INTRODUCTION

The field of mitochondrial dynamics is a tantalizing dichotomy: study of fission and fusion reveals a vast diversity of potential targets, yet the fragility of the system makes targeting a sensitive endeavor. Mitochondria readily vacillate between the unique processes of fission and fusion; the former results in a newly formed daughter mitochondrion and the latter in conglomerates. Mitochondria often employ these mechanisms for repair and cell survival as a response to external stress conditions and disruptions in cellular milieu (31, 53). Directly, fission and fusion are responsible for the creation of discrete daughter mitochondria and for integration and exchange of mitochondrial proteins, respectively (23). These contrasting processes are tightly regulated and enable fine-tuning of calcium homeostasis, energy metabolism, cell proliferation, and redox signaling.

Prior to the surge in the study of mitochondrial dynamics, evidence implicating mitochondrial dysregulation in pathogenesis was scarce except for genetic mutation (2). More specifically, the majority of mitochondrial proteins are nuclear-encoded; as a result, monogenic mutations were one of the few connections elucidated between mitochondria and pathology in early years of study (2). Now, over the past decade, the study of mitochondrial dynamics has evolved into a bustling field of its own. Several studies have chipped away at the mysteries of fission and fusion, allowing the identification of proteins involved in the process and concurrently shedding light on some unexpected involvement in disease pathogenesis. Considerable evidence shows that perturbation of this integral duo can play a role in several pathologies (24).

Fortunately, significant advancements have been made in the understanding of mitochondrial dynamics. Among the proteins identified in recent years, the most notable belong to the family of dynamin-related GTPases. Several proteins are critical for the regulation of fission and fusion, including dynamin-related protein 1 (Drp1), mitochondrial fusion protein 1 (Mfn1) and 2 (Mfn2), and optic atrophy 1 (Opa1) protein (8, 12, 53). This machinery will be discussed in greater detail in the sections below. Notably, Drp1 is of particular interest among these two dynamic processes as it is the integral facilitator of mitochondrial fission, but can also promote fusion-based activity (2, 24). Inactive Drp1 is normally found in the cytosol, which shifts mitochondrial balance towards fusion; conversely, activated Drp1 promotes mitochondrial fission by translocating to the mitochondrial outer membrane (MOM) (2, 24, 58, 62). From there, Drp1 GTPase activity causes constriction of its multimeric ringlike structure around the membrane, eventually giving way to a pair of discrete daughter mitochondria (12).

It is for this unique involvement in both fission and fusion that Drp1 is implicated in diseases such as pulmonary arterial hypertension, asthma, ischemia-reperfusion (I/R), Parkinson’s disease, Alzheimer’s disease, and various other lung diseases (2, 9, 30, 32, 46). The purpose of this review is to address the importance of Drp1 as a therapeutic target and thereby justify additional research to understand its role in the pathogenesis of various diseases (43). Insufficient attention is dedicated to the study of vital mediators like Drp1 and their surrounding mechanisms despite the growing significance of mitochondrial dynamics and the ever-increasing range of therapeutic targets therein.

SUMMARY OF MITOCHONDRIAL FISSION

The cellular environment is a rapidly changing bionetwork where each proteinaceous component plays a key role in the adaptation to new stimuli. Fluctuations in mitochondrial population are just that: an adaptation critical to the survival and flourishment of the cell. Mitochondrial fission increases the number of discrete mitochondria and effectively reorganizes the mitochondrial network; understandably, this has a wide variety of effects on the amalgamate function of mitochondria in response to cellular cues (24, 42). This is further supported by the noteworthy revelation that even nonproliferating neurons still require fission for survival (62). It has also been suggested that fission participates in an intimate relationship with apoptosis via influencing Bax-mediated cytochrome c release (62). Although the complexities of this interaction remain unclear, studies suggest that Drp1 colocalizes with clusters of Bax (Fig. 1) (7). Researchers also discovered that fission plays a prominent role in mitophagy, a process that encapsulates damaged mitochondria in autophagic vacuoles. Investigators are now testing the extent to which mitochondrial dynamics influence formation of autophagosomes in disease conditions (33). Notably, a 2015 study determined that Drp1 deficiency led to an accumulation of autophagosomes and reactive oxygen species (ROS) in human umbilical cord vein endothelial cells and in aged mice (33). Drp1 silencing also resulted in phenotypic changes, premature senescence, and endothelial dysfunction (33). These studies suggest that Drp1 may have a wider impact on cell survival than previously imagined.

