A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells
Contributed by Cynthia Kenyon, February 1, 2011 (sent for review November 26, 2010)
Abstract
Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916–919 and Cruts M, et al. (2006) Nature 442:920–924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287–297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.
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Frontotemporal lobar degeneration (FTLD) comprises a group of neurodegenerative syndromes that are characterized by behavioral changes, progressive aphasia, and/or semantic language deficits (1). Pathologically, the syndromes are characterized by frontal and temporal lobe atrophy and by intraneuronal deposition of aggregated tau, TDP-43, or FUS proteins (2–4). Several genetic mutations have been associated with the development of FTLD, including mutations affecting MAPT, VCP, CHMP2B, FUS, and progranulin (2, 5–11).
Progranulin mutations are present in 5% to 10% of all patients with FTLD (11, 12). Unlike mutations affecting tau, APP, α-synuclein, and huntingtin, which result in decreased protein solubility and formation of toxic oligomers (13), FTLD-associated progranulin mutations are not gain-of-function mutations, and progranulin itself is not found in neuronal inclusions. Instead, disease-associated progranulin mutations result in nonsense-mediated decay of progranulin mRNA or an inability to secrete the mutant protein (14). Individuals with these mutations exhibit less than half the normal blood and cerebrospinal fluid levels of progranulin (15–17). As the disease phenotype is inherited in an autosomal-dominant fashion, progranulin mutations appear to result in haploinsufficiency. In addition, variations in the human progranulin gene may also increase the risk for developing ALS, Alzheimer's disease, and Parkinson disease (18–21).
Progranulin (also known as GRN, acrogranin, proepithelin, granulin–epithelin precursor, and PC cell-derived growth factor) is a highly conserved glycoprotein involved in a variety of biological processes, including embryogenesis, inflammation, and wound healing (22). Increased progranulin secretion is associated with more aggressive forms of breast, brain, renal, and other tumors (22). Progranulin is secreted by many cell types including neurons and glia, and can be cleaved into 6-kDa cysteine-rich granulins whose functions are unclear, although they may oppose the activity of the proprotein (23, 24). In cultured neurons and neuronal cell lines, progranulin knockdown can adversely affect neurite outgrowth and long-term neuronal survival under serum-deprived conditions (25, 26).
The toxic insults to neurons that ultimately result in neuronal death and neurodegeneration in humans are generally thought to occur in a cell-autonomous fashion, although evidence is accumulating that microglia, the resident phagocytic and immune effector cells of the CNS, may be involved in neuronal killing (27). Here, by using Caenorhabditis elegans and ex vivo macrophage studies, we describe an unexpected role for progranulin. We find that loss of progranulin accelerates the clearance of apoptotic cells in both worms and mammals. Others have shown that the apoptotic cell-clearance machinery can remove cells that are dying but not yet dead, raising the possibility that loss of progranulin causes damaged neurons to be engulfed before they can be repaired.
Results
C. elegans PGRN-1 Protein Is Likely Secreted from Neurons and Intestine.
Many human disease proteins have conserved functions in invertebrates. Because of this, and because of the many molecular, genetic, and cell biological approaches available in C. elegans, we chose to study the function of progranulin in the nematode. The C. elegans progranulin gene (pgrn-1) encodes a protein with three predicted granulin domains, compared with the 7.5 granulin domains in human progranulin (Fig. S1). We generated a transgenic C. elegans line expressing a fluorescent mCherry protein that acts as a transcriptional reporter for pgrn-1 expression. In developing C. elegans larvae, mCherry protein was present in intestinal cells and select neurons (Fig. 1 A–F and Fig. S2) and was excluded from muscle cells in a pattern reminiscent of human progranulin expression (23). We tracked the timing of progranulin expression throughout development. C. elegans embryos are laid as eggs, hatch, and proceed through four larval stages to adulthood. The total time from embryo to adulthood takes approximately 3 d. The mCherry transcriptional reporter was first visible in the embryonic intestinal precursor cells at midgastrulation stage approximately 180 min after the first embryonic cleavage (Fig. 1 A and B and Fig. S3 A and B). We observed neuronal mCherry expression in L1 larvae, and neuronal and intestinal mCherry expression persisted throughout larval development and into adulthood (Fig. 1 C–F).
Fig. 1.
