, Joan X.
      Journal of Neurochemistry (2000), 75 (3), 991-1003CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
      A rapid and simple procedure is presented to obtain nearly pure populations of human neuron-like cells from the SH-SY5Y neuroblastoma cell line. Sequential exposure of SH-SY5Y cells to retinoic acid and brain-derived neurotrophic factor in serum-free medium yields homogeneous populations of cells with neuronal morphol., avoiding the presence of other neural crest derivs. that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5-bromo-2'-deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain-derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron-specific enolase, and growth-assocd. protein-43 as well as neuronal polarity markers such as tau and microtubule-assocd. protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the neg. staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large-scale biochem. and mol. studies due to its susceptibility to genetic manipulation and the availability of an unlimited amt. of cells.
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    23. 23
      Forster, J. I.; Köglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A. Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability. J. Biomol. Screen 2016, 21, 496– 509,  DOI: 10.1177/1087057115625190
      23
      Characterization of differentiated SH-SY5Y as neuronal screening model reveals increased oxidative vulnerability
      Forster, J. I.; Koglsberger, S.; Trefois, C.; Boyd, O.; Baumuratov, A. S.; Buck, L.; Balling, R.; Antony, P. M. A.
      Journal of Biomolecular Screening (2016), 21 (5), 496-509CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
      The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell lines in neuroscience and neuroblastoma research. However undifferentiated SH-SY5Y cells share few properties with mature neurons. In this study we present an optimized neuronal differentiation protocol for SH-SY5Y that requires only two work steps and 6 days. After differentiation the cells present increased levels of ATP and plasma membrane activity but reduced expression of energetic stress response genes. Differentiation results in reduced mitochondrial membrane potential and decreased robustness toward perturbations with -hydroxydopamine. We are convinced that the presented differentiation method will leverage genetic and chem. high-throughput screening projects targeting pathways that are involved in the selective vulnerability of neurons with high energetic stress levels.
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    24. 24
      Xicoy, H.; Wieringa, B.; Martens, G. J. M. The SH-SY5Y Cell Line in Parkinson’s Disease Research: A Systematic Review. Mol. Neurodegener. 2017, 12, 10,  DOI: 10.1186/s13024-017-0149-0
      24
      The SH-SY5Y cell line in Parkinson's disease research: a systematic review
      Xicoy, Helena; Wieringa, Be; Martens, Gerard J. M.
      Molecular Neurodegeneration (2017), 12 (), 10/1-10/11CODEN: MNOEAZ; ISSN:1750-1326. (BioMed Central Ltd.)
      Parkinson's disease (PD) is a devastating and highly prevalent neurodegenerative disease for which only symptomatic treatment is available. In order to develop a truly effective disease-modifying therapy, improvement of our current understanding of the mol. and cellular mechanisms underlying PD pathogenesis and progression is crucial. For this purpose, standardization of research protocols and disease models is necessary. As human dopaminergic neurons, the cells mainly affected in PD, are difficult to obtain and maintain as primary cells, current PD research is mostly performed with permanently established neuronal cell models, in particular the neuroblastoma SH-SY5Y lineage. This cell line is frequently chosen because of its human origin, catecholaminergic (though not strictly dopaminergic) neuronal properties, and ease of maintenance. However, there is no consensus on many fundamental aspects that are assocd. with its use, such as the effects of culture media compn. and of variations in differentiation protocols. Here we present the outcome of a systematic review of scientific articles that have used SH-SY5Y cells to explore PD. We describe the cell source, culture conditions, differentiation protocols, methods/approaches used to mimic PD and the preclin. validation of the SH-SY5Y findings by employing alternative cellular and animal models. Thus, this overview may help to standardize the use of the SH-SY5Y cell line in PD research and serve as a future user's guide.
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    25. 25
      Bright, N. A.; Davis, L. J.; Luzio, J. P. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity. Curr. Biol. 2016, 26, 2233– 2245,  DOI: 10.1016/j.cub.2016.06.046
      25
      Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity
      Bright, Nicholas A.; Davis, Luther J.; Luzio, J. Paul
      Current Biology (2016), 26 (17), 2233-2245CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      The endocytic delivery of macromols. from the mammalian cell surface for degrdn. by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochem. to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake expts., we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equil. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.
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    26. 26
      Cabukusta, B.; Neefjes, J. Mechanisms of Lysosomal Positioning and Movement. Traffic 2018, 19, 761– 769,  DOI: 10.1111/tra.12587
      26
      Mechanisms of lysosomal positioning and movement
      Cabukusta, Birol; Neefjes, Jacques
      Traffic (Oxford, United Kingdom) (2018), 19 (10), 761-769CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
      A review. Lysosomes are highly dynamic organelles that can move rapidly throughout the cell. They distribute in a rather immobile pool located around the microtubule-organizing center in a "cloud," and a highly dynamic pool in the cell periphery. Their spatiotemporal characteristics allow them to carry out multiple biol. functions, such as cargo degrdn., antigen presentation and plasma membrane repair. Therefore, it is not surprising that lysosomal dysfunction underlies various diseases, including cancer, neurodegenerative and autoimmune diseases. In most of these biol. events, the involvement of lysosomes is dependent on their ability to move throughout the cytoplasm, to find and fuse to the correct compartments to receive and deliver substrates for further handling. These dynamics are orchestrated by motor proteins moving along cytoskeletal components. The complexity of the mechanisms responsible for controlling lysosomal transport has recently been appreciated and has yielded novel insights into interorganellar communication, as well as lipid-protein interplay. In this review, we discuss the current understanding of the mechanisms of lysosomal transport and the mol. machineries that control this mobility.
