Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.     

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1

We showed earlier that 15 deoxy Δ^12,14 prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.     

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.     

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.     

线粒体融合、分裂与神经变性疾病

线粒体是一种处于高度运动状态的频繁地进行融合与分裂的细胞器.在生理状态下,线粒体的融合与分裂处于一种平衡的状态,这种平衡受线粒体融合蛋白1/2（Mfn1/2）、视神经萎缩蛋白1（OPA1）和动力相关蛋白1（Drp1）的调节. Mfn1/2介导线粒体外膜的融合,而OPA1则参与线粒体内膜的融合,这些蛋白受泛素化和蛋白水解的调控. Drp1参与线粒体的分裂过程,受多种翻译后修饰的调节,如磷酸化、泛素化、SUMO化和S 硝基化.对于神经元来说,线粒体融合分裂的动态平衡对保证神经元末梢长距离运输和能量平均分布是非常重要的.因此,线粒体融合分裂异常可能是许多神经变性疾病的致病因素之一.对线粒体融合而言,Mfn2错义突变将导致遗传性运动感觉神经病2型（CMT2A）;OPA1错义突变将引起显性遗传性视神经萎缩(ADOA),而就线粒体分裂而言,Drp1突变与多系统功能障碍的新生儿致死性相关.     

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.     

Spatial and temporal association of Bax with mitochondrial fission sites,Drp1, and Mfn2 during apoptosis

We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.     

Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins

Mitochondria are dynamic organelles that undergo continual fusion and fission to maintain their morphology and functions, but the mechanism involved is still not clear. Here, we investigated the effect of mitochondrial oxidative stress triggered by high-fluence low-power laser irradiation (HF-LPLI) on mitochondrial dynamics in human lung adenocarcinoma cells (ASTC-a-1) and African green monkey SV40-transformed kidney fibroblast cells (COS-7). Upon HF-LPLI-triggered oxidative stress, mitochondria displayed a fragmented structure, which was abolished by exposure to dehydroascorbic acid, a reactive oxygen species scavenger, indicating that oxidative stress can induce mitochondrial fragmentation. Further study revealed that HF-LPLI caused mitochondrial fragmentation by inhibiting fusion and enhancing fission. Mitochondrial translocation of the profission protein dynamin-related protein 1 (Drp1) was observed following HF-LPLI, demonstrating apoptosis-related activation of Drp1. Notably, overexpression of Drp1 increased mitochondrial fragmentation and promoted HF-LPLI-induced apoptosis through promoting cytochrome c release and caspase-9 activation, whereas overexpression of mitofusin 2 (Mfn2), a profusion protein, caused the opposite effects. Also, neither Drp1 overexpression nor Mfn2 overexpression affected mitochondrial reactive oxygen species generation, mitochondrial depolarization, or Bax activation. We conclude that mitochondrial oxidative stress mediated through Drp1 and Mfn2 causes an imbalance in mitochondrial fission-fusion, resulting in mitochondrial fragmentation, which contributes to mitochondrial and cell dysfunction.     

Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons

Mitochondrial dysfunction is an underpinning event in many neurodegenerative disorders. Less clear, however, is how mitochondria become injured during neuronal demise. Nitric oxide (NO) evokes rapid mitochondrial fission in cortical neurons. Interestingly, proapoptotic Bax relocates from the cytoplasm into large foci on mitochondrial scission sites in response to nitrosative stress. Antiapoptotic Bcl-xL does not prevent mitochondrial fission despite its ability to block Bax puncta formation on mitochondria and to mitigate neuronal cell death. Mitofusin 1 (Mfn1) or dominant-negative dynamin-related protein 1(K38A) (Drp1(k38A)) inhibits mitochondrial fission and Bax accumulation on mitochondria induced by exposure to an NO donor. Although NO is known to cause a bioenergetic crisis, lowering ATP by glycolytic or mitochondrial inhibitors neither induces mitochondrial fission nor Bax foci formation on mitochondria. Taken together, these data indicate that the mitochondrial fission machinery acts upstream of the Bcl-2 family of proteins in neurons challenged with nitrosative stress.     