Mechanistically, fission is a straightforward process that is fairly well understood, but still has a few points of uncertainty. Generally, the literature agrees that both fission and fusion can be triggered by a variety of stressors, including UV radiation, oxidative stress, and starvation. More specifically, several damage response pathways can promote mitochondrial fission through phosphorylation of Drp1 (Fig. 2); once Drp1 is activated via phosphorylation, fission protein 1 (Fis1) and mitochondrial fission factor (Mff) recruit Drp1 to the MOM, although some further clarification on this process is still necessary (Fig. 1) (40, 68). Following recruitment to the membrane, Drp1 forms a multimer that spirals around the target mitochondria and is assisted by various other molecules (discussed in detail later) to ultimately divide the mitochondria into two daughter organelles. These two resultant daughter mitochondria generally possess unequal, and often extreme, membrane polarization (62). Normally, the daughter mitochondria that are damaged will fail to stabilize their polarity, tending instead to depolarize completely and be targeted for mitophagy (62).

While it is understood that Mff forms a heterodimer with Drp1 for mitochondrial recruitment, the exact role of Fis1 in either recruitment or activation of Drp1, or whether it is necessary altogether, remains a hazy and controversial subject in mitochondrial dynamics (Fig. 1) (24). Notably, one study demonstrated that conditional knockout of Fis1 in colon carcinoma cells did not abolish mitochondrial fission (40). Using mouse embryonic fibroblasts and HeLa cells, another study provided evidence that two mitochondrial elongation factor proteins, MiD49 and MiD51, are in fact more critical for recruiting Drp1 than Mff and Fis1 (24, 41). They found that MiD49 and MiD51 can target Drp1 to the mitochondrial membrane independent of both Mff and Fis1 (Fig. 1) (41). This strongly suggests that perhaps our understanding of fission machinery requires more detailed analyses in certain conditions.

SUMMARY OF MITOCHONDRIAL FUSION

In contrast to the mitophagy-driven reparative process of mitochondrial fission, fusion employs the principles of recombination to preserve mitochondrial DNA and buoy energy production. Mitochondria accomplish DNA preservation by fusing a wild-type mitochondrion with a damaged or mutant one; in doing so, the cell can potentially mitigate further injury by suppressing the mutant DNA (2, 24, 58, 62). In this sense, the mixing of wild-type and damaged mitochondrial DNA is used as a reparative mechanism in the event of external or internal stress, such as elevated levels of reactive oxygen species (ROS). In addition to conservation of mitochondrial DNA, fusion can also function to meet high-energy demands during cellular stress (2, 58, 62). Forming extensive networks of mitochondria, fusion effectively increases oxidative phosphorylation (62). However, excessive or prolonged fusion of mitochondria (hyperfusion) can deprive the cell of the benefits of mitophagy: thus, mitochondrial fusion exists in tight regulation with fission (2). It has been reported that inhibition of Drp1, and thus mitochondrial fission, can lead to a preference for mitochondrial fusion that promotes cell cycle arrest at the G₂-M phase (2). Formation of extensive mitochondrial networks is a powerful function for cell survival; yet, research still holds several unanswered questions regarding the pathophysiological relationship between fission and fusion, and to what extent they may safely be targeted as therapeutic avenues.