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    27. 27
      Johnson, D. E.; Ostrowski, P.; Jaumouillé, V.; Grinstein, S. The Position of Lysosomes within the Cell Determines Their Luminal PH. J. Cell Biol. 2016, 212, 677– 692,  DOI: 10.1083/jcb.201507112
      27
      The position of lysosomes within the cell determines their luminal pH
      Johnson, Danielle E.; Ostrowski, Philip; Jaumouille, Valentin; Grinstein, Sergio
      Journal of Cell Biology (2016), 212 (6), 677-692CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      We examd. the luminal pH of individual lysosomes using quant. ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+-ATPase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered compn. of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b dets. the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 d. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is assocd. with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.
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    28. 28
      Gulnik, S.; Baldwin, E. T.; Tarasova, N.; Erickson, J. Human Liver Cathepsin D. J. Mol. Biol. 1992, 227, 265– 270,  DOI: 10.1016/0022-2836(92)90696-H
      28
      Human liver cathepsin D. Purification, crystallization and preliminary x-ray diffraction analysis of a lysosomal enzyme
      Gulnik, Sergei; Baldwin, Eric T.; Tarasova, Nadezhda; Erickson, John
      Journal of Molecular Biology (1992), 227 (1), 265-70CODEN: JMOBAK; ISSN:0022-2836.
      The 2-chain form of active cathepsin D, a glycosylated, lysosomal aspartic proteinase, was isolated from human liver. Isoelec. focusing revealed 2 major species of enzyme that differed by approx. 0.2 pI unit. Crystals suitable for x-ray diffraction anal. were prepd. from acidic solns. using pptn. with ammonium sulfate. The hexagonal crystals diffracted x-rays to beyond 3.1 Å resoln. and belonged to space group P61 (or P65) with cell consts. a = b = 125.9 Å, c = 104.1 Å, γ = 120.0°. The crystals likely contain 2 mols. in the asym. unit, giving a solvent content of 56% (v/w). Biochem. anal. of crystals indicated that both isoforms were present in approx. equimolar proportions. Full structure detn. of the enzyme is underway.
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    29. 29
      Chen, C.-S.; Chen, W.-N. U.; Zhou, M.; Arttamangkul, S.; Haugland, R. P. Probing the Cathepsin D Using a BODIPY FL–Pepstatin A: Applications in Fluorescence Polarization and Microscopy. J. Biochem. Biophys. Methods 2000, 42, 137– 151,  DOI: 10.1016/S0165-022X(00)00048-8
      29
      Probing the cathepsin D using a BODIPY FL-pepstatin A: applications in fluorescence polarization and microscopy
      Chen, C.-S.; Chen, W.-N. U.; Zhou, M.; Arttamangkul, S.; Haugland, R. P.
      Journal of Biochemical and Biophysical Methods (2000), 42 (3), 137-151CODEN: JBBMDG; ISSN:0165-022X. (Elsevier Science Ireland Ltd.)
      Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathol. progressions during tumor formation and oxidn. stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL-pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D.
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    30. 30
      Phatnani, H.; Maniatis, T. Astrocytes in Neurodegenerative Disease. Cold Spring Harbor Perspect. Biol. 2015, 7, a020628,  DOI: 10.1101/cshperspect.a020628
      There is no corresponding record for this reference.
    31. 31
      Leng, K.; Rooney, B.; Kim, H.; Xia, W.; Koontz, M.; Krawczyk, M.; Zhang, Y.; Ullian, E. M.; Fancy, S. P. J.; Schrag, M. S.; Lippmann, E. S.; Kampmann, M. CRISPRi Screens in Human Astrocytes Elucidate Regulators of Distinct Inflammatory Reactive States; preprint. Neuroscience 2021,  DOI: 10.1101/2021.08.23.457400
      There is no corresponding record for this reference.
    32. 32
      Rooney, B.; Leng, K.; McCarthy, F.; Rose, I. V. L.; Herrington, K. A.; Bax, S.; Chin, M. Y.; Fathi, S.; Leonetti, M.; Kao, A. W.; Elias, J. E.; Kampmann, M. MTOR Controls Neurotoxic Lysosome Exocytosis in Inflammatory Reactive Astrocytes; preprint. Cell Biol. 2021,  DOI: 10.1101/2021.09.11.459904
      There is no corresponding record for this reference.
    33. 33
      Apsel, B.; Blair, J. A.; Gonzalez, B.; Nazif, T. M.; Feldman, M. E.; Aizenstein, B.; Hoffman, R.; Williams, R. L.; Shokat, K. M.; Knight, Z. A. Targeted Polypharmacology: Discovery of Dual Inhibitors of Tyrosine and Phosphoinositide Kinases. Nat. Chem. Biol. 2008, 4, 691– 699,  DOI: 10.1038/nchembio.117
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      Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases
      Apsel, Beth; Blair, Jimmy A.; Gonzalez, Beatriz; Nazif, Tamim M.; Feldman, Morri E.; Aizenstein, Brian; Hoffman, Randy; Williams, Roger L.; Shokat, Kevan M.; Knight, Zachary A.