We also generated a translational reporter with an RFP fluorescent tag fused to PGRN-1. In contrast to the transcriptional reporter, this PGRN-1::RFP fusion protein was not found in discrete tissues. Instead, it was visible in what appeared to be intestinal organelles, in the pseudocoelomic cavity, and in scavenging coelomocytes, suggesting that, as in mammals (14), progranulin is secreted (Fig. 1 G–J and Fig. S3 C–F).
Progranulin Loss-of-Function Mutant Has a Normal Lifespan.
The pgrn-1(tm985) allele contains a 347-bp deletion encompassing part of the progranulin promoter, all of exon 1, and part of the first intron (Fig. S1). Animals carrying this mutation did not produce progranulin mRNA as assayed by quantitative RT-PCR (Fig. S4A). Thus, pgrn-1(tm985) is likely to be a null allele. pgrn-1 mutants appeared grossly normal, and both the homozygous and heterozygous mutants exhibited a normal lifespan (Fig. S4B and Tables S1 and S2). Likewise, overexpression of progranulin did not significantly alter lifespan (Fig. S4B and Table S3). However, the homozygous mutant had approximately 20% reduction in total number of progeny (Fig. S4C).
Programmed Cell Death Is Altered in pgrn-1 Mutants.
C. elegans development is characterized by an invariant pattern of cell division and cell death (28). The initiation of PGRN-1 production and secretion coincided temporally with the first programmed cell deaths in C. elegans (Fig. 1 A and B). Because of this temporal link between PGRN-1 production and the onset of programmed cell death, and because progranulin haploinsufficiency has been linked to human neurodegenerative disease, we investigated whether pgrn-1 mutants exhibited defective regulation of programmed cell death. In developing nematodes and the adult nematode germ line, cells dying via the process of apoptosis can be visualized by Nomarski microscopy as refractile bodies or “corpses.” We counted apoptotic corpses in embryos at several developmental stages and found significantly fewer apoptotic bodies in pgrn-1 mutants compared with control animals (Fig. 2A). To verify that the cell death defect was a result of progranulin deficiency, we reintroduced the C. elegans progranulin gene and observed partial rescue of the mutant phenotype (Fig. S4D). We speculate that the incomplete rescue may be because expression of pgrn-1 from a transgene does not fully mimic the expression of the endogenous gene. In addition to cell deaths that occur as part of the developmental program, apoptosis can be triggered in the germ line by UV irradiation. We found that pgrn-1 mutations did not affect UV-induced germ cell death (Fig. 2B).
Fig. 2.
Reduced numbers of apoptotic bodies in embryos could be caused by a failure of cells to die or by an increase in the rate of apoptotic cell clearance. To distinguish these possibilities, we used fluorescent markers that label the sisters of apoptotic cells that survive. These fluorescent markers are known to label “undead” cells in ced-3 caspase mutants in which cell death is blocked. If we saw “extra” cells, this would suggest that some cells normally programmed to die did not. First, we used an ODR-1::RFP reporter that normally is present in AWB, AWC, I1, and ASI neurons (29). I1 and ASI have sister cells that die (28). The same number of ODR-1::RFP fluorescent cell bodies were observed in pgrn-1 mutants and controls, suggesting that cell death occurs normally in the I1 and ASI lineage (Fig. 2C). We also used a LIN-11::GFP reporter which is seen in approximately five extra ventral cord motor neurons in strong ced-3(lf) mutants (30). Once again, pgrn-1 mutants had similar numbers of LIN-11::GFP fluorescent cell bodies compared with controls (Fig. 2D). Finally, we looked for extra cells in the anterior pharynx, where, in strong ced-3(lf) mutants, approximately 12 extra cells are found (31). Once again, pgrn-1 mutants had the same number of cells in the anterior pharynx as control worms (Fig. 2E). Taken together, our inability to find extra cells suggests that cells that normally die still do so in pgrn-1 mutants.
Loss of pgrn-1 Accelerates the Clearance of Dying Cells.
