Identification of Novel Therapeutic Targets for Polyglutamine Diseases That Target Mitochondrial Fragmentation

Huntington’s disease (HD) is one of at least nine polyglutamine diseases caused by a trinucleotide CAG repeat expansion, all of which lead to age-onset neurodegeneration. Mitochondrial dynamics and function are disrupted in HD and other polyglutamine diseases. While multiple studies have found beneficial effects from decreasing mitochondrial fragmentation in HD models by disrupting the mitochondrial fission protein DRP1, disrupting DRP1 can also have detrimental consequences in wild-type animals and HD models. In this work, we examine the effect of decreasing mitochondrial fragmentation in a neuronal C. elegans model of polyglutamine toxicity called Neur-67Q. We find that Neur-67Q worms exhibit mitochondrial fragmentation in GABAergic neurons and decreased mitochondrial function. Disruption of drp-1 eliminates differences in mitochondrial morphology and rescues deficits in both movement and longevity in Neur-67Q worms. In testing twenty-four RNA interference (RNAi) clones that decrease mitochondrial fragmentation, we identified eleven clones—each targeting a different gene—that increase movement and extend lifespan in Neur-67Q worms. Overall, we show that decreasing mitochondrial fragmentation may be an effective approach to treating polyglutamine diseases and we identify multiple novel genetic targets that circumvent the potential negative side effects of disrupting the primary mitochondrial fission gene drp-1.

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Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

Cell Death and Disease ◽

10.1038/s41419-021-03752-2 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Anthony R. Anzell ◽

Garrett M. Fogo ◽

Zoya Gurm ◽

Sarita Raghunayakula ◽

Joseph M. Wider ◽

...

Keyword(s):

Cortical Neurons ◽

Ischemia Reperfusion Injury ◽

Mitochondrial Dynamics ◽

Ischemia Reperfusion ◽

Mitochondrial Fission ◽

Conditional Knockout ◽

Mitochondrial Morphology ◽

Primary Neurons ◽

Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

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Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients

Biomolecules ◽

10.3390/biom10030450 ◽

2020 ◽

Vol 10 (3) ◽

pp. 450 ◽

Cited By ~ 1

Author(s):

Takeshi Tokuyama ◽

Asei Hirai ◽

Isshin Shiiba ◽

Naoki Ito ◽

Keigo Matsuno ◽

...

Keyword(s):

Mitochondrial Disease ◽

Mitochondrial Myopathy ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Critical Role ◽

Leigh Syndrome ◽

Mitochondrial Morphology ◽

Mitochondrial Fragmentation ◽

Cellular Toxicity ◽

Morphologic Changes

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.

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Dynamin-related protein 1 mediates low glucose-induced endothelial dysfunction in human arterioles

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00499.2016 ◽

2017 ◽

Vol 312 (3) ◽

pp. H515-H527 ◽

Cited By ~ 17

Author(s):

Michael J. Tanner ◽

Jingli Wang ◽

Rong Ying ◽

Tisha B. Suboc ◽

Mobin Malik ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Cardiovascular Complications ◽