      Nature Chemical Biology (2008), 4 (11), 691-699CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      The clin. success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of mols. that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chem. synthesis, X-ray crystallog. and kinome-level biochem. profiling, we identified compds. that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these mols. is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that compd. I blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These mols. demonstrate the feasibility of accessing a chem. space that intersects two families of oncogenes.
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    34. 34
      Bhagwat, S. V.; Gokhale, P. C.; Crew, A. P.; Cooke, A.; Yao, Y.; Mantis, C.; Kahler, J.; Workman, J.; Bittner, M.; Dudkin, L.; Epstein, D. M.; Gibson, N. W.; Wild, R.; Arnold, L. D.; Houghton, P. J.; Pachter, J. A. Preclinical Characterization of OSI-027, a Potent and Selective Inhibitor of MTORC1 and MTORC2: Distinct from Rapamycin. Mol. Cancer Ther. 2011, 10, 1394– 1406,  DOI: 10.1158/1535-7163.MCT-10-1099
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      Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin
      Bhagwat, Shripad V.; Gokhale, Prafulla C.; Crew, Andrew P.; Cooke, Andy; Yao, Yan; Mantis, Christine; Kahler, Jennifer; Workman, Jennifer; Bittner, Mark; Dudkin, Lorina; Epstein, David M.; Gibson, Neil W.; Wild, Robert; Arnold, Lee D.; Houghton, Peter J.; Pachter, Jonathan A.
      Molecular Cancer Therapeutics (2011), 10 (8), 1394-1406CODEN: MCTOCF; ISSN:1535-7163. (American Association for Cancer Research)
      The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clin. validated target. MTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metab., proliferation, and survival. Rapamycin and its analogs partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clin. utility in certain cancers. Here, we report the preclin. characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochem. IC50 values of 22 nmol/L and 65 nmol/L, resp. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kβ, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analog of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concn.-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clin. trials in cancer patients.
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    35. 35
      Falcon, B. L.; Barr, S.; Gokhale, P. C.; Chou, J.; Fogarty, J.; Depeille, P.; Miglarese, M.; Epstein, D. M.; McDonald, D. M. Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual MTORC1/MTORC2 Inhibitors. Cancer Res. 2011, 71, 1573– 1583,  DOI: 10.1158/0008-5472.CAN-10-3126
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      Reduced VEGF Production, Angiogenesis, and Vascular Regrowth Contribute to the Antitumor Properties of Dual mTORC1/mTORC2 Inhibitors
      Falcon, Beverly L.; Barr, Sharon; Gokhale, Prafulla C.; Chou, Jeyling; Fogarty, Jennifer; Depeille, Philippe; Miglarese, Mark; Epstein, David M.; McDonald, Donald M.
      Cancer Research (2011), 71 (5), 1573-1583CODEN: CNREA8; ISSN:0008-5472. (American Association for Cancer Research)
      The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiol. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examd. the effects of mTORC1/2 dual inhibition on VEGF prodn., tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF prodn. in tumors in a manner assocd. with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral prodn. of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist. Cancer Res; 71(5); 1573-83.
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    36. 36
      Kim, Y. C.; Guan, K.-L. MTOR: A Pharmacologic Target for Autophagy Regulation. J. Clin. Invest. 2015, 125, 25– 32,  DOI: 10.1172/JCI73939
      36
      mTOR: a pharmacologic target for autophagy regulation
      Kim Young Chul; Guan Kun-Liang
      The Journal of clinical investigation (2015), 125 (1), 25-32 ISSN:.
      mTOR, a serine/threonine kinase, is a master regulator of cellular metabolism. mTOR regulates cell growth and proliferation in response to a wide range of cues, and its signaling pathway is deregulated in many human diseases. mTOR also plays a crucial role in regulating autophagy. This Review provides an overview of the mTOR signaling pathway, the mechanisms of mTOR in autophagy regulation, and the clinical implications of mTOR inhibitors in disease treatment.
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    37. 37
      Yim, W. W.-Y.; Mizushima, N. Lysosome Biology in Autophagy. Cell Discov. 2020, 6, 1– 12,  DOI: 10.1038/s41421-020-0141-7
      There is no corresponding record for this reference.
    38. 38
      Zhou, J.; Tan, S.-H.; Nicolas, V.; Bauvy, C.; Yang, N.-D.; Zhang, J.; Xue, Y.; Codogno, P.; Shen, H.-M. Activation of Lysosomal Function in the Course of Autophagy via MTORC1 Suppression and Autophagosome-Lysosome Fusion. Cell Res. 2013, 23, 508– 523,  DOI: 10.1038/cr.2013.11
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      Activation of lysosomal function in the course of autophagy via mTORC1 suppression and autophagosome-lysosome fusion
      Zhou, Jing; Tan, Shi-Hao; Nicolas, Valerie; Bauvy, Chantal; Yang, Nai-Di; Zhang, Jianbin; Xue, Yuan; Codogno, Patrice; Shen, Han-Ming
      Cell Research (2013), 23 (4), 508-523CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
      Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy. In this study, we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal function is assocd. with the suppression of mTOR complex 1 (mTORC1), but not mTORC2, and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function. Third, we examd. the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation. Finally, Atg5 or Atg7 deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, suggesting that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Taken together, this study demonstrates that in the course of autophagy, lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXltFCmsLg%253D&md5=f4b34cb44f2f3773f249f23128e49f72
    39. 39
      Jung, C. H.; Jun, C. B.; Ro, S.-H.; Kim, Y.-M.; Otto, N. M.; Cao, J.; Kundu, M.; Kim, D.-H. ULK-Atg13-FIP200 Complexes Mediate MTOR Signaling to the Autophagy Machinery. Mol. Biol. Cell 2009, 20, 1992– 2003,  DOI: 10.1091/mbc.E08-12-1249
      39
      ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery
      Jung, Chang Hwa; Jun, Chang Bong; Ro, Seung-Hyun; Kim, Young-Mi; Otto, Neil Michael; Cao, Jing; Kundu, Mondira; Kim, Do-Hyung
      Molecular Biology of the Cell (2009), 20 (7), 1992-2003CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      Autophagy, the starvation-induced degrdn. of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homolog of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
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    40. 40
      Klionsky, D. J.; Abdelmohsen, K.; Abe, A.; Abedin, M. J.; Abeliovich, H.; Arozena, A. A.; Adachi, H.; Adams, C. M.; Adams, P. D. Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy (3rd Edition). Autophagy 2016, 12, 1– 222,  DOI: 10.1080/15548627.2016.1139264
      40
      Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
      Autophagy (2016), 12 (1), 1-222 ISSN:.