Apoptotic corpses are visible from the time they are generated until they are engulfed and degraded by neighboring cells. We next tested whether the reduced cell corpse number in pgrn-1 embryos was caused by a change in the kinetics of apoptosis: either delayed initiation of cell death or faster engulfment and clearance of the dying cell. To do this, we used four-dimensional Nomarski time-lapse video microscopy to follow the first 13 programmed cell deaths in the AB cell lineage (32). By using this technique, we found striking alterations in the kinetics of cell death (Fig. 3). The time from which a cell is born until it begins to show morphological characteristics of apoptosis (i.e., rounding of the cell, cytoplasmic condensation, nuclear swelling) is referred to as the “time to die.” The time from which initial morphological signs of apoptosis are seen until the time at which the dying cell is completely eliminated and disappears is referred to as the “time to clearance” (Fig. 3A). We observed a striking change in the mean time to clearance, which was decreased by nearly half in pgrn-1 mutants (harmonic mean of 5.7 min in pgrn-1 mutants vs. 10.7 min in WT; P < 0.0036; Fig. 3B and Table S4) This indicates that, when the cell death program has been initiated, corpse clearance is accelerated in pgrn-1 mutants.
Fig. 3.
We also measured the time to die in WT and pgrn-1 mutant animals. In WT animals, the time to die for apoptotic cells is distributed in a Gaussian manner. In pgrn-1 mutants, the distribution was skewed and no longer Gaussian (P < 0.0001). However, the mean time to die of the two strains was not significantly different (harmonic mean: control, 20.2 min vs. pgrn-1 mutants, 21.7 min; Fig. 3 C and D and Table S4). Many mutations have been described that change programmed cell death specification or slow the process of cell corpse engulfment; however, this is a mutation that accelerates the process of cell corpse clearance (i.e., time to clearance) and narrows the time window of cell death initiation (i.e., time to die). Although the effect of narrowed initiation time on number of apoptotic corpses is unclear, more rapid clearance can explain the reduced apoptotic cell corpse numbers observed during embryonic development.
How Might C. elegans Progranulin Influence the Kinetics of Programmed Cell Death?
We did not observe PGRN-1 production from dying cells or their engulfing sister cells during the embryonic stage in which this altered progression of cell death and clearance took place (Fig. S3 A and B). In fact, the only cells that produced PGRN-1 at this time were intestinal precursor cells. As progranulin is secreted (Fig. 1 G–J and Fig. S3 C–F), it is likely to act cell-non autonomously—on dying cells, engulfing cells, or both—to delay the clearance of apoptotic cells.
Studies in C. elegans have established two partially redundant pathways in engulfing cells that converge on the small GTPase CED-10 to promote phagocytosis of apoptotic cells. One pathway contains CED-1, 6, and 7 and DYN-1 and the other contains CED-2, 5, and 12, MIG-2, and UNC-73 (33). Mutations affecting these pathways slow the clearance of apoptotic cells by neighboring engulfing cells. To determine if progranulin was acting through these engulfment pathways, we generated double mutants between pgrn-1 and cell-engulfment mutants. As loss-of-function mutations in pgrn-1 accelerate corpse clearance, we would expect that the number of corpses observed in double mutants to be decreased if pgrn-1 were acting independently of an engulfment pathway. This was seen with a mutation in abl-1, which also accelerates corpse clearance (34). In engulfment mutants, uncleared corpses from cells that die during embryogenesis are visible in newly hatched L1 worms. We counted corpses in these newly hatched L1 heads and found that loss of progranulin did not affect the number of corpses (Table S5). Because loss of either of these partially redundant engulfment pathways abrogated the effect of pgrn-1, WT progranulin appears to require fully functional engulfment machinery to exert its effects on the rate of apoptotic cell clearance.
Macrophages from Progranulin KO Mice Display Enhanced Phagocytosis.
Many biological processes discovered in C. elegans are conserved in mammals. To determine whether this might be the case with progranulin's effect on the kinetics of apoptotic cell engulfment, we asked whether peritoneal macrophages from progranulin-deficient animals differed from WT in their rates of phagocytosis. Macrophages are involved in the clearance of apoptotic cells (35), express progranulin (36), and can cross the blood–brain barrier to become indistinguishable from resident microglia, the phagocytic cells of the CNS (37). We cultured peritoneal macrophages from WT (i.e., +/+) and progranulin-KO littermates in heat-inactivated serum for several days and then exposed them to one of several targets. First, we exposed them to FITC-labeled polystyrene beads. We found that, compared with WT macrophages, progranulin-KO macrophages engulfed latex beads more rapidly (Fig. 4A). We then tested substrates likely internalized via receptor-mediated endocytosis, yeast cell wall β-glucan particles, and, most significantly, apoptotic thymocytes. Compared with Pgrn+/+ controls, Pgrn−/− macrophages had increased levels of phagocytosis of yeast cell wall particles and apoptotic thymocytes (Fig. 4 B–D). Together, these data suggest that, as in C. elegans, loss of progranulin influences phagocytosis by macrophages, including engulfment of apoptotic cells.