Stage-specific enhanced expression of mitochondrial fusion and fission factors during spermatogenesis in rat testis

The mitochondrial fusion factors mitofusins 1 and 2 (Mfn1 and Mfn2) and the fission factor dynamin-related protein 1 (Drp1) were found to be highly expressed in the pubertal and adult rat testis by Northern blot analysis. Immunohistochemistry using specific antisera to Mfn2 and Drp1 revealed a pronounced expression of the fusion and fission factors in the round and elongating spermatids in the seminiferous tubules, suggesting that at precise steps of spermiogenesis (i.e., steps 8-12), spermatid mitochondria are rapidly homogenized by frequent fusion and division. Although physiological relevance of this phenomenon remains to be clarified, a role is proposed for it as an effective means of achieving complete and homogeneous ubiquitination of mitochondria, which has recently been demonstrated to be a mechanism for the elimination of paternal mitochondria during fertilization, based on the fact that the timing of expression of Mfn2 and Drp1 coincides well with that reported for a spermatid-specific ubiquitin-conjugating enzyme.     

Mitochondrial biogenesis in the axons of vertebrate peripheral neurons

Mitochondria are widely distributed via regulated transport in neurons, but their sites of biogenesis remain uncertain. Most mitochondrial proteins are encoded in the nuclear genome, and evidence has suggested that mitochondrial DNA (mtDNA) replication occurs mainly or entirely in the cell body. However, it has also become clear that nuclear-encoded mitochondrial proteins can be translated in the axon and that components of the mitochondrial replication machinery reside there as well. We assessed directly whether mtDNA replication can occur in the axons of chick peripheral neurons labeled with 5-bromo-2'-deoxyuridine (BrdU). In axons that were physically separated from the cell body or had disrupted organelle transport between the cell bodies and axons, a significant fraction of mtDNA synthesis continued. We also detected the mitochondrial fission protein Drp1 in neurons by immunofluorescence or expression of GFP-Drp1. Its presence and distribution on the majority of axonal mitochondria indicated that a substantial number had undergone recent division in the axon. Because the morphology of mitochondria is maintained by the balance of fission and fusion events, we either inhibited Drp1 expression by RNAi or overexpressed the fusion protein Mfn1. Both methods resulted in significantly longer mitochondria in axons, including many at a great distance from the cell body. These data indicate that mitochondria can replicate their DNA, divide, and fuse locally within the axon; thus, the biogenesis of mitochondria is not limited to the cell body.     

Human MIEF1 recruits Drp1 to mitochondrial outer membranes and promotes mitochondrial fusion rather than fission

Mitochondrial morphology is controlled by two opposing processes: fusion and fission. Drp1 (dynamin-related protein 1) and hFis1 are two key players of mitochondrial fission, but how Drp1 is recruited to mitochondria and how Drp1-mediated mitochondrial fission is regulated in mammals is poorly understood. Here, we identify the vertebrate-specific protein MIEF1 (mitochondrial elongation factor 1; independently identified as MiD51), which is anchored to the outer mitochondrial membrane. Elevated MIEF1 levels induce extensive mitochondrial fusion, whereas depletion of MIEF1 causes mitochondrial fragmentation. MIEF1 interacts with and recruits Drp1 to mitochondria in a manner independent of hFis1, Mff (mitochondrial fission factor) and Mfn2 (mitofusin 2), but inhibits Drp1 activity, thus executing a negative effect on mitochondrial fission. MIEF1 also interacts with hFis1 and elevated hFis1 levels partially reverse the MIEF1-induced fusion phenotype. In addition to inhibiting Drp1, MIEF1 also actively promotes fusion, but in a manner distinct from mitofusins. In conclusion, our findings uncover a novel mechanism which controls the mitochondrial fusion-fission machinery in vertebrates. As MIEF1 is vertebrate-specific, these data also reveal important differences between yeast and vertebrates in the regulation of mitochondrial dynamics.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to IR‐783‐induced apoptosis in human breast cancer cells

IR‐783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR‐783 has not been well demonstrated. In this study, we showed that IR‐783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA‐MB‐231 cells to IR‐783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR‐783 induced dynamin‐related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission‐1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR‐783‐mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR‐783 markedly inhibited tumour growth and induced apoptosis in an MDA‐MB‐231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR‐783 induces apoptosis in human breast cancer cells by increasing Drp1‐mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti‐breast cancer effects of IR‐783 and provided novel perspectives for the application of IR‐783 in the treatment of breast cancer.     