Mitochondrial Morphology ◽

Vascular Relaxation ◽

Mitochondrial Fragmentation ◽

Low Glucose

Intensive glycemic regulation has resulted in an increased incidence of hypoglycemia. Hypoglycemic burden correlates with adverse cardiovascular complications and contributes acutely and chronically to endothelial dysfunction. Prior data indicate that mitochondrial dysfunction contributes to hypoglycemia-induced endothelial dysfunction, but the mechanisms behind this linkage remain unknown. We attempt to determine whether clinically relevant low-glucose (LG) exposures acutely induce endothelial dysfunction through activation of the mitochondrial fission process. Characterization of mitochondrial morphology was carried out in cultured endothelial cells by using confocal microscopy. Isolated human arterioles were used to explore the effect LG-induced mitochondrial fission has on the formation of detrimental reactive oxygen species (ROS), bioavailability of nitric oxide (NO), and endothelial-dependent vascular relaxation. Fluorescence microscopy was employed to visualize changes in mitochondrial ROS and NO levels and videomicroscopy applied to measure vasodilation response. Pharmacological disruption of the profission protein Drp1 with Mdivi-1 during LG exposure reduced mitochondrial fragmentation among vascular endothelial cells (LG: 0.469; LG+Mdivi-1: 0.276; P = 0.003), prevented formation of vascular ROS (LG: 2.036; LG+Mdivi-1: 1.774; P = 0.005), increased the presence of NO (LG: 1.352; LG+Mdivi-1: 1.502; P = 0.048), and improved vascular dilation response to acetylcholine (LG: 31.6%; LG+Mdivi-1; 78.5% at maximum dose; P < 0.001). Additionally, decreased expression of Drp1 via siRNA knockdown during LG conditions also improved vascular relaxation. Exposure to LG imparts endothelial dysfunction coupled with altered mitochondrial phenotypes among isolated human arterioles. Disruption of Drp1 and subsequent mitochondrial fragmentation events prevents impaired vascular dilation, restores mitochondrial phenotype, and implicates mitochondrial fission as a primary mediator of LG-induced endothelial dysfunction. NEW & NOTEWORTHY Acute low-glucose exposure induces mitochondrial fragmentation in endothelial cells via Drp1 and is associated with impaired endothelial function in human arterioles. Targeting of Drp1 prevents fragmentation, improves vasofunction, and may provide a therapeutic target for improving cardiovascular complications among diabetics. Listen to this article’s corresponding podcast @ http://ajpheart.podbean.com/e/mitochondrial-dynamics-impact-endothelial-function/ .

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Cyclin-dependent kinases regulate splice-specific targeting of dynamin-related protein 1 to microtubules

The Journal of Cell Biology ◽

10.1083/jcb.201210045 ◽

2013 ◽

Vol 201 (7) ◽

pp. 1037-1051 ◽

Cited By ~ 68

Author(s):

Stefan Strack ◽

Theodore J. Wilson ◽

J. Thomas Cribbs

Keyword(s):

Alternative Splicing ◽

Splice Variants ◽

Mitochondrial Fission ◽

Alternative Exon ◽

Mitochondrial Morphology ◽

Related Protein ◽

Fusion Reactions ◽

Mitochondrial Fragmentation ◽

Cyclin Dependent Kinases ◽

And Function

Fission and fusion reactions determine mitochondrial morphology and function. Dynamin-related protein 1 (Drp1) is a guanosine triphosphate–hydrolyzing mechanoenzyme important for mitochondrial fission and programmed cell death. Drp1 is subject to alternative splicing of three exons with previously unknown functional significance. Here, we report that splice variants including the third but excluding the second alternative exon (x01) localized to and copurified with microtubule bundles as dynamic polymers that resemble fission complexes on mitochondria. A major isoform in immune cells, Drp1-x01 required oligomeric assembly and Arg residues in alternative exon 3 for microtubule targeting. Drp1-x01 stabilized and bundled microtubules and attenuated staurosporine-induced mitochondrial fragmentation and apoptosis. Phosphorylation of a conserved Ser residue adjacent to the microtubule-binding exon released Drp1-x01 from microtubules and promoted mitochondrial fragmentation in a splice form–specific manner. Phosphorylation by Cdk1 contributed to dissociation of Drp1-x01 from mitotic microtubules, whereas Cdk5-mediated phosphorylation modulated Drp1-x01 targeting to interphase microtubules. Thus, alternative splicing generates a latent, cytoskeletal pool of Drp1 that is selectively mobilized by cyclin-dependent kinase signaling.

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Squamosamide Derivative FLZ Diminishes Aberrant Mitochondrial Fission by Inhibiting Dynamin-Related Protein 1

Frontiers in Pharmacology ◽

10.3389/fphar.2021.588003 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hanyu Yang ◽

Lu Wang ◽

Caixia Zang ◽

Xu Yang ◽

Xiuqi Bao ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Dopaminergic Neurons ◽

Motor Coordination ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Mitochondrial Morphology ◽