In terms of mechanism, fusion seems to be a simpler interaction of proteins. Three dynamin proteins are essential for the process of mitochondrial fusion in mammals: Mfn1, Mfn2, and Opa1 (62). Both Mfn1 and Mfn2 are located on the MOM, whereas Opa1 is located on the mitochondrial inner membrane (MIM) (2, 24). The process of fusion begins with Mfn1 and Mfn2 tethering the MOMs of adjacent mitochondria through the use of specific mitochondrial targeting sequences (2). Mfn2 also creates a linkage between the mitochondria and the endoplasmic reticulum (ER) (2). Interestingly, Opa1 plays an important role as a secondary regulator of mitochondrial fusion by allowing for partial fusion between mitochondria. In this case, a network of mitochondria can be formed wherein the outer membranes are fused, but the contents within the inner membrane remain isolated (62). The exact implications and benefits of this remain hazy, but it is clear that proteolytic modulation of Opa1 levels in mitochondria, which typically occurs as a response to changes in membrane potential, is a deliberate method of cellular regulation (2, 58, 62). It has also been shown that Opa1 is involved in cytochrome c release and that decreased levels of soluble Opa1 can be found in cells where Drp1 is inhibited (24). Similarly, the well-known serine-threonine kinase PTEN-induced kinase 1 (PINK1) and its regulatory ubiquitin E3 ligase partner, Parkin, have been shown to play a critical role in the regulation of mitochondrial fusion through ubiquitination of Mfn1 and Mfn2 (to inhibit fusion) as well as ubiquitination of Drp1 (to prevent fission) (2, 24).

SUMMARY OF DRP1 FUNCTION AND REGULATION IN NORMAL PHYSIOLOGY

Drp1 is considered to be the primary regulator of mitochondrial fission and is heavily regulated by a wide array of signaling pathways (12). The structure of Drp1 is composed of four conserved regions integral to its tight regulation: GTPase domain, middle domain, variable domain, and GTPase effector domain (GED) (Fig. 2) (18). The middle and GED domains are responsible for mitochondrial targeting (determined by phosphorylation) as well as for the assembly of the Drp1 multimeric collar structure that constricts the mitochondrial membrane (18). Several serine residues throughout the protein act as phosphorylation control points that can be selectively regulated by a variety of kinases, such as the Drp1-inhibitive phosphorylation of Ser⁶⁵⁶ or Ser⁶³⁷ by protein kinase A (PKA), to impact the translocation and activity of Drp1 (Fig. 2) (2, 13).

Following the phosphorylation of Drp1 on the appropriate serine residue(s), Drp1 can function to promote either fission or fusion; if Drp1 is active, the site of fission will first be marked (42). Current research suggests that the fission site is typically marked alongside an interface between the ER and mitochondria (42). Researchers have demonstrated that this interaction with the ER is indeed necessary for Drp1-mediated mitochondrial fission. A current theory relies on mitochondrial diameter: as a consequence of the mitochondrial diameter exceeding that of an assembled Drp1 multimer (by a factor of 10), an initial constriction event, or at least external assistance in some form, is required for Drp1 hydrolysis activity (11, 42). Evidence suggests that actin filaments along the ER-mitochondria contact site facilitate the initial constriction event or aid Drp1 multimer formation (Fig. 1) (11, 42). Subsequently, Drp1 GTPase activity is triggered and results in a series of conformational changes that constrict the mitochondrion (18).

As opposed to ER dependency, some studies have shown that the mitochondrial lipid cardiolipin is a specific binding partner of Drp1 that facilitates its self-assembly (49, 55). Cardiolipin is a dimeric phospholipid involved in stabilization of membrane curvature and exists in a cooperative relationship with Drp1 (55). This association permits coordination of membrane remodeling and fission through stimulation of Drp1 GTPase activity. Such regulation is typical in cardiac diseases, where calcium-signaling pathways play a prominent role in the regulation of Drp1 via molecules such as calcineurin and cardiolipin. An understanding of the interplay between distinct pathways that involve calcium signaling, and other stress response mechanisms, may reveal previously unknown control points in mitochondrial dynamics.

DRP1 IN CARDIAC PATHOLOGIES

Mitochondrial dysregulation is a key contributor to the exacerbation of several cardiac issues, including I/R (50, 64). In cardiac diseases, changes in mitochondrial network structure, particularly via fission, lead to overproduction of ROS, which can exacerbate the disease (50). Regulation of mitochondrial fission is intrinsically tied to ROS production and management. ROS response pathways, such as calcineurin or protein kinase Cδ (PKCδ) and cyclin-dependent kinase 1 (Cdk1), can activate Drp1 via dephosphorylation of Ser⁶³⁷ or phosphorylation of Ser⁶¹⁶ and induce cardiomyocyte death (Fig. 2) (64). More specifically, calcineurin, a serine/threonine protein phosphatase involved in calcium homeostasis, cardiac morphogenesis, and cardiac hypertrophy, can facilitate dephosphorylation Drp1 on Ser⁶³⁷ (Fig. 2) (50). It was demonstrated that administration of the calcineurin inhibitor FK506, following myocardial I/R, effectively inhibits Drp1 dephosphorylation and thus mitochondrial translocation (50).