Dynamin-related protein 1 is required for normal mitochondrial bioenergetic and synaptic function in CA1 hippocampal neurons

Disrupting particular mitochondrial fission and fusion proteins leads to the death of specific neuronal populations; however, the normal functions of mitochondrial fission in neurons are poorly understood, especially in vivo, which limits the understanding of mitochondrial changes in disease. Altered activity of the central mitochondrial fission protein dynamin-related protein 1 (Drp1) may contribute to the pathophysiology of several neurologic diseases. To study Drp1 in a neuronal population affected by Alzheimer''s disease (AD), stroke, and seizure disorders, we postnatally deleted Drp1 from CA1 and other forebrain neurons in mice (CamKII-Cre, Drp1^lox/lox (Drp1cKO)). Although most CA1 neurons survived for more than 1 year, their synaptic transmission was impaired, and Drp1cKO mice had impaired memory. In Drp1cKO cell bodies, we observed marked mitochondrial swelling but no change in the number of mitochondria in individual synaptic terminals. Using ATP FRET sensors, we found that cultured neurons lacking Drp1 (Drp1KO) could not maintain normal levels of mitochondrial-derived ATP when energy consumption was increased by neural activity. These deficits occurred specifically at the nerve terminal, but not the cell body, and were sufficient to impair synaptic vesicle cycling. Although Drp1KO increased the distance between axonal mitochondria, mitochondrial-derived ATP still decreased similarly in Drp1KO boutons with and without mitochondria. This indicates that mitochondrial-derived ATP is rapidly dispersed in Drp1KO axons, and that the deficits in axonal bioenergetics and function are not caused by regional energy gradients. Instead, loss of Drp1 compromises the intrinsic bioenergetic function of axonal mitochondria, thus revealing a mechanism by which disrupting mitochondrial dynamics can cause dysfunction of axons.Mitochondrial dynamics – the balance between mitochondrial fission and fusion – regulates mitochondrial quality control by segregating poorly functioning mitochondria for degradation while mixing the contents of healthy mitochondria.^{1, 2} In neurons, fission uniquely facilitates movement of mitochondria down narrow distal axons.^{3, 4} Disruptions of this movement, and of other neuron-specific functions, may explain why systemic mutations in mitochondrial fusion and fission proteins specifically cause death of neurons. However, the roles and requirements of these proteins also differ between neuronal types.¹ For example, mutations in the fusion protein optic atrophy 1 cause degeneration of retinal ganglion neurons,⁵ and mutations in the fusion protein mitofusin-2 or the fission protein ganglioside-induced differentiation-associated protein 1 cause peripheral neuropathy (Charcot-Marie-Tooth types 2A and 4A^{6, 7}).There are several potential reasons why specific neurons have unique requirements for fission–fusion proteins. First, the functions of these proteins may be more critical in vulnerable neuronal populations. Recently, we showed that most midbrain DA neurons are uniquely vulnerable to loss of the central mitochondrial fission protein dynamin-related protein 1 (Drp1),⁴ a GTPase recruited to fission sites on the outer mitochondrial membrane.¹ Loss of Drp1 depletes axonal mitochondria, which is followed by axonal degeneration and neuronal death. However, a subpopulation of midbrain DA neurons survive, despite losing their axonal mitochondria, suggesting that they have lower needs for energy or other mitochondrial functions in their axons.⁴ Do unique requirements for mitochondrial dynamics underlie differential neuronal vulnerability? Do resistant neurons compensate with other fission or fusion mechanisms? Do the functions of fission differ between neurons? Notably, Drp1 may also have mitochondria-independent functions in synaptic vesicle release.⁸ Addressing these issues could help elucidate the physiological functions of mitochondrial dynamics in the nervous system and reveal how shifts in the fission–fusion balance contribute to selective neuronal death in neurodegenerative diseases, including Huntington''s disease, Parkinson''s disease and Alzheimer''s disease (AD),^{1, 4} and in other neurologic disorders, including stroke and epilepsy.^{9, 10, 11}To understand mitochondrial dynamics, it would be useful to know why mitochondrial fission is needed in the nervous system in the first place, and how loss of fission affects mitochondrial functions in specific cell types. Notably, Drp1 knockout did not change respiration or ATP levels in resuspended mouse embryonic fibroblasts (MEFs),^{12, 13} indicating that mitochondrial fission is not required for respiration in these cells. However, neuronal respiration may be more sensitive to Drp1 loss. Indeed, Drp1 loss markedly decreased the number of mitochondria in axons and the cell body in midbrain DA neurons in vivo,⁴ and reduced staining of complex I and IV activity in cerebellar neurons in vivo.¹⁴ However, it is unclear whether these changes translate into decreased ATP levels in neurons and, if so, whether this decrease compromises neuronal function. Furthermore, Drp1 loss caused cell death in cerebellar and most midbrain DA neurons,^{4, 14} which challenges our ability to dissociate the specific effects of Drp1 loss on mitochondrial function from other non-specific changes that accompany cell death.To learn how disrupting mitochondrial fission contributes to selective neurodegeneration, we studied the function of Drp1 in CA1 hippocampal neurons and its role in mitochondrial bioenergetics. Surprisingly, despite losing Drp1, most CA1 neurons survived for more than 1 year in vivo, although their function was compromised, leading to deficits in synaptic transmission and memory. To begin to understand how loss of Drp1 causes neuronal dysfunction, we examined the role of Drp1 in mitochondrial bioenergetics. We found that Drp1 is required to maintain normal mitochondrial-derived ATP levels specifically in axons (but not the cell body), and that the loss of this function is unrelated to the distribution of mitochondria within axons.     