      There is no expanded citation for this reference.
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    41. 41
      Menzies, F. M.; Moreau, K.; Puri, C.; Renna, M.; Rubinsztein, D. C. Measurement of Autophagic Activity in Mammalian Cells. Curr. Protoc. Cell Biol. 2012, 54, 15.16.1– 15.16.25,  DOI: 10.1002/0471143030.cb1516s54
      There is no corresponding record for this reference.
    42. 42
      Bjørkøy, G.; Lamark, T.; Pankiv, S.; Øvervatn, A.; Brech, A.; Johansen, T. Monitoring Autophagic Degradation of P62/SQSTM1. Methods Enzymol. 2009, 452, 181– 197,  DOI: 10.1016/S0076-6879(08)03612-4
      42
      Monitoring autophagic degradation of p62/SQSTM1
      Bjorkoy Geir; Lamark Trond; Pankiv Serhiy; Overvatn Aud; Brech Andreas; Johansen Terje
      Methods in enzymology (2009), 452 (), 181-97 ISSN:.
      The p62 protein, also called sequestosome 1 (SQSTM1), is a ubiquitin-binding scaffold protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative diseases and proteinopathies of the liver. The protein is able to polymerize via an N-terminal PB1 domain and can interact with ubiquitinated proteins via the C-terminal UBA domain. Also, p62/SQSTM1 binds directly to LC3 and GABARAP family proteins via a specific sequence motif. The protein is itself degraded by autophagy and may serve to link ubiquitinated proteins to the autophagic machinery to enable their degradation in the lysosome. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 may be used as a marker to study autophagic flux. Here, we present several protocols for monitoring autophagy-mediated degradation of p62 using Western blots, pulse-chase measurement of p62 half-life, immunofluorescence and immuno-electron microscopy, as well as live cell imaging with a pH-sensitive mCherry-GFP double tag. We also present data on species-specificity and map the epitopes recognized by several commercially available anti-p62 antibodies.
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    43. 43
      van der Poel, S.; Wolthoorn, J.; van den Heuvel, D.; Egmond, M.; Groux-Degroote, S.; Neumann, S.; Gerritsen, H.; van Meer, G.; Sprong, H. Hyperacidification of Trans-Golgi Network and Endo/Lysosomes in Melanocytes by Glucosylceramide-Dependent V-ATPase Activity. Traffic 2011, 12, 1634– 1647,  DOI: 10.1111/j.1600-0854.2011.01263.x
      43
      Hyperacidification of trans-Golgi network and endo/lysosomes in melanocytes by glucosylceramide-dependent V-ATPase activity
      van der Poel, Selene; Wolthoorn, Jasja; van den Heuvel, Dave; Egmond, Maarten; Groux-Degroote, Sophie; Neumann, Sylvia; Gerritsen, Hans; van Meer, Gerrit; Sprong, Hein
      Traffic (Oxford, United Kingdom) (2011), 12 (11), 1634-1647CODEN: TRAFFA; ISSN:1398-9219. (Wiley-Blackwell)
      Sphingolipids are considered to play a key role in protein sorting and membrane trafficking. In melanocytic cells, sorting of lysosomal and melanosomal proteins requires the sphingolipid glucosylceramide (GlcCer). This sorting information is located in the lumenal domain of melanosomal proteins. We found that two processes dependent on lumenal pH, protein sialylation and lysosomal acid lipase (LAL) activity were aberrant in GM95 melanocyte cells, which do not produce glycosphingolipids. Using fluorescence lifetime imaging microscopy (FLIM), we found that the lumenal pH in the trans-Golgi network and lysosomes of wild-type melanocyte MEB4 cells are >1 pH unit lower than GM95 cells and fibroblasts. In addn. to the lower pH found in vivo, the in vitro activity of the proton pump, the vacuolar-type H+-translocating ATPase (V-ATPase), was twofold higher in MEB4 compared to GM95 cells. The apparent Ki for inhibition of the V-ATPase by concanamycin A and archazolid A, which share a common binding site on the c-ring, was lower in glycosphingolipid-deficient GM95 cells. No difference between the MEB4 and GM95 cells was found for the V-ATPase inhibitors apicularen A and salicylihalimide. We conclude that hyperacidification in MEB4 cells requires glycosphingolipids and propose that low pH is necessary for protein sorting and melanosome biogenesis. Furthermore, we suggest that glycosphingolipids are indirectly involved in protein sorting and melanosome biogenesis by stimulating the proton pump, possibly through binding of GlcCer. These expts. establish, for the first time, a link between pH, glycosphingolipids and melanosome biogenesis in melanocytic MEB4 cells, to suggest a role for glycosphingolipids in hyperacidification in melanocytes.
