Mitochondrial Fission GTPase Research Articles

Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.

Abstract A43: Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML

Abstract Background: RAS/RAF/ERK pathway activation is associated with primary or secondary resistance to venetoclax in AML. Oncogenic RAS activates mitochondrial fission through ERK1/2 mediated phosphorylation of the mitochondrial fission GTPase Drp1. Although the deletion of Drp1 inhibits KRAS driven pancreatic cancer, the role of RAS-driven mitochondrial dynamics in AML is unclear. Inhibiting ERK1/2 may, therefore, shed light on AML cells' dependence on RAS driven mitochondrial dynamics and metabolism. Objectives: We assessed the anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and further investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., 2018), sensitized intrinsically resistant and NRAS-mutant OCI-AML3 cells to venetoclax with combination index (CI) of 0.008. Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax (75% apoptosis versus 22% by venetoclax, CI=0.6). Bulk and CD34+CD38- leukemia initiating cells (LICs) were also eliminated in primary AML samples by the combination (CI:0.03-0.23). Colony formation assay and CyTOF using NRAS-mutant PDX showed impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to ERK1/2 inhibition and combination treatment, suggesting impact on stemness. ERK1/2 inhibition using ASTX029 (Heightman et al., 2021) alone and in combination with venetoclax reduced leukemia burden in-vivo and extended survival (p<0.05). Mitochondrial ROS mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML. ERK1/2 inhibition decreased pDrp1-Ser616 levels along with an increase in mitochondrial length (p<0.001) and mitochondrial ROS (p<0.01), suggesting impaired mitochondrial fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Overexpression of a phospho-mimetic i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells decreased apoptosis and reversed synergy between ERK1/2 and Bcl-2 inhibition as compared to wild type and phospho-null (Ser616Ala) Drp1 (p<0.001). Of note, overexpression of Drp1-phospho-mimetic led to decreased mitochondrial length, suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, significantly decreased maximal oxygen consumption rate, ATP production and ROS production. Moreover, combination treatment failed to significantly induce ROS production in cells expressing phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Conclusion: Inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. In summary, these data support combining ERK1/2 and Bcl-2 inhibitors in the treatment of AML and warrant further clinical investigation. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Anna Zal, Christopher Hindley, Kim-Hien Dao, Martin Sims, Tomasz Zal, Vivian Ruvolo, Michael Andreeff, Gautam Borthakur. Targeting ERK1/2 overcomes resistance to venetoclax by altering Drp1-dependent mitochondrial fission and metabolism in AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A43.

Opa1 and Drp1 reciprocally regulate cristae morphology, ETC function, and NAD+ regeneration in KRas-mutant lung adenocarcinoma

Oncogenic KRas activates mitochondrial fission through Erk-mediated phosphorylation of the mitochondrial fission GTPase Drp1. Drp1 deletion inhibits tumorigenesis of KRas-driven pancreatic cancer, but the role of mitochondrial dynamics in other Ras-driven malignancies is poorly defined. Here we show that invitro and invivo growth of KRas-driven lung adenocarcinoma is unaffected by deletion of Drp1 but is inhibited by deletion of Opa1, the GTPase that regulates inner membrane fusion and proper cristae morphology. Mechanistically, Opa1 knockout disrupts cristae morphology and inhibits electron transport chain (ETC) assembly and activity, which inhibits tumor cell proliferation through loss of NAD+ regeneration. Simultaneous inactivation of Drp1 and Opa1 restores cristae morphology, ETC activity, and cell proliferation indicating that mitochondrial fission activity drives ETC dysfunction induced by Opa1 knockout. Our results support a model in which mitochondrial fission events disrupt cristae structure, and tumor cells with hyperactive fission activity require Opa1 activity to maintain ETC function.