有氧运动改善AD模型APP/PS1双转基因小鼠中枢神经元线粒体融合分裂动态平衡

线粒体融合分裂平衡是线粒体动力学的需要。本研究观察12周规律有氧运动对APP/PS1双转基因小鼠中枢神经元线粒体融合分裂动态平衡的影响。本研究采用3月龄雄性APP/PS1小鼠（AD模型）随机分为AD安静组（AS）、AD运动组（AE）,同月龄雄性C57BL/6J小鼠做正常对照组（CS）。AE组进行12周规律跑台运动,5 d/周,60 min/d。前10 min运动速度12 m/min,后50 min运动速度15 m/min,跑台坡度为0°。八臂迷宫实验检测小鼠工作记忆错误频率和参考记忆错误频率;Western印迹检测小鼠皮层、海马组织中线粒体分裂蛋白Drp1和Fis1的含量,以及Drp1的活性（p-Drp1-Ser616）、线粒体融合蛋白Mfn1、Mfn2、Opa1的表达水平;透射电镜观察皮层、海马线粒体形态结构、健康线粒体比率及线粒体平均直径。本研究证实AS组较CS组工作记忆错误频率显著提高（P<0.05）,12周有氧运动显著降低工作记忆错误频率（P<0.05）。AS组小鼠皮层Fis1蛋白和海马脑区Drp1、Fis1蛋白表达水平及皮层、海马脑区Drp1蛋白的活性增加（P<0.05）。而皮层Mfn1和海马Mfn1、Mfn2蛋白表达水平显著降低（P<0.05）。12周有氧运动显著减低Fis1、Drp1蛋白表达及Drp1蛋白的活性,提高Mfn1、Mfn2蛋白表达水平（P<0.05）。AS组小鼠皮层、海马线粒体多呈现球形,部分线粒体膜结构消失,线粒体嵴结构紊乱。且AS组较CS组小鼠健康线粒体比率降低、直径缩短。12周规律有氧运动可明显改善线粒体形态和结构,提高健康线粒体比率及直径。本研究提示,12周规律有氧运动可有效抑制皮层、海马脑区线粒体分裂蛋白Drp1和 Fis1的表达,降低Drp1的活性（p-Drp1-Ser616）,上调线粒体融合蛋白Mfn1、Mfn2的蛋白表达水平,改善线粒体形态和结构以促进线粒体质量控制,是有氧运动改善AD模型空间学习记忆能力的分子机制之一。     