Fig. 4.
Discussion
We have identified a function for progranulin in modulating the kinetics of programmed cell death that may be relevant for neuron loss in neurodegenerative diseases. We showed that lack of progranulin caused apoptotic cells to be cleared more rapidly in C. elegans. This type of change in the kinetics of programmed cell death would have been difficult to observe in mammals. However, we have extended this finding to mouse macrophages, which also exhibit enhanced engulfment of latex beads, yeast cell wall, and, importantly, apoptotic cells. A defect in programmed cell death kinetics could gradually contribute to neuronal loss over the lifetime of human progranulin-mutation carriers. A neurodegenerative phenotype was not observed in C. elegans, but this may be because, unlike vertebrates, adult worms do not exhibit programmed cell death in somatic tissues.
Apoptosis was once thought to be an irreversible process, such that, when programmed cell death had been initiated, death of a cell was inevitable. Recent studies have found, however, that even after a cell initiates the program of cell death, the process can be reversed (30, 32, 38, 39). Additionally, the process of apoptosis is not entirely cell autonomous, as engulfing cells actively promote cell death (30, 32). Because lack of progranulin in C. elegans causes apoptotic cells to be cleared more quickly than if progranulin is present, one normal function of progranulin appears to be to regulate the rate of cell clearance. Others have shown that expression of human progranulin in an immortalized mouse neuronal cell line can decrease the percentage of cells undergoing apoptosis after serum deprivation even in the absence of any “engulfing” cells (26). Our results with the use of cultured primary macrophages now also implicate the engulfing cell in regulating the kinetics of cell engulfment and death in response to progranulin. It is not clear how progranulin acts biochemically to influence the rate of cell engulfment. Our studies in worms suggest that progranulin acts through the normal cell engulfment machinery to affect the rate of apoptotic cell clearance, as the slower engulfment that occurs in the absence of this machinery is not affected by loss of progranulin. In mammals, progranulin can activate specific kinases such as MAPK and AKT, and it interacts with sortilin, a membrane protein that can complex with pro-NGF and p75NTR to activate proapoptotic signals (40–42). It would be interesting to ask, by using mutants, whether progranulin acts through any of these proteins to influence the rate of cell engulfment.
Based on these findings, we propose a model in which inadequate progranulin levels may promote neurodegeneration by disrupting the kinetics of programmed cell death (Fig. 5). Under conditions in which WT levels of progranulin are produced, a cell that undergoes a sublethal insult has adequate time to repair itself and survive. If insufficient progranulin is present, the rate at which an injured cell is recognized and/or engulfed by phagocytic cells is accelerated, and the damaged cell has less time to recover. Such changes in the kinetics of cell death could cause a shift in the dynamic equilibrium toward survival or death of individual cells that could ultimately result in cumulative neuronal loss. Likewise, precocious clearance could conceivably prevent cells damaged by toxic neurodegenerative proteins from recovering. This model could help explain how identical mutations within a family can manifest in disease with differing phenotypes and ages of onset, as extent and location of neuronal injury could vary dramatically from individual to individual. In addition, our findings suggest an additional mechanism by which progranulin overexpression by tumor cells may promote tumor growth; namely, by inhibiting the activity of surveillance phagocytes and promoting tumor immune evasion.
Fig. 5.
Although this model to explain the effect of progranulin deficiency on development of FTLD remains speculative, several groups have recently implicated abnormal microglial activity in FTLD and related diseases. While our studies were in progress, progranulin-deficient mice were shown to exhibit an exaggerated inflammatory response after infection (43) as well as an age-dependent increased accumulation and activation of microglia (43–45). Our findings with cultured macrophages would be consistent with this type of enhanced microglial activity. Moreover, they suggest that progranulin loss alters the activity of engulfing cells even in the absence of progranulin-deficient neurons. Enhanced microglial action has also been observed in a mouse model of Alzheimer's disease. Here, microglia were shown to increase in number and migration velocity around neurons just before their elimination (46). Finally, in a mouse model of ALS, a neurodegenerative disorder linked to FTLD by overlapping genetic and pathological changes, expression of mutant SOD1 protein in microglia and not neurons is the major determinant of disease progression (47, 48). These and other studies implicate microglial function in the pathogenesis of neurodegenerative disease. As loss of progranulin changes macrophage engulfment characteristics, and reduced progranulin is linked to FTLD, our findings suggest a potentially new mechanism by which phagocytic cells may contribute to neuron loss and neurodegeneration. Nonetheless, the direct effect of reduced progranulin on microglia in vivo remains to be demonstrated in mouse models of progranulin deficiency.