ERK1/2 Inhibition Overcomes Resistance to Venetoclax in AML By Altering Drp1-Dependent Mitochondrial Fission and Metabolism

Background: Mutational (RAS, PTPN11 etc.) or non-mutational activation of RAS/RAF/ERK pathway is associated with primary or secondary resistance to venetoclax in AML therapy (Zhang et al., Sig Transduct Target Ther. 2022). While upregulation of alternate antiapoptotic proteins e.g. Mcl-1 has been implicated in resistance to venetoclax (Zhang et al., Sig Transduct Target Ther. 2022) role of mitochondrial dynamics (fission/fusion) in venetoclax resistance has not been well explored. Oncogenic RAS activates downstream ERK1/2 phosphorylation and mitochondrial fission through ERK1/2-mediated phosphorylation of the dynamin-like mitochondrial fission GTPase Drp1 (Nagdas et al., Cell Rep. 2019). Drp1 deletion inhibits tumorigenesis of KRAS driven pancreatic cancer, but the importance of RAS driven changes in mitochondrial dynamics in AML is poorly defined. ERK1/2 inhibition may therefore provide insights into the dependence of AML cells on altered mitochondrial dynamics and metabolism driven by oncogenic RAS. Objectives: We assessed the synergistic anti-leukemia efficacy of concomitant Bcl2 and ERK1/2 inhibition in preclinical models of venetoclax resistance and in primary patient samples (n=8). Further, we investigated the role of ERK1/2/Drp1-dependent mitochondrial fission in mediating resistance to venetoclax through altered metabolism. Results: ERK1/2 inhibition using Compound 27 (Heightman et al., J Med Chem. 2018) a close analog of ASTX029, which is currently under clinical investigation in solid tumors (NCT03520075), was highly synergistic with venetoclax at inducing apoptosis in AML cells. NRAS-mutant OCI-AML3 and THP1 cells, that are intrinsically resistant to venetoclax, were sensitized to venetoclax by ERK1/2 inhibition with a combination index (CI) of 0.008 (cell death ~90% for combination versus 20% with venetoclax alone). Synergy was also confirmed in OCI-AML2 cells with acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax alone (CI=0.6). The combination was also effective in eliminating bulk and CD34+CD38- leukemia initiating cells (LICs) in primary AML samples (CI:0.03-0.23). Colony formation assay and single cell level CyTOF analysis using an NRAS-mutant PDX showed markedly impaired clonogenic potential (p=0.0002) and decreased expression of cMyc and CD44 in LICs in response to single agent ERK1/2 inhibition and combination treatment, indicating a potential impact on stemness. Mitochondrial reactive oxygen species (ROS) mitigation, Drp1-mediated mitochondrial fission and glycolysis are critical to maintain stemness in AML (Schimmer et al., Cell Stem Cell. 2018). As a result of ERK1/2 inhibition, pDrp1 (Ser616) levels decreased, and mitochondrial length (p<0.001) and mitochondrial ROS levels (p<0.01) were increased, suggesting impaired fission. In OCI-AML3 cells, combination treatment caused downregulation of Drp1 mRNA and decreased phosphorylation of Drp1 at Ser616. Rescue with overexpression of active phospho-mimetic form of Drp1 i.e. Drp1-Ser616Glu-Ser637Ala in OCI-AML3 cells resulted in decreased apoptosis and reversed the synergy between ERK1/2 and Bcl-2 inhibition when compared to wild type and phospho-null (Ser616Ala) Drp1 overexpression (p<0.001, Fig 1a). Of note, overexpression of Drp1-Ser616Glu-Ser637Ala phospho-mimetic led to decreased mitochondrial length (Fig 1b), suggesting enhanced fission with a distinct metabolic phenotype including reduced basal respiration, decreased maximal oxygen consumption rate (p<0.05) and ATP production (p<0.05), as well as reduced ROS production (p<0.01). Moreover, combination treatment failed to significantly induce ROS production in cells expressing the phospho-mimetic Drp1 as opposed to those expressing phospho-null or wildtype Drp1. Collectively, these results indicate that inhibition of ERK1/2/Drp1dependent mitochondrial fission and metabolic alterations may be a potential mechanism of synergistic tumor cell killing for the combination treatment. Conclusion: ERK1/2 inhibition alters Drp1 dependent mitochondrial dynamics and metabolism, resulting in mitochondrial defects and overcoming resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibiting mitochondrial fission rescues degeneration in hereditary spastic paraplegia neurons.