Over the past decade, several studies revealed the significance of Drp1 in cardiomyopathies. One research group demonstrated that loss of Drp1 in postnatal cardiomyocytes is associated with mouse death at 6 wk of age (54). Although mechanistically inconclusive, the same study then proceeded to provide evidence that Drp1 deficiency leads to elevated myocardial fibrosis and prolific cardiomyocyte necrosis was confirmed by Evans blue staining (54). Another study noted that downregulation of Drp1 in 16-wk-old cardiac-specific conditional (heterozygous) Drp1-knockout mice exacerbates development of cardiac hypertrophy, dysfunction, and mitochondrial dysregulation when subjected to transverse aortic constriction (52).

As demonstrated, the implication of Drp1 deficiency in cardiac dysfunction is widespread and has several plausible mechanisms of action. In one study, loss of Drp1 in mice led to decreased contraction, heart rate, and left ventricular function (27). The same group also found that knockout of Drp1 in cardiomyocytes is associated with inhibition of autophagic flux, and thus accumulation of p62, a mitophagy-associated adaptor protein that is recruited to the mitochondria (27). A number of studies link Drp1 loss to increased heart size and/or enlarged or interconnected mitochondria in cardiomyocytes (27, 50, 54). Similar to the function of calcineurin, one study demonstrates that the serine/threonine kinase Pim-1 also acts to regulate translocation of Drp1 (15). According to this study, Pim-1 overexpression in neonatal rat cardiomyocytes elevated phosphorylation of Drp1 on Ser⁶³⁷ and prevented its translocation to the mitochondria (Fig. 2) (15). They also found that prevention of Drp1 translocation is associated with a decrease in mitochondrial fragmentation during I/R (15).

Mitochondrial fission is necessary for maintenance of homeostasis as well as for control of ROS production and autophagic flux. However, administration of Drp1 inhibitors, namely the calcineurin inhibitor Mdivi-1, improves cell function, particularly in cardiomyocytes in cardiac I/R. Interestingly, in neonatal murine ventricular cardiomyocytes, Mdivi-1-mediated Drp1 inhibition preserved mitochondrial network morphology and prevented I/R-induced mitochondrial fragmentation (50). This suggests that Drp1 elevation during disease conditions may exacerbate disease and decrease cell survival. Many studies on Drp1 in cancer support the evidence found in cardiac pathologies, illustrating that Drp1 modulation is a viable means of attenuating cellular damage and that mitochondrial fragmentation can be used as an indicator of disease progression.

DRP1 IN CANCER

For many years, Drp1 has been a focal point for studies on mitochondrial dynamics in a diverse set of cancers, including melanomas, brain tumors, pancreatic tumors, and several others (4, 17, 29, 60). The literature is consistent in that mitochondrial morphology changes in response to, and may even impact, tumor growth (29). As a result, studies suggest that the balance of the mitochondrial network can influence both metastasis and the metabolic processes of cancer. One study demonstrates through histology of human thyroid tissues that Drp1, along with other mitochondrial dynamics machinery, is significantly elevated, suggesting a link between mitochondrial dynamics and tumor malignancy (17). Thyroid cancer cells, both XTC.UC1 (oncocytic) and TPC1 (nononcocytic) were found to have increased levels of Drp1, but expectedly the oncocytic cell line demonstrated a substantially higher level of Drp1 expression (17). The study also demonstrated that thyroid cancer cells tend to disrupt the balance of the mitochondrial network with a decided preference for fission and could be slowed by the inhibition of Drp1 via Mdivi-1 administration (17). This preference could be, at least in part, a response to an increased need for metabolism in accordance with the Warburg effect seen in cancer (an abnormal propensity for ATP production via glycolysis) (17).