As polymorphisms in the human progranulin gene are associated with increased risk of ALS, Alzheimer's disease, and Parkinson disease (18–21), our findings here could have implications on the development of neurodegenerative disease more generally. A change in the regulation of programmed cell death kinetics represents an attractive and potentially important hypothesis to explain how mutations in the progranulin gene may result in neurodegeneration and suggests future avenues for investigation in the pathogenesis of neurodegenerative diseases.
Materials and Methods
Strains.
C. elegans were cultured at 20 °C according to standard procedures (49). The pgrn-1(tm985) strain was provided by the Mitani Laboratory at the Tokyo Women's Medical University (Tokyo, Japan) and out-crossed four times to our laboratory's N2 strain. Other published strains were obtained from the Caenorhabditis Genetics Center at the University of Minnesota. SI Materials and Methods provides additional strain information.
Assays.
Somatic cell and germ line corpses were visualized directly on a Zeiss Axioplan 2 microscope fitted with Nomarski optics as described previously (28). C. elegans embryos, larvae, and adults were mounted on 2% agarose pads in M9 with 2.5 mM levamisole. Apoptotic corpses were counted in a blinded fashion. To measure the kinetics of cell corpse formation and clearance, four-dimensional time-lapse Nomarski microscopy was performed as described previously (50). Results are shown in scatterplots and histogram form. SI Materials and Methods provides additional details on phagocytosis assays.
Acknowledgments
We thank the Caenorhabditis Genetics Center at the University of Minnesota and the Mitani and Ashrafi laboratories for strains and reagents. We thank Kaveh Ashrafi, Shai Shaham, Michael Hengartner, Bruce Miller, Suneil Koliwad, Laura Mitic, Kenyon laboratory members, and members of the Consortium for Frontotemporal Dementia Research (CFR) for helpful advice. A.W.K. was supported by a Hillblom Foundation Postdoctoral Fellowship and by the CFR. L.H.M. and R.V.F. were supported by CFR and by National Institutes of Health (NIH) Grant P50 AG023501 and NIH/National Center for Research Resources Grant C06 RR018928 for animal facilities. A.d.L. and J.C. were supported by the Riojasalud Foundation and by Spanish Ministry of Science and Innovation Grant BFU2010-21794. This work was supported by NIH Grants K08 NS59604 (to A.W.K.) and R01 AG11816 (to C.K.). C.K. is an American Cancer Society Professor and director of the University of California, San Francisco, Hillblom Foundation for the Biology of Aging.
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Published online: February 28, 2011
Published in issue: March 15, 2011
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Acknowledgments
We thank the Caenorhabditis Genetics Center at the University of Minnesota and the Mitani and Ashrafi laboratories for strains and reagents. We thank Kaveh Ashrafi, Shai Shaham, Michael Hengartner, Bruce Miller, Suneil Koliwad, Laura Mitic, Kenyon laboratory members, and members of the Consortium for Frontotemporal Dementia Research (CFR) for helpful advice. A.W.K. was supported by a Hillblom Foundation Postdoctoral Fellowship and by the CFR. L.H.M. and R.V.F. were supported by CFR and by National Institutes of Health (NIH) Grant P50 AG023501 and NIH/National Center for Research Resources Grant C06 RR018928 for animal facilities. A.d.L. and J.C. were supported by the Riojasalud Foundation and by Spanish Ministry of Science and Innovation Grant BFU2010-21794. This work was supported by NIH Grants K08 NS59604 (to A.W.K.) and R01 AG11816 (to C.K.). C.K. is an American Cancer Society Professor and director of the University of California, San Francisco, Hillblom Foundation for the Biology of Aging.
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The authors declare no conflict of interest.
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