Hereditary spastic paraplegias are characterized by lower limb spasticity resulting from degeneration of long corticospinal axons. SPG11 is one of the most common autosomal recessive hereditary spastic paraplegias, and the SPG11 protein spatacsin forms a complex with the SPG15 protein spastizin and heterotetrameric AP5 adaptor protein complex, which includes the SPG48 protein AP5Z1. Using the integration-free episomal method, we established SPG11 patient-specific induced pluripotent stem cells (iPSCs) from patient fibroblasts. We differentiated SPG11 iPSCs, as well as SPG48 iPSCs previously established, into cortical projection neurons and examined protective effects by targeting mitochondrial dynamics using P110, a peptide that selectively inhibits mitochondrial fission GTPase Drp1. P110 treatment mitigates mitochondrial fragmentation, improves mitochondrial motility, and restores mitochondrial health and ATP levels in SPG11 and SPG48 neurons. Neurofilament aggregations are increased in SPG11 and SPG48 axons, and these are also suppressed by P110. Similarly, P110 mitigates neurofilament disruption in both SPG11 and SPG48 knockdown cortical projection neurons, confirming the contribution of hereditary spastic paraplegia gene deficiency to subsequent neurofilament and mitochondrial defects. Strikingly, neurofilament aggregations in SPG11 and SPG48 deficient neurons double stain with ubiquitin and autophagy related proteins, resembling the pathological hallmark observed in SPG11 autopsy brain sections. To confirm the cause-effect relationship between the SPG11 mutations and disease phenotypes, we knocked-in SPG11 disease mutations to human embryonic stem cells (hESCs) and differentiated these stem cells into cortical projection neurons. Reduced ATP levels and accumulated neurofilament aggregations along axons are observed, and both are mitigated by P110. Furthermore, rescue experiment with expression of wild-type SPG11 in cortical projection neurons derived from both SPG11 patient iPSCs and SPG11 disease mutation knock-in hESCs leads to rescue of mitochondrial dysfunction and neurofilament aggregations in these SPG11 neurons. Finally, in SPG11 and SPG48 long-term cultures, increased release of phosphoNF-H, a biomarker for nerve degeneration, is significantly reduced by inhibiting mitochondrial fission pharmacologically using P110 and genetically using Drp1 shRNA. Taken together, our results demonstrate that impaired mitochondrial dynamics underlie both cytoskeletal disorganization and axonal degeneration in SPG11 and SPG48 neurons, highlighting the importance of targeting these pathologies therapeutically.

Fis1 phosphorylation by Met promotes mitochondrial fission and hepatocellular carcinoma metastasis

Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as β-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesiclesare formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acidwas blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-basedcell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology

Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

Loss of the Mitochondrial Fission GTPase Drp1 Contributes to Neurodegeneration in a Drosophila Model of Hereditary Spastic Paraplegia

Mitochondrial morphology, distribution and function are maintained by the opposing forces of mitochondrial fission and fusion, the perturbation of which gives rise to several neurodegenerative disorders. The large guanosine triphosphate (GTP)ase dynamin-related protein 1 (Drp1) is a critical regulator of mitochondrial fission by mediating membrane scission, often at points of mitochondrial constriction at endoplasmic reticulum (ER)-mitochondrial contacts. Hereditary spastic paraplegia (HSP) subtype SPG61 is a rare neurodegenerative disorder caused by mutations in the ER-shaping protein Arl6IP1. We have previously reported defects in both the ER and mitochondrial networks in a Drosophila model of SPG61. In this study, we report that knockdown of Arl6IP1 lowers Drp1 protein levels, resulting in reduced ER–mitochondrial contacts and impaired mitochondrial load at the distal ends of long motor neurons. Increasing mitochondrial fission, by overexpression of wild-type Drp1 but not a dominant negative Drp1, increases ER–mitochondrial contacts, restores mitochondrial load within axons and partially rescues locomotor deficits. Arl6IP1 knockdown Drosophila also demonstrate impaired autophagic flux and an accumulation of ubiquitinated proteins, which occur independent of Drp1-mediated mitochondrial fission defects. Together, these findings provide evidence that impaired mitochondrial fission contributes to neurodegeneration in this in vivo model of HSP.