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    44. 44
      Lenk, G. M.; Park, Y. N.; Lemons, R.; Flynn, E.; Plank, M.; Frei, C. M.; Davis, M. J.; Gregorka, B.; Swanson, J. A.; Meisler, M. H.; Kitzman, J. O. CRISPR Knockout Screen Implicates Three Genes in Lysosome Function. Sci. Rep. 2019, 9, 9609,  DOI: 10.1038/s41598-019-45939-w
      44
      CRISPR knockout screen implicates three genes in lysosome function
      Lenk Guy M; Park Young N; Lemons Rosemary; Flynn Emma; Plank Margaret; Frei Christen M; Meisler Miriam H; Kitzman Jacob O; Davis Michael J; Gregorka Brian; Swanson Joel A
      Scientific reports (2019), 9 (1), 9609 ISSN:.
      Defective biosynthesis of the phospholipid PI(3,5)P2 underlies neurological disorders characterized by cytoplasmic accumulation of large lysosome-derived vacuoles. To identify novel genetic causes of lysosomal vacuolization, we developed an assay for enlargement of the lysosome compartment that is amenable to cell sorting and pooled screens. We first demonstrated that the enlarged vacuoles that accumulate in fibroblasts lacking FIG4, a PI(3,5)P2 biosynthetic factor, have a hyperacidic pH compared to normal cells'. We then carried out a genome-wide knockout screen in human HAP1 cells for accumulation of acidic vesicles by FACS sorting. A pilot screen captured fifteen genes, including VAC14, a previously identified cause of endolysosomal vacuolization. Three genes not previously associated with lysosome dysfunction were selected to validate the screen: C10orf35, LRRC8A, and MARCH7. We analyzed two clonal knockout cell lines for each gene. All of the knockout lines contained enlarged acidic vesicles that were positive for LAMP2, confirming their endolysosomal origin. This assay will be useful in the future for functional evaluation of patient variants in these genes, and for a more extensive genome-wide screen for genes required for endolysosome function. This approach may also be adapted for drug screens to identify small molecules that rescue endolysosomal vacuolization.
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    45. 45
      De Duve, C.; De Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P.; Van Hoof, F. o. Lysosomotropic Agents. Biochem. Pharmacol. 1974, 23, 2495– 2531,  DOI: 10.1016/0006-2952(74)90174-9
      45
      Lysosomotropic agents
      De Duve, Christian; De Barsy, Thierry; Poole, Brian; Trouet, Andre; Tulkens, Paul; Van Hoof, Francois
      Biochemical Pharmacology (1974), 23 (18), 2495-531CODEN: BCPCA6; ISSN:0006-2952.
      A review of the nature, mechanisms of entry, and therapeutic possibilities of lysosomotropic agents, i.e., substances that are taken up selectively into lysosomes.
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    46. 46
      Kuzu, O. F.; Toprak, M.; Noory, M. A.; Robertson, G. P. Effect of Lysosomotropic Molecules on Cellular Homeostasis. Pharmacol. Res. 2017, 117, 177– 184,  DOI: 10.1016/j.phrs.2016.12.021
      46
      Effect of lysosomotropic molecules on cellular homeostasis
      Kuzu, Omer F.; Toprak, Mesut; Noory, M. Anwar; Robertson, Gavin P.
      Pharmacological Research (2017), 117 (), 177-184CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
      Weak bases that readily penetrate through the lipid bilayer and accumulate inside the acidic organelles are known as lysosomotropic mols. Many lysosomotropic compds. exhibit therapeutic activity and are commonly used as antidepressant, antipsychotic, antihistamine, or antimalarial agents. Interestingly, studies also have shown increased sensitivity of cancer cells to certain lysosomotropic agents and suggested their mechanism of action as a promising approach for selective destruction of cancer cells. However, their chemotherapeutic utility may be limited due to various side effects. Hence, understanding the homeostatic alterations mediated by lysosomotropic compds. has significant importance for revealing their true therapeutic potential as well as toxicity. In this review, after briefly introducing the concept of lysosomotropism and classifying the lysosomotropic compds. into two major groups according to their cytotoxicity on cancer cells, we focused on the subcellular alterations mediated by class-II lysosomotropic compds. Briefly, their effect on intracellular cholesterol homeostasis, autophagy and lysosomal sphingolipid metab. was discussed. Accordingly, class-II lysosomotropic mols. inhibit intracellular cholesterol transport, leading to the accumulation of cholesterol inside the late endosomal-lysosomal cell compartments. However, the accumulated lysosomal cholesterol is invisible to the cellular homeostatic circuits, hence class-II lysosomotropic mols. also upregulate cholesterol synthesis pathway as a downstream event. Considering the fact that Niemann-Pick disease, a lysosomal cholesterol storage disorder, also triggers similar pathol. abnormalities, this review combines the knowledge obtained from the Niemann-Pick studies and lysosomotropic compds. Taken together, this review is aimed at allowing readers a better understanding of subcellular alterations mediated by lysosomotropic drugs, as well as their potential therapeutic and/or toxic activities.