Protective Effects ◽

Related Protein ◽

Mitochondrial Fragmentation

Mitochondrial dysfunction is involved in the pathogenesis of Parkinson’s disease (PD). Mitochondrial morphology is dynamic and precisely regulated by mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation, which can result in cell death, is controlled by the mitochondrial fission protein, dynamin-related protein 1 (Drp1). Our previous results demonstrated that FLZ could correct mitochondrial dysfunction, but the effect of FLZ on mitochondrial dynamics remain uncharacterized. In this study, we investigated the effect of FLZ and the role of Drp1 on 1-methyl-4-phenylpyridinium (MPP+)–induced mitochondrial fission in neurons. We observed that FLZ blocked Drp1, inhibited Drp1 enzyme activity, and reduced excessive mitochondrial fission in cultured neurons. Furthermore, by inhibiting mitochondrial fission and ROS production, FLZ improved mitochondrial integrity and membrane potential, resulting in neuroprotection. FLZ curtailed the reduction of synaptic branches of primary cultured dopaminergic neurons caused by MPP+ exposure, reduced abnormal fission, restored normal mitochondrial distribution in neurons, and exhibited protective effects on dopaminergic neurons. The in vitro research results were validated using an MPTP-induced PD mouse model. The in vivo results revealed that FLZ significantly reduced the mitochondrial translocation of Drp1 in the midbrain of PD mice, which, in turn, reduced the mitochondrial fragmentation in mouse substantia nigra neurons. FLZ also protected dopaminergic neurons in PD mice and increased the dopamine content in the striatum, which improved the motor coordination ability of the mice. These findings elucidate this newly discovered mechanism through which FLZ produces neuroprotection in PD.

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Blocked O-GlcNAc cycling alters mitochondrial morphology, function, and mass

Scientific Reports ◽

10.1038/s41598-021-01512-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Elizabeth O. Akinbiyi ◽

Lara K. Abramowitz ◽

Brianna L. Bauer ◽

Maria S. K. Stoll ◽

Charles L. Hoppel ◽

...

Keyword(s):

Cellular Localization ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Mitochondrial Morphology ◽

Post Translational Modification ◽

Mitochondrial Content ◽

Protein Levels ◽

Cellular Environment ◽

And Function ◽

The Impact

AbstractO-GlcNAcylation is a prevalent form of glycosylation that regulates proteins within the cytosol, nucleus, and mitochondria. The O-GlcNAc modification can affect protein cellular localization, function, and signaling interactions. The specific impact of O-GlcNAcylation on mitochondrial morphology and function has been elusive. In this manuscript, the role of O-GlcNAcylation on mitochondrial fission, oxidative phosphorylation (Oxphos), and the activity of electron transport chain (ETC) complexes were evaluated. In a cellular environment with hyper O-GlcNAcylation due to the deletion of O-GlcNAcase (OGA), mitochondria showed a dramatic reduction in size and a corresponding increase in number and total mitochondrial mass. Because of the increased mitochondrial content, OGA knockout cells exhibited comparable coupled mitochondrial Oxphos and ATP levels when compared to WT cells. However, we observed reduced protein levels for complex I and II when comparing normalized mitochondrial content and reduced linked activity for complexes I and III when examining individual ETC complex activities. In assessing mitochondrial fission, we observed increased amounts of O-GlcNAcylated dynamin-related protein 1 (Drp1) in cells genetically null for OGA and in glioblastoma cells. Individual regions of Drp1 were evaluated for O-GlcNAc modifications, and we found that this post-translational modification (PTM) was not limited to the previously characterized residues in the variable domain (VD). Additional modification sites are predicted in the GTPase domain, which may influence enzyme activity. Collectively, these results highlight the impact of O-GlcNAcylation on mitochondrial dynamics and ETC function and mimic the changes that may occur during glucose toxicity from hyperglycemia.