GFP fluorescence tagging alters dynamin-related protein\xa01 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. However, the consequences of appending the bulky GFP moiety to the protein of interest are rarely investigated. Here, using a powerful combination of quantitative fluorescence spectroscopic and imaging techniques, we have examined the oligomerization dynamics of the GFP-tagged mitochondrial fission GTPase dynamin-related protein 1 (Drp1) both in vitro and in vivo. We find that GFP-tagged Drp1 exhibits impaired oligomerization equilibria in solution that corresponds to a greatly diminished cooperative GTPase activity in comparison to native Drp1. Consequently, GFP-tagged Drp1 constitutes aberrantly stable, GTP-resistant supramolecular assemblies both in vitro and in vivo, neither of which reflects a more dynamic native Drp1 oligomerization state. Indeed, GFP-tagged Drp1 is detected more frequently per unit length over mitochondria in Drp1-null mouse embryonic fibroblasts (MEFs) compared to wild-type (wt) MEFs, indicating that the drastically reduced GTP turnover restricts oligomer disassembly from the mitochondrial surface relative to mixed oligomers comprising native and GFP-tagged Drp1. Yet, GFP-tagged Drp1 retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo. These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp1 higher-order oligomerization over membranes is sufficient for mitochondrial fission.

Nitric Oxide-Dependent Protein Post-Translational Modifications Impair Mitochondrial Function and Metabolism to Contribute to Neurodegenerative Diseases.

Significance: Most brains affected by neurodegenerative diseases manifest mitochondrial dysfunction as well as elevated production of reactive oxygen species and reactive nitrogen species (RNS), contributing to synapse loss and neuronal injury. Recent Advances: Excessive production of RNS triggers nitric oxide (NO)-mediated post-translational modifications of proteins, such as S-nitrosylation of cysteine residues and nitration of tyrosine residues. Proteins thus affected impair mitochondrial metabolism, mitochondrial dynamics, and mitophagy in the nervous system. Critical Issues: Identification and better characterization of underlying molecular mechanisms for NO-mediated mitochondrial dysfunction will provide important insights into the pathogenesis of neurodegenerative disorders. In this review, we highlight recent discoveries concerning S-nitrosylation of the tricarboxylic acid cycle enzymes, mitochondrial fission GTPase dynamin-related protein 1, and mitophagy-related proteins Parkin and phosphatase and tensin homolog-induced putative kinase protein 1. We delineate signaling cascades affected by pathologically S-nitrosylated proteins that diminish mitochondrial function in neurodegenerative diseases. Future Directions: Further elucidation of the pathological events resulting from aberrant S-nitrosothiol or nitrotyrosine formation may lead to new therapeutic approaches to ameliorate neurodegenerative disorders.

Drp1 Promotes KRas-Driven Metabolic Changes to Drive Pancreatic Tumor Growth

SUMMARYMitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.