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    47. 47
      Silva, M. C.; Nandi, G. A.; Tentarelli, S.; Gurrell, I. K.; Jamier, T.; Lucente, D.; Dickerson, B. C.; Brown, D. G.; Brandon, N. J.; Haggarty, S. J. Prolonged Tau Clearance and Stress Vulnerability Rescue by Pharmacological Activation of Autophagy in Tauopathy Neurons. Nat. Commun. 2020, 11, 3258,  DOI: 10.1038/s41467-020-16984-1
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      Prolonged tau clearance and stress vulnerability rescue by pharmacological activation of autophagy in tauopathy neurons
      Silva, M. Catarina; Nandi, Ghata A.; Tentarelli, Sharon; Gurrell, Ian K.; Jamier, Tanguy; Lucente, Diane; Dickerson, Bradford C.; Brown, Dean G.; Brandon, Nicholas J.; Haggarty, Stephen J.
      Nature Communications (2020), 11 (1), 3258CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Tauopathies are neurodegenerative diseases assocd. with accumulation of abnormal tau protein in the brain. Patient iPSC-derived neuronal cell models replicate disease-relevant phenotypes ex vivo that can be pharmacol. targeted for drug discovery. Here, we explored autophagy as a mechanism to reduce tau burden in human neurons and, from a small-mol. screen, identify the mTOR inhibitors OSI-027, AZD2014 and AZD8055. These compds. are more potent than rapamycin, and robustly downregulate phosphorylated and insol. tau, consequently reducing tau-mediated neuronal stress vulnerability. MTORC1 inhibition and autophagy activity are directly linked to tau clearance. Notably, single-dose treatment followed by washout leads to a prolonged redn. of tau levels and toxicity for 12 days, which is mirrored by a sustained effect on mTORC1 inhibition and autophagy. This new insight into the pharmacodynamics of mTOR inhibitors in regulation of neuronal autophagy may contribute to development of therapies for tauopathies.
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    48. 48
      Wang, M.-X.; Cheng, X.-Y.; Jin, M.; Cao, Y.-L.; Yang, Y.-P.; Wang, J.-D.; Li, Q.; Wang, F.; Hu, L.-F.; Liu, C.-F. TNF Compromises Lysosome Acidification and Reduces α-Synuclein Degradation via Autophagy in Dopaminergic Cells. Exp. Neurol. 2015, 271, 112– 121,  DOI: 10.1016/j.expneurol.2015.05.008
      48
      TNF compromises lysosome acidification and reduces α-synuclein degradation via autophagy in dopaminergic cells
      Wang, Mei-Xia; Cheng, Xiao-Yu; Jin, Mengmeng; Cao, Yu-Lan; Yang, Ya-Ping; Wang, Jian-Da; Li, Qian; Wang, Fen; Hu, Li-Fang; Liu, Chun-Feng
      Experimental Neurology (2015), 271 (), 112-121CODEN: EXNEAC; ISSN:0014-4886. (Elsevier Inc.)
      Tumor necrosis factor-α (TNF) is increasingly implicated as a crit. pro-inflammatory cytokine involved in chronic inflammation and neurodegeneration of Parkinson's disease (PD). However, the cellular and mol. events that lead to dopaminergic neuron degeneration are not fully understood. In this study, we demonstrated that microglia-released and recombinant TNF disrupted α-synuclein (α-SYN) degrdn. and caused its accumulation in PC12 cells and midbrain neurons. At subtoxic doses, recombinant TNF was found to increase the no. of LC3 puncta dots and LC3II protein level, assocd. with the increases of P62 protein level. Inhibition of lysosomal degrdn. with Bafilomycin A1 pretreatment abrogated the TNF-induced elevation in LC3II protein level whereas autophagy inhibitor 3-methyladenine did not affect it. Moreover, TNF led to a marked increase in the no. of yellow LC3 dots with a marginal elevation in red-only dots in RFP-GFP-tandem fluorescent LC3 (tf-LC3) transfected PC12 cells, implying the impairment in autophagic flux. Furthermore, TNF treatment reduced lysosomal acidification, as LysoTracker Red fluorescence and LysoSensor fluorescence shift from blue to yellow was markedly decreased in TNF-treated PC12 cells. Co-treatment with mammalian target of rapamycin kinase complex 1 (mTORC1) inhibitor PP242, which activated transcription factor EB (TFEB) signaling and lysosome biogenesis, partially rescued the accumulation of α-SYN in PC12 cells and midbrain neurons. Taken together, our results demonstrated that at subtoxic levels, TNF was able to impair autophagic flux and result in α-SYN accumulation by compromising lysosomal acidification in dopaminergic cells. This may represent a novel mechanism for TNF-induced dopaminergic neuron degeneration in PD.
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    49. 49
      Vogler, C.; Rosenberg, H. S.; Williams, J. C.; Butler, I.; Opitz, J. M.; Bernstein, J. Electron Microscopy in the Diagnosis of Lysosomal Storage Diseases. Am. J. Med. Genet. Suppl. 1987, 28, 243– 255,  DOI: 10.1002/ajmg.1320280529
      There is no corresponding record for this reference.
    50. 50
      Jung, M.; Choi, H.; Mun, J. Y. The Autophagy Research in Electron Microscopy. Appl. Microsc. 2019, 49, 11,  DOI: 10.1186/s42649-019-0012-6
      50
      The autophagy research in electron microscopy
      Jung Minkyo; Mun Ji Young; Choi Hyosun
      Applied microscopy (2019), 49 (1), 11 ISSN:.