Ras signaling plays a role in the development and progression of human cancer, especially pancreatic cancer (29). A study on the role of Drp1 in relation to Ras signaling found that Drp1 is phosphorylated on Ser⁶¹⁶ (activated) by extracellular signal regulated kinase 2 (ERK2) in both pancreatic cancer cells lines (HEK-TtH cells expressing HRasG12V) and in patient tumor tissue (Fig. 2) (29). The study demonstrates that the MAPK pathway may be responsible for the upregulation of Drp1 and that knockdown of Drp1 attenuates tumor growth in BxPC3 cells (29).

In brain tumor initiating cells (BTICs), one group further validated the literature’s evidence that mitochondrial fragmentation is a key commonality among various cancer types (60). Moreover, similar to the mechanism in pancreatic cancer, Drp1 phosphorylation in BTICs is a significant control point for mitochondrial morphology and network organization (60). Using both lentiviral silencing and pharmacologic inhibition via Mdivi-1 to target Drp1 in BTICs resulted in significantly dampened tumor growth and was associated with a loss of tumor phenotypes (60). This same study tied Drp1 regulation to cyclin-dependent kinases in BTICs and calcium mechanisms in non-BTIC tumor cells (60). They show that CDK5 is the predominant regulator of Drp1 activation and that two calcium/calmodulin-dependent protein kinases hold Drp1 activity under tight regulatory control via phosphorylation of Ser⁶³⁷ (Fig. 2) (60).

DRP1 IN NEURAL PATHOLOGIES

Although neural cells do not proliferate, Drp1 and mitochondrial fission are still essential. Studies show that Drp1 is vital for neuronal survival and brain development (22, 35, 51, 59). Drp1 protein levels in Alzheimer’s disease were found to be significantly elevated in patients throughout Braak stages I–VI when compared with the levels of fusion proteins (35). Another group studied the effects of Drp1 conditional knockout on mice, showing that excitatory postsynaptic potentials were appreciably decreased, suggesting its importance in synaptic function (51). The same study showed that Drp1 loss is associated with changes in mitochondrial morphology and decreased energy production in axons (51). Furthermore, Drp1 deficiency was found to disrupt mitochondrial cristae structure and lead to enlarged mitochondria in dendritic cells (51). Intriguingly, mitochondrial fission impairment in axons causes dysregulation of mitochondrial bioenergetics, which leads to decreased ATP production associated with early loss of axons in neurodegenerative disease conditions (51). Additionally, as a result of this Drp1-loss-mediated disruption, synaptic transmission-inhibition can exacerbate neurodegenerative diseases (51).

In contrast to axons, hippocampal neurons are surprisingly unaffected by the loss of Drp1: hippocampal neurons can survive more than a year without Drp1, whereas Drp1 loss causes midbrain dopaminergic neurons to die within one month and Purkinje cells to die within six months (51). This suggests that some cell types may utilize pathways other than Drp1-mediated fission and/or do not require fission as frequently as other cell types. An in vitro study on HT-22 cells demonstrated that siRNA silencing of Drp1 halted glutamate-induced mitochondrial fragmentation (22). The study found that inhibition of Drp1 is capable of attenuating neuronal cell death by interrupting a proapoptotic pathway implicated in cerebral ischemia (22). Administration of the Drp1 inhibitor Mdivi-1 preserved healthy nuclear morphology and reduced mean infarct volume in oxygen-glucose deprived primary cortical neurons (22).

Mitochondrial fragmentation is also associated with enhanced amyloid-β toxicity both in vitro and in vivo in Alzheimer’s disease (59, 61). Studies show that mitochondria experience several negative changes during Alzheimer’s pathogenesis including disruption of mitochondrial dynamics, elevated ROS production, and increased membrane permeability (59). One study identified that glycogen synthase kinase-3β (GSK3β), an enzyme implicated in neural development and Alzheimer’s disease, exhibits control over Drp1-mediated mitochondrial fragmentation via phosphorylation on Ser⁴⁰ and Ser⁴⁴ of Drp1 (Fig. 2) (61). Blockage of GSK3β prevents phosphorylation of Drp1 in neurons and thwarts translocation of Drp1 to the mitochondria (61).