DRP-1 functions independently of mitochondrial structural perturbations to facilitate BH3 mimetic-mediated apoptosis

Maintenance of mitochondrial integrity is critical for normal cellular homoeostasis. Most cells respond to stress stimuli and undergo apoptosis by perturbing mitochondrial structure and function to release proteins, such as cytochrome c, which are essential for the execution of the intrinsic apoptotic cascade. Cancer cells evade these events by overexpressing the anti-apoptotic BCL-2 family of proteins on mitochondrial membranes. Inhibitors of the anti-apoptotic BCL-2 family proteins, also known as BH3 mimetics, antagonise the pro-survival functions of these proteins and result in rapid apoptosis. Although the precise mechanism by which BH3 mimetics induce apoptosis has been well characterised, not much is known in terms of the structural changes that occur in mitochondria during apoptosis. Using a panel of highly selective BH3 mimetics and a wide range of cell lines, we demonstrate that BH3 mimetics induce extensive mitochondrial fission, accompanied by swelling of the mitochondrial matrix and rupture of the outer mitochondrial membrane. These changes occur in a BAX/ BAK-dependent manner. Although a major mitochondrial fission GTPase, DRP-1, has been implicated in mitochondrial apoptosis, our data demonstrate that DRP-1 might function independently/downstream of BH3 mimetic-mediated mitochondrial fission to facilitate the release of cytochrome c and apoptosis. Moreover, downregulation of DRP-1 prevented cytochrome c release and apoptosis even when OPA1, a protein mediating mitochondrial fusion, was silenced. Although BH3 mimetic-mediated displacement of BAK and other BH3-only proteins from BCL-XL and MCL-1 was unaffected by DRP-1 downregulation, it prevented BAK activation significantly, thus placing DRP-1 as one of the most critical players, along with BAX and BAK, that governs BH3 mimetic-mediated cytochrome c release and apoptosis.

ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects

Mitochondrial fragmentation and bioenergetic failure manifest in Huntington’s disease (HD), a fatal neurodegenerative disease. The factors that couple mitochondrial fusion/fission with bioenergetics and their impacts on neurodegeneration however remain poorly understood. Our proteomic analysis identifies mitochondrial protein ATAD3A as an interactor of mitochondrial fission GTPase, Drp1, in HD. Here we show that, in HD, ATAD3A dimerization due to deacetylation at K135 residue is required for Drp1-mediated mitochondrial fragmentation. Disturbance of ATAD3A steady state impairs mtDNA maintenance by disrupting TFAM/mtDNA binding. Blocking Drp1/ATAD3A interaction with a peptide, DA1, abolishes ATAD3A oligomerization, suppresses mitochondrial fragmentation and mtDNA lesion, and reduces bioenergetic deficits and cell death in HD mouse- and patient-derived cells. DA1 treatment reduces behavioral and neuropathological phenotypes in HD transgenic mice. Our findings demonstrate that ATAD3A plays a key role in neurodegeneration by linking Drp1-induced mitochondrial fragmentation to defective mtDNA maintenance, suggesting that DA1 might be useful for developing HD therapeutics.

Dynamin-like protein 1 cleavage by calpain in Alzheimer's disease.

Abnormal mitochondrial dynamics contributes to mitochondrial dysfunction in Alzheimer's disease (AD), yet the underlying mechanism remains elusive. In the current study, we reported that DLP1, the key mitochondrial fission GTPase, is a substrate of calpain which produced specific N‐terminal DLP1 cleavage fragments. In addition, various AD‐related insults such as exposure to glutamate, soluble amyloid‐β oligomers, or reagents inducing tau hyperphosphorylation (i.e., okadaic acid) led to calpain‐dependent cleavage of DLP1 in primary cortical neurons. DLP1 cleavage fragments were found in cortical neurons of CRND8 APP transgenic mice which can be inhibited by calpeptin, a potent small molecule inhibitor of calpain. Importantly, these N‐terminal DLP1 fragments were also present in the human brains, and the levels of both full‐length and N‐terminal fragments of DLP1 and the full‐length and calpain‐specific cleavage product of spectrin were significantly reduced in AD brains along with significantly increased calpain. These results suggest that calpain‐dependent cleavage is at least one of the posttranscriptional mechanisms that contribute to the dysregulation of mitochondrial dynamics in AD.