      Autophagy, a highly conserved process of eukaryotic cellular recycling, plays an important role in cell survival and maintenance. Dysfunctional autophagy contributes to the pathologies of many human diseases. Many studies have attempted to clarify the process of autophagy. Here, we review morphological studies of autophagy involving electron microscopy.
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    51. 51
      Kim, J.; Kundu, M.; Viollet, B.; Guan, K.-L. AMPK and MTOR Regulate Autophagy through Direct Phosphorylation of Ulk1. Nat. Cell Biol. 2011, 13, 132– 141,  DOI: 10.1038/ncb2152
      51
      AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1
      Kim, Joungmok; Kundu, Mondira; Viollet, Benoit; Guan, Kun-Liang
      Nature Cell Biology (2011), 13 (2), 132-141CODEN: NCBIFN; ISSN:1465-7392. (Nature Publishing Group)
      Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metab. to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a mol. mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homolog of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. This study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
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    52. 52
      Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lélias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A Small Molecule-Kinase Interaction Map for Clinical Kinase Inhibitors. Nat. Biotechnol. 2005, 23, 329– 336,  DOI: 10.1038/nbt1068
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      A small molecule-kinase interaction map for clinical kinase inhibitors
      Fabian, Miles A.; Biggs, William H.; Treiber, Daniel K.; Atteridge, Corey E.; Azimioara, Mihai D.; Benedetti, Michael G.; Carter, Todd A.; Ciceri, Pietro; Edeen, Philip T.; Floyd, Mark; Ford, Julia M.; Galvin, Margaret; Gerlach, Jay L.; Grotzfeld, Robert M.; Herrgard, Sanna; Insko, Darren E.; Insko, Michael A.; Lai, Andiliy G.; Lelias, Jean-Michel; Mehta, Shamal A.; Milanov, Zdravko V.; Velasco, Anne Marie; Wodicka, Lisa M.; Patel, Hitesh K.; Zarrinkar, Patrick P.; Lockhart, David J.
      Nature Biotechnology (2005), 23 (3), 329-336CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Kinase inhibitors show great promise as a new class of therapeutics. Here the authors describe an efficient way to det. kinase inhibitor specificity by measuring binding of small mols. to the ATP site of kinases. The authors have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clin. development, against a panel of 119 protein kinases. The authors find that specificity varies widely and is not strongly correlated with chem. structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. The authors also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small mol.-protein interaction map for clin. compds. across a large no. of related proteins.
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    53. 53
      Elzinga, B. M.; Nyhan, M. J.; Crowley, L. C.; O’Donovan, T. R.; Cahill, M. R.; McKenna, S. L. Induction of Autophagy by Imatinib Sequesters Bcr-Abl in Autophagosomes and down-Regulates Bcr-Abl Protein. Am. J. Hematol. 2013, 88, 455– 462,  DOI: 10.1002/ajh.23428
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      Induction of autophagy by Imatinib sequesters Bcr-Abl in autophagosomes and down-regulates Bcr-Abl protein
      Elzinga, Baukje M.; Nyhan, Michelle J.; Crowley, Lisa C.; O'Donovan, Tracey R.; Cahill, Mary R.; McKenna, Sharon L.
      American Journal of Hematology (2013), 88 (6), 455-462CODEN: AJHEDD; ISSN:0361-8609. (Wiley-Liss, Inc.)
      Chronic Myeloid Leukemia (CML) is a disease of hematopoietic stem cells which harbor the chimeric gene Bcr-Abl. Expression levels of this constitutively active tyrosine kinase are crit. for response to tyrosine kinase inhibitor treatment and also disease progression, yet the regulation of protein stability is poorly understood. We have previously demonstrated that imatinib can induce autophagy in Bcr-Abl expressing cells. Autophagy has been assocd. with the clearance of large macromol. signaling complexes and abnormal proteins, however, the contribution of autophagy to the turnover of Bcr-Abl protein in imatinib treated cells is unknown. In this study, we show that following imatinib treatment, Bcr-Abl is sequestered into vesicular structures that co-localize with the autophagy marker LC3 or GABARAP. This assocn. is inhibited by siRNA mediated knockdown of autophagy regulators (Beclin 1/ATG7). Pharmacol. inhibition of autophagy also reduced Bcr-Abl/LC3 co-localization in both K562 and CML patient cells. Bcr-Abl protein expression was reduced with imatinib treatment. Inhibition of both autophagy and proteasome activity in imatinib treated cells was required to restore Bcr-Abl protein levels to those of untreated cells. This ability to down-regulate Bcr-Abl protein levels through the induction of autophagy may be an addnl. and important feature of the activity of imatinib. 88:455-462, 2013. © 2013 Wiley Periodicals, Inc.
