Oxidative stress-mediated activation of extracellular signal-regulated kinase contributes to mild cognitive impairment-related mitochondrial dysfunction

Mild cognitive impairment (MCI) occurs during the predementia stage of Alzheimer disease (AD) and is characterized by a decline in cognitive abilities that frequently represents a transition between normal cognition and AD dementia. Its pathogenesis is not well understood. Here, we demonstrate the direct consequences and potential mechanisms of oxidative stress and mitochondrial dynamic and functional defects in MCI-derived mitochondria. Using a cytoplasmic hybrid (cybrid) cell model in which mitochondria from MCI or age-matched non-MCI subjects were incorporated into a human neuronal cell line depleted of endogenous mitochondrial DNA, we evaluated the mitochondrial dynamics and functions, as well as the role of oxidative stress in the resultant cybrid lines. We demonstrated that increased expression levels of mitofusin 2 (Mfn2) are markedly induced by oxidative stress in MCI-derived mitochondria along with aberrant mitochondrial functions. Inhibition of oxidative stress rescues MCI-impaired mitochondrial fusion/fission balance as shown by the suppression of Mfn2 expression, attenuation of abnormal mitochondrial morphology and distribution, and improvement in mitochondrial function. Furthermore, blockade of MCI-related stress-mediated activation of extracellular signal-regulated kinase (ERK) signaling not only attenuates aberrant mitochondrial morphology and function but also restores mitochondrial fission and fusion balance, in particular inhibition of overexpressed Mfn2. Our results provide new insights into the role of the oxidative stress–ERK–Mfn2 signal axis in MCI-related mitochondrial abnormalities, indicating that the MCI phase may be targetable for the development of new therapeutic approaches that improve mitochondrial function in age-related neurodegeneration.     

Leucine-rich repeat kinase 2 disturbs mitochondrial dynamics via Dynamin-like protein

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the leading causes of genetically inherited Parkinson's disease (PD) identified so far. The underlying mechanism whereby missense alterations in LRRK2 initiate neurodegeneration remains largely unclear. Mitochondrial dysfunction has been recognized to contribute to the pathogenesis of both sporadic and familial PD. The pathogenic gain-of-function mutant form of LRRK2, LRRK2 G2019S, is associated with elevated kinase activity and PD. Here we show that LRRK2 G2019S can cause defects in the morphology and dynamics of mitochondria in cortical neurons. In neurons, endogenous LRRK2 and the mitochondrial fission factor Dynamin like protein 1 (DLP1) interact with and partially co-localize with each other. DLP1 plays an essential role in LRRK2-induced mitochondrial fission. In support of this, expression of LRRK2 leads to the translocation of DLP1 from the cytosol to the mitochondria and knockdown of DLP1 expression inhibits LRRK2-induced mitochondrial fission. In addition, co-expression of LRRK2 and DLP1 induces mitochondrial clearance. Furthermore, we have found that expression of LRRK2 leads to increased reactive oxygen species levels in cells. Taken together, our results provide insights into the pathobiology of LRRK2 and suggest that LRRK2 G2019S may induce neuronal dysfunction or cell death by disturbing normal mitochondrial fission/fusion dynamics and function.     

Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer's disease

Mitochondria play critical roles in neuronal function and almost all aspects of mitochondrial function are altered in Alzheimer neurons. Emerging evidence shows that mitochondria are dynamic organelles that undergo continuous fission and fusion, the balance of which not only controls mitochondrial morphology and number, but also regulates mitochondrial function and distribution. In this review, after a brief overview of the basic mechanisms involved in the regulation of mitochondrial fission and fusion and how mitochondrial dynamics affects mitochondrial function, we will discuss in detail our and others' recent work demonstrating abnormal mitochondrial morphology and distribution in Alzheimer's disease (AD) models and how these abnormalities may contribute to mitochondrial and synaptic dysfunction in AD. We propose that abnormal mitochondrial dynamics plays a key role in causing the dysfunction of mitochondria that ultimately damage AD neurons.     

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.     

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca²⁺) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca²⁺ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca²⁺ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca²⁺ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca²⁺ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca²⁺ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca²⁺-mediated ROS generation in cardiac myocytes. These results suggest that Ca²⁺, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.     

Disruption of fusion results in mitochondrial heterogeneity and dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.     