Srv2 Is a Pro-fission Factor that Modulates Yeast Mitochondrial Morphology and Respiration by Regulating Actin Assembly

SummaryDynamic processes such as fusion, fission, and trafficking are important in the regulation of cellular organelles, with an abundant literature focused on mitochondria. Mitochondrial dynamics not only help shape its network within cells but also are involved in the modulation of respiration and integrity. Disruptions of mitochondrial dynamics are associated with neurodegenerative disorders. Although proteins that directly bind mitochondria to promote membrane fusion/fission have been studied intensively, machineries that regulate dynamic mitochondrial processes remain to be explored. We have identified an interaction between the mitochondrial fission GTPase Dnm1/DRP1 and the actin-regulatory protein Srv2/CAP at mitochondria. Deletion of Srv2 causes elongated-hyperfused mitochondria and reduces the reserved respiration capacity in yeast cells. Our results further demonstrate that the irregular network morphology in Δsrv2 cells derives from disrupted actin assembly at mitochondria. We suggest that Srv2 functions as a pro-fission factor in shaping mitochondrial dynamics and regulating activity through its actin-regulatory effects.

A Metabolic Checkpoint for the Yeast-to-Hyphae Developmental Switch Regulated by Endogenous Nitric Oxide Signaling

The yeast Candida albicans colonizes several sites in the human body and responds to metabolic signals in commensal and pathogenic states. The yeast-to-hyphae transition correlates with virulence, but how metabolic status is integrated with this transition is incompletely understood. We used the putative mitochondrial fission inhibitor mdivi-1 to probe the crosstalk between hyphal signaling and metabolism. Mdivi-1 repressed C.albicans hyphal morphogenesis, but the mechanism was independent of its presumed target, the mitochondrial fission GTPase Dnm1. Instead, mdivi-1 triggered extensive metabolic reprogramming, consistent with metabolic stress, and reduced endogenous nitric oxide (NO) levels. Limiting endogenous NO stabilized the transcriptional repressor Nrg1 and inhibited the yeast-to-hyphae transition. We establish a role for endogenous NO signaling in C.albicans hyphal morphogenesis and suggest that NO regulates a metabolic checkpoint for hyphal growth. Furthermore, identifying NO signaling as an mdivi-1 target could inform its therapeutic applications in human diseases.

Impaired mitochondrial dynamics underlie axonal defects in hereditary spastic paraplegias.

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

Loss of laforin or malin results in increased Drp1 level and concomitant mitochondrial fragmentation in Lafora disease mouse models

Lafora disease (LD) is an autosomal recessive form of a fatal disorder characterized by the myoclonus epilepsy, ataxia, psychosis, dementia, and dysarthria. A hallmark of LD is the presence of abnormal glycogen inclusions called Lafora bodies in the affected tissues including the neurons. LD can be caused by defects either in the laforin phosphatase coded by the EPM2A gene or in the malin E3 ubiquitin ligase coded by the NHLRC1 gene. The mouse models of LD, created by the targeted disruption of the LD genes, display several neurodegenerative changes. Prominent among them are the autophagic defects, abnormally large lysosomes, neurofibrillary tangles, amyloid beta deposits, and abnormal mitochondria. However, whether or not such neurodegenerative changes are a direct effect of the loss of laforin/malin was not unequivocally established. Here, we show that laforin- or malin-deficient neurons and fibroblasts display a significantly higher number of fragmented mitochondria. Loss of laforin or malin resulted in increased levels of the mitochondrial fission GTPase Drp1, its enhanced mitochondrial targeting, and increased intracellular calcium levels. Intriguingly, laforin and malin display opposite effects on the cellular level of parkin, an ubiquitin ligase of Drp1; loss of laforin led to reduced levels of parkin while the loss of malin resulted in increased parkin levels. Laforin and malin, however, interact with and positively regulate the activity of parkin, thus explaining the molecular basis of increased Drp1 levels in LD tissues. Our results suggest that laforin and malin are novel regulators of mitochondrial quality control pathway and that the mitochondrial dysfunction resulting from the increased Drp1 levels could underlie neuropathology in LD.

Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.