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    54. 54
      Iershov, A.; Nemazanyy, I.; Alkhoury, C.; Girard, M.; Barth, E.; Cagnard, N.; Montagner, A.; Chretien, D.; Rugarli, E. I.; Guillou, H.; Pende, M.; Panasyuk, G. The Class 3 PI3K Coordinates Autophagy and Mitochondrial Lipid Catabolism by Controlling Nuclear Receptor PPARα. Nat. Commun. 2019, 10, 1566,  DOI: 10.1038/s41467-019-09598-9
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      The class 3 PI3K coordinates autophagy and mitochondrial lipid catabolism by controlling nuclear receptor PPARα
      Iershov Anton; Nemazanyy Ivan; Alkhoury Chantal; Girard Muriel; Pende Mario; Panasyuk Ganna; Iershov Anton; Nemazanyy Ivan; Alkhoury Chantal; Girard Muriel; Pende Mario; Panasyuk Ganna; Iershov Anton; Nemazanyy Ivan; Alkhoury Chantal; Girard Muriel; Pende Mario; Panasyuk Ganna; Nemazanyy Ivan; Girard Muriel; Barth Esther; Rugarli Elena I; Cagnard Nicolas; Montagner Alexandra; Chretien Dominique; Chretien Dominique; Guillou Herve
      Nature communications (2019), 10 (1), 1566 ISSN:.
      The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.
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    55. 55
      Wang, S.; Li, J.; Du, Y.; Xu, Y.; Wang, Y.; Zhang, Z.; Xu, Z.; Zeng, Y.; Mao, X.; Cao, B. The Class I PI3K Inhibitor S14161 Induces Autophagy in Malignant Blood Cells by Modulating the Beclin 1/Vps34 Complex. J. Pharmacol. Sci. 2017, 134, 197– 202,  DOI: 10.1016/j.jphs.2017.07.001
      55
      The Class I PI3K inhibitor S14161 induces autophagy in malignant blood cells by modulating the Beclin 1/Vps34 complex
      Wang, Siyu; Li, Jie; Du, Yanyun; Xu, Yujia; Wang, Yali; Zhang, Zubin; Xu, Zhuan; Zeng, Yuanying; Mao, Xinliang; Cao, Biyin
      Journal of Pharmacological Sciences (Amsterdam, Netherlands) (2017), 134 (4), 197-202CODEN: JPSTGJ; ISSN:1347-8613. (Elsevier B.V.)
      S14161 is a pan-Class I PI3K inhibitor that induces blood cancer cell death, but its mechanism is largely unknown. In the present study, we evaluated the role of S14161 in autophagy, an emerging event in cell destination. Multiple myeloma cell lines RPMI-8226, OPM2, KMS11 and leukemia cell line K562 were treated with S14161. The results showed that S14161 induced autophagy as demonstrated by increased LC3-II and decreased p62, which were prevented by autophagy inhibitors including 3-methyladenine and bafilomycin A1. Mechanistic studies showed that S14161 had no effects on Vps34 expression, but increased Beclin 1 and decreased Bcl-2, two major regulators of autophagy. Furthermore, S14161 dissocd. the Beclin 1/Bcl-2 complex and enhanced the formation of Beclin 1/Vps34 complex. Moreover, S14161 inhibited the mTORC1 signaling transduction. S14161 downregulated activation of mTOR and its two crit. targets 4E-BP1 and p70S6K, suggesting S14161 inhibited protein synthesis. Taken together, these results demonstrated that Class I PI3K regulates autophagy by modulating protein synthesis and the Beclin 1 signaling pathway. This finding helps understanding the roles of PI3K in autophagy and cancer treatment.
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    56. 56
      Yogalingam, G.; Pendergast, A. M. Abl Kinases Regulate Autophagy by Promoting the Trafficking and Function of Lysosomal Components. J. Biol. Chem. 2008, 283, 35941– 35953,  DOI: 10.1074/jbc.M804543200
      56
      Abl Kinases Regulate Autophagy by Promoting the Trafficking and Function of Lysosomal Components
      Yogalingam, Gouri; Pendergast, Ann Marie
      Journal of Biological Chemistry (2008), 283 (51), 35941-35953CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Autophagy is a lysosome-dependent degradative pathway that regulates the turnover of intracellular organelles, parasites, and long-lived proteins. Deregulation of autophagy results in a variety of pathol. conditions, but little is known regarding the mechanisms that link normal cellular and pathol. signals to the regulation of distinct stages in the autophagy pathway. Here we uncover a novel role for the Abl family kinases in the regulation of the late stages of autophagy. Inhibition, depletion, or knockout of of the Abl family kinases, Abl and Arg, resulted in a dramatic redn. in the intracellular activities of the lysosomal glycosidases α-galactosidase, α-mannosidase and neuraminidase. Inhibition of Abl kinases also reduced the processing of the precursor forms of cathepsin D and cathepsin L to their mature, lysosomal forms, which coincided with the impaired turnover of long-lived cytosolic proteins and accumulation of autophagosomes. Furthermore, defective lysosomal degrdn. of long-lived proteins in the absence of Abl kinase signaling was accompanied by a perinuclear redistribution of lysosomes and increased glycosylation and stability of lysosome-assocd. membrane proteins, which are known to be substrates for lysosomal enzymes and play a role in regulating lysosome mobility. Our findings reveal a role for Abl kinases in the regulation of late-stage autophagy and have important implications for therapies that employ pharmacol. inhibitors of the Abl kinases.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsVylsrfJ&md5=430f737a320e8813cdb91bb80c717a80

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.1c00804.

    • Assay performance for negative controls, hit selection for lysosomal alkalinizers, hit confirmation for top alkaline hit, mTORC1/2 immunoblots of cells treated with rapamycin and torin1, ULK1Ser555 immunoblots of cells treated with mTOR inhibitors, and methods describing the population- and object-based analysis pipelines (PDF)


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