Sirt3 modulate renal ischemia-reperfusion injury through enhancing mitochondrial fusion and activating the ERK-OPA1 signaling pathway

Mitochondrial fusion is linked to heart and liver ischemia-reperfusion (IR) insult. Unfortunately, there is no report to elucidate the detailed influence of mitochondrial fusion in renal IR injury. This study principally investigated the mechanism by which mitochondrial fusion protected kidney against IR injury. Our results indicated that sirtuin 3 (Sirt3) was inhibited after renal IR injury in vivo and in vitro. Overexpression of Sirt3 improved kidney function, modulated oxidative injury, repressed inflammatory damage, and reduced tubular epithelial cell apoptosis. The molecular investigation found that Sirt3 overexpression attenuated IR-induced mitochondrial damage in renal tubular epithelial cells, as evidenced by decreased reactive oxygen species production, increased antioxidants sustained mitochondrial membrane potential, and inactivated mitochondria-initiated death signaling. In addition, our information also illuminated that Sirt3 maintained mitochondrial homeostasis against IR injury by enhancing optic atrophy 1 (OPA1)-triggered fusion of mitochondrion. Inhibition of OPA1-induced fusion repressed Sirt3 overexpression-induced kidney protection, leading to mitochondrial dysfunction. Further, our study illustrated that OPA1-induced fusion could be affected through ERK; inhibition of ERK abolished the regulatory impacts of Sirt3 on OPA1 expression and mitochondrial fusion, leading to mitochondrial damage and tubular epithelial cell apoptosis. Altogether, our results suggest that renal IR injury is closely associated with Sirt3 downregulation and mitochondrial fusion inhibition. Regaining Sirt3 and/or activating mitochondrial fission by modifying the ERK-OPA1 cascade may represent new therapeutic modalities for renal IR injury.     

Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.     

Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS‐ERK activation

Mitochondrial dynamics control mitochondrial functions as well as their morphology. However, the role of mitochondrial dynamics in melanogenesis is largely unknown. Here, we show that mitochondrial dynamics regulate melanogenesis by modulating the ROS‐ERK signaling pathway. Genetic and chemical inhibition of Drp1, a mitochondrial fission protein, increased melanin production and mitochondrial elongation in melanocytes and melanoma cells. In contrast, down‐regulation of OPA1, a mitochondria fusion regulator, suppressed melanogensis but induced massive mitochondrial fragmentation in hyperpigmented cells. Consistently, treatment with CCCP, a mitochondrial fission chemical inducer, also efficiently repressed melanogenesis. Furthermore, we found that ROS production and ERK phosphorylation were increased in cells with fragmented mitochondria. And inhibition of ROS or ERK suppressed the antimelanogenic effect of mitochondrial fission in α‐MSH‐treated cells. In addition, the activation of ROS‐ERK pathway by mitochondrial fission induced phosphorylation of serine73 on MITF accelerating its proteasomal degradation. In conclusion, mitochondrial dynamics may regulate melanogenesis by modulating ROS‐ERK signaling pathway.     

Control of mitochondrial morphology through differential interactions of mitochondrial fusion and fission proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria.     

Cyclical mitochondrial deltapsiM fluctuations linked to electron transport, F0F1 ATP-synthase and mitochondrial Na+/Ca+2 exchange are reduced in Alzheimer's disease cybrids

Reduced complex IV, increased oxidative stress and beta amyloid peptide secretion in Alzheimer's disease (AD) can be replicated in cybrid models. We characterized cyclical mitochondrial deltapsiM fluctuations ('flickering') in neuroblastoma cells and AD/CTL cybrids. Flickering was blocked by ATP-synthase inhibition, was not observed in rho0 cells and was not blocked by antioxidant treatment. Flickering was not affected by the Ca(+2) uniporter antagonist Ru360 but was eliminated by BAPTA or CGP37137 blockade of the mitochondrial Na(+)/Ca(+2) exchanger. AD cybrid mitochondria showed reduced flickering. Flickering seems to represent coupling of deltapsiM to F0F1 ATP-synthase; reduction of flickering in AD cybrids suggests dysfunction of this coupling.     

DLP1‐dependent mitochondrial fragmentation and redistribution mediate prion‐associated mitochondrial dysfunction and neuronal death

Mitochondrial malfunction is a universal and critical step in the pathogenesis of many neurodegenerative diseases including prion diseases. Dynamin‐like protein 1 (DLP1) is one of the key regulators of mitochondrial fission. In this study, we investigated the role of DLP1 in mitochondrial fragmentation and dysfunction in neurons using in vitro and in vivo prion disease models. Mitochondria became fragmented and redistributed from axons to soma, correlated with increased mitochondrial DLP1 expression in murine primary neurons (N2a cells) treated with the prion peptide PrP^106–126 in vitro as well as in prion strain‐infected hamster brain in vivo. Suppression of DLP1 expression by DPL1 RNAi inhibited prion‐induced mitochondrial fragmentation and dysfunction (measured by ADP/ATP ratio, mitochondrial membrane potential, and mitochondrial integrity). We also demonstrated that DLP1 RNAi is neuroprotective against prion peptide in N2a cells as shown by improved cell viability and decreased apoptosis markers, caspase 3 induced by PrP^106–126. On the contrary, overexpression of DLP1 exacerbated mitochondrial dysfunction and cell death. Moreover, inhibition of DLP1 expression ameliorated PrP^106–126‐induced neurite loss and synaptic abnormalities (i.e., loss of dendritic spine and PSD‐95, a postsynaptic scaffolding protein as a marker of synaptic plasticity) in primary neurons, suggesting that altered DLP1 expression and mitochondrial fragmentation are upstream events that mediate PrP^106–126‐induced neuron loss and degeneration. Our findings suggest that DLP1‐dependent mitochondrial fragmentation and redistribution plays a pivotal role in PrP^Sc‐associated mitochondria dysfunction and neuron apoptosis. Inhibition of DLP1 may be a novel and effective strategy in the prevention and treatment of prion diseases.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