DRP1 IN RENAL, CARDIORENAL, AND PULMONARY DISEASES

Under stressed conditions, such as in cardiac and neurodegenerative diseases, notable fluctuations of Drp1 can lead to a variety of outcomes with regard to mitochondrial stability. As is a general trend with many pathologies, after some mechanisms of a disease or specific molecular axis become more elucidated, research in more specialized fields begins to occur. For Drp1, studies on its involvement in renal, cardiorenal, and pulmonary pathologies are perfect examples. Although cardiorenal may draw upon the wealth of knowledge found in cardiac studies, all three fields remain relatively new to the exploration of mitochondrial dynamics.

One group demonstrated that azide treatment of rat proximal tubular cells led to Drp1 translocation to the mitochondria and excessive changes in mitochondrial morphology in a study of acute renal failure (pertinent to renal ischemia-reperfusion and cisplatin-induced nephrotoxicity) (6). The group attempted to attenuate mitochondrial fragmentation by inhibiting the caspase cascade, but the mitochondrial outcome remained unchanged despite successful prevention of apoptosis (6). Interestingly, the researchers employed a dominant-negative point mutant of Drp1 in vitro and found that it successfully attenuated mitochondrial fragmentation and prevented cytochrome c release despite azide challenge. This result was further corroborated in primary proximal tubular cells using Drp1 siRNA (6).

The cross-talk between the heart and kidney during disease provides a very unique set of conditions for the study of mitochondrial dynamics. In C57BL/6 mice, one study demonstrated (supporting the in vivo studies of the previously discussed article) that renal I/R was associated with significantly increased mitochondrial fragmentation and cytochrome c release in cardiomyocytes (6, 56). Notably, the study reports that of the mitochondrial dynamics machinery examined, only Drp1 was significantly upregulated in cardiac tissues following mouse renal I/R (56). As in the previous renal article discussed, Mdivi-1 pharmacotherapy remained the go-to treatment for attenuating mitochondrial dynamics. In this study, Mdivi-1 suppresses Drp1 translocation and cytochrome c release in cardiomyocytes isolated from mice with renal I/R (56).

With regard to pulmonary diseases, one study on pulmonary arterial hypertension (PAH) stands out. This study demonstrates that activation of hypoxia-inducible factor-1α (HIF-1α) in PAH is associated with increased mitochondrial fragmentation and elevated Drp1 levels in human pulmonary arterial smooth muscle cells (PASMCs) (38). Remarkably, inhibition of Cdk1 in human PAH PASMCs was found to reduce the phosphorylation of Ser⁶¹⁶ and decrease mitochondrial Drp1 activity (Fig. 2) (38). This is similar to the phosphorylation status of Ser⁶³⁷ discussed previously, which can also regulate translocation of Drp1 to the mitochondria (Fig. 2) (50, 60). Another study demonstrates that inhibition of Drp1 using Mdivi-1 led to disruption of cell cycle progression in vascular smooth muscle cells, which has wide implications in disease survivability (38). Within the cell, alterations in Drp1 can result in drastic changes in the mitochondrial network, namely elongation with a loss of Drp1 and fragmentation with an excess of Drp1, effectively stymieing cell growth and mitochondrial repair (1).

A wide range of stressors in pulmonology are known to cause or be associated with mitochondrial dysregulation, such as oxidative stress, lipid peroxidation by-products (such as 4-hydroxy-2-nonenal), and cigarette smoke (1, 19, 20). In particular, cigarette smoke alters the structural organization of the mitochondrial network by influencing the balance of mitochondrial dynamics (1). In nonasthmatic human airway smooth muscle cells, exposure to cigarette smoke extract (1%) increases Drp1 mRNA and protein levels in a time- and dose-dependent manner (1). The same study attributes this relationship to a cigarette smoke extract-mediated alteration of kinases, such as ERK, 1-2, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and protein kinase C (PKC) (1). This is a reasonable connection considering the supported nature of Drp1 regulation as a GTPase.

THERAPEUTIC RELEVANCE OF DRP1

Mitochondrial proteins are relatively stable and involved in several layers of cellular regulation; thus, these proteins can be viewed as more accessible targets for therapy when compared with other cellular machinery. However, as mentioned, mitochondrial dynamics exist in a delicate balance, and a simple tipping of the scales can have devastating effects on autophagic flux, mitochondrial network organization, cellular bioenergetics, and even senescence. As a result, therapeutic approaches that involve modulation of mitochondrial dynamics necessitate a precise and highly controllable means of administration. Many studies have turned to the inhibition of Drp1 due to its relatively forgiving nature and inherently important involvement, as well as the accessibility of its two inhibitors: P110 and Mdivi-1 (Fig. 3).