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Adrenergic Regulation of Drp1-Driven Mitochondrial Fission in Cardiac Physio-Pathology

Antioxidants ◽

10.3390/antiox7120195 ◽

2018 ◽

Vol 7 (12) ◽

pp. 195 ◽

Cited By ~ 14

Author(s):

Bong Jhun ◽

Jin O-Uchi ◽

Stephanie Adaniya ◽

Michael Cypress ◽

Yisang Yoon

Keyword(s):

Mitochondrial Dysfunction ◽

Molecular Mechanisms ◽

Mitochondrial Fission ◽

Ros Generation ◽

Mitochondrial Morphology ◽

Mitochondrial Fragmentation ◽

Cellular Functions ◽

Post Translational Modifications ◽

Signaling Activation ◽

And Function

Abnormal mitochondrial morphology, especially fragmented mitochondria, and mitochondrial dysfunction are hallmarks of a variety of human diseases including heart failure (HF). Although emerging evidence suggests a link between mitochondrial fragmentation and cardiac dysfunction, it is still not well described which cardiac signaling pathway regulates mitochondrial morphology and function under pathophysiological conditions such as HF. Mitochondria change their shape and location via the activity of mitochondrial fission and fusion proteins. This mechanism is suggested as an important modulator for mitochondrial and cellular functions including bioenergetics, reactive oxygen species (ROS) generation, spatiotemporal dynamics of Ca2+ signaling, cell growth, and death in the mammalian cell- and tissue-specific manners. Recent reports show that a mitochondrial fission protein, dynamin-like/related protein 1 (DLP1/Drp1), is post-translationally modified via cell signaling pathways, which control its subcellular localization, stability, and activity in cardiomyocytes/heart. In this review, we summarize the possible molecular mechanisms for causing post-translational modifications (PTMs) of DLP1/Drp1 in cardiomyocytes, and further discuss how these PTMs of DLP1/Drp1 mediate abnormal mitochondrial morphology and mitochondrial dysfunction under adrenergic signaling activation that contributes to the development and progression of HF.

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Gag3p, an Outer Membrane Protein Required for Fission of Mitochondrial Tubules

The Journal of Cell Biology ◽

10.1083/jcb.151.2.333 ◽

2000 ◽

Vol 151 (2) ◽

pp. 333-340 ◽

Cited By ~ 122

Author(s):

Peter Fekkes ◽

Kelly A. Shepard ◽

Michael P. Yaffe

Keyword(s):

Outer Membrane ◽

Mitochondrial Fission ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Gene Products ◽

Mitochondrial Fragmentation ◽

Gene Encoding ◽

Peripheral Protein ◽

And Function ◽

Two Hybrid

Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.

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High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

10.1101/2021.01.14.426579 ◽

2021 ◽

Author(s):

Emma Cretin ◽

Priscilla Lopes ◽

Elodie Vimont ◽

Takashi Tatsuta ◽

Thomas Langer ◽

...

Keyword(s):

High Throughput ◽

Mitochondrial Disease ◽

Optic Atrophy ◽

Mitochondrial Dynamics ◽

Mitochondrial Fission ◽

Supervised Machine Learning ◽

Mitochondrial Morphology ◽

Phenotypic Screening ◽

Genetic Modifiers ◽

Mitochondrial Fragmentation

AbstractMutations affecting the mitochondrial fusion protein Optic Atrophy 1 (OPA1) cause autosomal dominant optic atrophy (DOA) – one of the most common form of mitochondrial disease. The majority of patients develop isolated optic atrophy, but about 20% of OPA1 mutation carriers manifest more severe neurological deficits as part of a “DOA+” phenotype. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae organization, oxidative phosphorylation, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. To better understand the genetic regulation of mitochondrial dynamics, we established a high-throughput imaging pipeline using supervised machine learning (ML) to perform unbiased, quantitative mitochondrial morphology analysis that was coupled with a bespoke siRNA library targeting the entire known mitochondrial proteome (1531 genes), providing a detailed phenotypic screening of human fibroblasts. In control fibroblasts, we identified known and novel genes whose depletion promoted elongation or fragmentation of the mitochondrial network. In DOA+ patient fibroblasts, we identified 91 candidate genes whose depletion prevents mitochondrial fragmentation, including the mitochondrial fission genes DNM1L, MIEF1, and SLC25A46, but also genes not previously linked to mitochondrial dynamics such as Phosphatidyl Glycerophosphate Synthase (PGS1), which belongs to the cardiolipin (CL) synthesis pathway. PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation. This study illustrates the power of a first-in-kind objective automated imaging approach to uncover genetic modifiers of mitochondrial disease through high-throughput phenotypic screening of patient fibroblasts.

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