S-Nitrosylation of Drp1 links excessive mitochondrial fission to neuronal injury in neurodegeneration

Neurons are known to use large amounts of energy for their normal function and activity. In order to meet this demand, mitochondrial fission, fusion, and movement events (mitochondrial dynamics) control mitochondrial morphology, facilitating biogenesis and proper distribution of mitochondria within neurons. In contrast, dysfunction in mitochondrial dynamics results in reduced cell bioenergetics and thus contributes to neuronal injury and death in many neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease. We recently reported that amyloid-β peptide, thought to be a key mediator of AD pathogenesis, engenders S-nitrosylation and thus hyperactivation of the mitochondrial fission protein Drp1. This activation leads to excessive mitochondrial fragmentation, bioenergetic compromise, and synaptic damage in models of AD. Here, we provide an extended commentary on our findings of nitric oxide-mediated abnormal mitochondrial dynamics.     

Mitochondrial dynamic alterations regulate melanoma cell progression

Research on mitochondrial fusion and fission (mitochondrial dynamics) has gained much attention in recent years, as it is important for understanding many biological processes, including the maintenance of mitochondrial functions, apoptosis, and cancer. The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. However, the role of mitochondrial dynamics in melanoma progression remains controversial and requires a mechanistic understanding to target the altered metabolism of cancer cells. Therefore, in our study, we disrupted mitochondrial fission with mdivi-1, the reported inhibitor of dynamin related protein 1 (Drp1), and knocked down Drp1 and Mfn2 to evaluate the effects of mitochondrial dynamic alterations on melanoma cell progression. Our confocal study results showed that mitochondrial fission was inhibited both in mdivi-1 and in Drp1 knockdown cells and, in parallel, mitochondrial fusion was induced. We also found that mitochondrial fission inhibition by mdivi-1 induced cell death in melanoma cells. However, silencing Drp1 and Mfn2 did not affect cell viability, but enhanced melanoma cell migration. We further show that dysregulated mitochondrial fusion by Mfn2 knockdowns suppressed the oxygen consumption rate of melanoma cells. Together, our findings suggest that mitochondrial dynamic alterations regulate melanoma cell migration and progression.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Attenuation of Doxorubicin-Induced Cardiotoxicity by mdivi-1: A Mitochondrial Division/Mitophagy Inhibitor

Doxorubicin is one of the most effective anti-cancer agents. However, its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Given the multiple beneficial effects of the mitochondrial division inhibitor (mdivi-1) in a variety of pathological conditions including heart failure and ischaemia and reperfusion injury, we investigated the effects of mdivi-1 on doxorubicin-induced cardiac dysfunction in naïve and stressed conditions using Langendorff perfused heart models and a model of oxidative stress was used to assess the effects of drug treatments on the mitochondrial depolarisation and hypercontracture of cardiac myocytes. Western blot analysis was used to measure the levels of p-Akt and p-Erk 1/2 and flow cytometry analysis was used to measure the levels p-Drp1 and p-p53 upon drug treatment. The HL60 leukaemia cell line was used to evaluate the effects of pharmacological inhibition of mitochondrial division on the cytotoxicity of doxorubicin in a cancer cell line. Doxorubicin caused a significant impairment of cardiac function and increased the infarct size to risk ratio in both naïve conditions and during ischaemia/reperfusion injury. Interestingly, co-treatment of doxorubicin with mdivi-1 attenuated these detrimental effects of doxorubicin. Doxorubicin also caused a reduction in the time taken to depolarisation and hypercontracture of cardiac myocytes, which were reversed with mdivi-1. Finally, doxorubicin caused a significant elevation in the levels of signalling proteins p-Akt, p-Erk 1/2, p-Drp1 and p-p53. Co-incubation of mdivi-1 with doxorubicin did not reduce the cytotoxicity of doxorubicin against HL-60 cells. These data suggest that the inhibition of mitochondrial fission protects the heart against doxorubicin-induced cardiac injury and identify mitochondrial fission as a new therapeutic target in ameliorating doxorubicin-induced cardiotoxicity without affecting its anti-cancer properties.     

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.     

Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with “bulge”-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous “fusion” and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.