It is accepted that Mdivi-1 acts as an allosteric inhibitor of GTPase assembly and prevents GTP hydrolysis and Drp1 activity (7). Of course, while a recent controversy about Mdivi-1 may impact the understanding of its mechanism (discussed below in recent advances in the study of drp1), the application of it in research may continue to evolve positively. In contrast to Mdivi-1, P110 is a Drp1-specific inhibitor that alters Drp1-multimer assembly and prevents translocation to the mitochondria (44). Arguably, P110 is one of the most intriguing recent developments surrounding Drp1. Unfortunately, the therapeutic potential of P110 has been infrequently studied due to its age relative to Mdivi-1 (literature on Mdivi-1 preceded P110 by roughly 4 years).

In relation to cardiac pathologies, administration of Mdivi-1 to adult rats with I/R preserved mitochondrial morphology and maintained the composition of the mitochondrial network (50). Similarly, one study identified that administration of P110 to rat neonatal cardiomyocytes inhibited Drp1 translocation to the mitochondria in oxidative I/R conditions (16). In a subsequent ex vivo model of myocardial infarction, administration of P110 reduced infarct size and facilitated restabilization of mitochondrial morphology and dynamics (16). In neuropathologies, administration of Mdivi-1 protects primary neurons against glutamate toxicity (22). Lastly, in vivo administration of Mdivi-1 decreases Drp1 translocation to the mitochondria and consequently attenuation of ischemic brain injury (22).

As opposed to the popular paradigm of Drp1 inhibition, some recent studies are finding that perhaps bolstering Drp1 function, and thus mitochondrial fission, is a more beneficial therapy for some diseases (10, 29, 66). One interesting study employs astaxanthin, a non-provitamin A carotenoid, in mice with bleomycin-induced pulmonary fibrosis (66). In this study, Astaxanthin reportedly resulted in decreased levels of myofibroblasts in lung tissues via apoptosis and increased mitochondrial fission via Drp1 and led to overall amelioration of fibrosis in lung tissues (66). This suggests that perhaps in some diseases, elevation of Drp1 may be more advantageous. Regardless of whether we target Drp1 for upregulation or downregulation, it is readily permutable, a characteristic that makes it both a propitious and difficult target for disease attenuation.

RECENT ADVANCES IN THE STUDY OF DRP1

Each year, researchers unveil novel roles for mitochondrial regulation in cell survival: exciting new discoveries that are grounded in the foundational literature that sparked them (Table 1 and Table 2). Likewise, the implications of mitochondrial Drp1 expand both in normal physiology and pathophysiology (39). A 2017 study focused on the beneficial role of Drp1-mediated mitochondrial fission in the aging of Drosophila melanogaster (45). The study found that induction of Drp1 is associated with increased levels of fission and mitophagy, which was critical to the maintenance of a healthy mitochondrial population (45). Using unique gene switches, the study pinpoints specific tissues for the induction of Drp1 in midlife flies (~30–37 days) and reports that this induction is associated with a significant increase in average and maximal lifespan (45). Although observing this relationship is a stirring revelation in D. melanogaster, identifying whether it is reproducible in complex organisms is a crucial next-step for this fascinating avenue in geroscience.

A few years ago, a riveting revelation was made concerning the upstream events of the Drp1 mechanism; a detailed in vitro study described a novel mitochondrial Spire family protein, Spire1C, as the potential mediator of the initial constriction event of the mitochondria at the ER (37). This study utilized U2OS (human osteosarcoma cell line) and Cos-7 (monkey kidney cell line) cells to demonstrate that Spire1C interacts with the ER anchored inverted formin 2 (INF2) protein to help facilitate polymerization of actin filaments (Fig. 1) (37). The actin filament bridges then allow the constriction of the mitochondria before Drp1-mediated fission (37). Another study examined the role of a protein called Cofilin1 in relation to Drp1-mediated dynamics (47). Using mouse embryonic fibroblasts, it found that Cofilin1 may act as a negative regulator of Drp1 through actin depolymerization (47). Further clarification of the role of Spire1C and Cofilin1 could unveil much needed therapeutic avenues for mitochondrial dysregulation and network stability.

A somewhat controversial study was published in early 2017 reporting a misconception of the popular “Drp1 inhibitor” Mdivi-1. According to this study, Mdivi-1 does not target Drp1 but instead alters the efficacy of mitochondrial Complex I (5). The authors provide bioenergetic evidence in neuronal cells (oxygen consumption rates) that Mdivi-1 in fact inhibits Complex I in the electron transport chain (5). Furthermore, they debunk the idea that Mdivi-1 inhibits Drp1 in neuronal cells by using immunofluorescence staining to show the unchanged morphology of mitochondria in neurons (5). The study also assesses the enzyme kinetics of Mdivi-1 showing that it is a poor uncompetitive inhibitor of Drp1 GTPase activity (5). Coupled with evidence that Mdivi-1 treatment has the same effect on immortalized mouse embryonic fibroblasts even in the absence of Drp1 (global knockout), the group builds a strong case against Mdivi-1 being a Drp1 inhibitor (5).

What does this mean for the future of Mdivi-1? Although this report goes against the common verbiage used for the popular inhibitor, it does not necessarily redefine the utilization of it. It is no secret that Mdivi-1 is widely accepted as a Drp1 inhibitor and, with great success, is also readily employed for remediating mitochondrial fragmentation. How is this possible? Drp1 possesses control, to some degree, over both fission and fusion, and depending on its concentration or activity levels, the mitochondrial response can vary greatly. It can be posited that perhaps Mdivi-1-mediated inhibition of Complex 1 targets the same processes but on a more macromolecular scale, or simply through interactions downstream of Drp1. In this case, perhaps loss of Drp1 leads to greater levels of fusion and loss of fission, and that this shift has a similar impact on bioenergetics as inhibition of Complex I.

However, this does not explain the studies that report Mdivi-1 impacts Drp1 translocation, and while the data presented in this controversial study are compelling for neurons and fibroblasts, there may be cell-specific intricacies not yet explored. It cannot be ignored that foundational studies of Mdivi-1 report direct mechanistic inhibition of Drp1, which is in stark contrast to this new evidence (14, 21, 57). Nevertheless, in the future, studies planning to utilize Mdivi-1 should be wary that it may not be directly targeting Drp1, and should opt instead for more specific targeting approaches. Drp1, and similar mitochondrial proteins, could represent the dawn of a new realm of targeted mitochondrial therapeutics that could impact a wide range of diseases.

CONCLUSIONS

As the gradual shift towards translational medicine occurs, we become enamored with the prospects of vital molecules such as Drp1. There is a sound understanding of the physiological role of Drp1 in fission and its impact on the whole of mitochondrial dynamics, but much remains a mystery in terms of pathologies. The paradigm that mitochondrial dysregulation is a key contributor to a number of diseases is relatively new in several fields, including hepatology, endocrinology, and pulmonology. However, the intricacies of fission and its upstream regulators remain largely unknown due to the complexity of the cross-talk involved in mitochondrial dynamics.

Although the regulation of Drp1 is in a delicate balance with fission, fusion, cell proliferation, mitophagy, and mitochondrial bioenergetics, it remains a fascinating new avenue for translational research. When compounded with the availability of strongly supported inhibitors, Drp1 evolves into a protein that should receive more attention. Developing an understanding of Drp1 in pathology will help uncover the mysteries of mitochondrial dynamics and may also simultaneously reveal new avenues of precise treatment for a spectrum of deleterious diseases.

GRANTS

N. Kolliputi was funded by the American Heart Association National Scientist Development Grant (09SDG2260957), National Institutes of Health National Heart, Lung, and Blood Institute Grants R01 HL-105932 and R56 HL-105932, and the Joy McCann Culverhouse endowment to the Division of Allergy and Immunology.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.T.B. prepared figures; M.T.B. drafted manuscript; M.T.B., M.D.A., and R.F.L. edited and revised manuscript; N.K. approved final version of manuscript.