Role of the lipid transport protein StarD7 in mitochondrial dynamics

Mitochondria are dynamic organelles crucial for cell function and survival implicated in oxidative energy production whose central functions are tightly controlled by lipids. StarD7 is a lipid transport protein involved in the phosphatidylcholine (PC) delivery to mitochondria. Previous studies have shown that StarD7 knockdown induces alterations in mitochondria and endoplasmic reticulum (ER) with a reduction in PC content, however whether StarD7 modulates mitochondrial dynamics remains unexplored. Here, we generated HTR-8/SVneo stable cells expressing the precursor StarD7.I and the mature processed StarD7.II isoforms. We demonstrated that StarD7.I overexpression altered mitochondrial morphology increasing its fragmentation, whereas no changes were observed in StarD7.II-overexpressing cells compared to the control (Ct) stable cells. StarD7.I (D7.I) stable cells were able to transport higher fluorescent PC analog to mitochondria than Ct cells, yield mitochondrial fusions, maintained the membrane potential, and produced lower levels of reactive oxygen species (ROS). Additionally, the expression of Dynamin Related Protein 1 (Drp1) and Mitofusin (Mfn2) proteins were increased, whereas the amount of Mitofusin 1 (Mfn1) decreased. Moreover, transfections with plasmids encoding Drp1-K38A, Drp1-S637D or Drp1-S637A mutants indicated that mitochondrial fragmentation in D7.I cells occurs in a fission-dependent manner via Drp1. In contrast, StarD7 silencing decreased Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level. These cells increased ROS levels and presented donut-shape mitochondria, indicative of metabolic stress. Altogether our findings provide novel evidence indicating that alterations in StarD7.I expression produce significant changes in mitochondrial morphology and dynamics.     

Fis1, Mff,MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.     

Mitofusins ‘bridge’ the gap between oxidative stress and mitochondrial hyperfusion

During stress conditions, mitochondria can undergo hyperfusion to protect the cell. A recent study in EMBO reports identifies a new mechanism by which mitofusins can be activated to initiate mitochondrial during oxidative stress.EMBO reports (2012) 13, 909–915; doi:10.1038/embor.2012.128Mitochondria are dynamic organelles that undergo fission and fusion events, but we are only beginning to understand some of the reasons and the machineries involved in these processes [1]. Fission allows distribution of mitochondria to daughter cells following mitosis. Small mitochondria are more efficiently tracked along the cytoskeleton, which also allows a quality control mechanism to exist where fragmented ‘old'' mitochondria can be turned over by autophagy [2]. Fusion enables mitochondrial contents to be mixed between neighbouring organelles. Fission and fusion events must be tightly regulated, and whilst they are opposing processes, they seem to act in concert together—for example, fusion events often lead to a subsequent fission event at the same site. In this issue of EMBO reports, McBride and colleagues [3] identify a new mechanism by which mitofusins can be activated to initiate mitochondrial fusion under conditions of oxidative stress.…GSSG stimulates mitochondrial fusion whereas GSH inhibits itFission involves surface receptors and adaptors (Fis1, Mff, MiD49 and MiD51/MIEF1) and the cytosolic dynamin-related protein Drp1. Fusion involves the dynamin GTPases mitofusin (Mfn) 1 and 2 at the outer and Opa1 at the inner membrane. Mitofusins form homo- and hetero-oligomers to tether adjacent mitochondria together. How the outer membranes fuse before Opa1 acts to fuse the inner membranes is not known. Moreover, the mechanisms regulating their fusion activity are not well-understood. Mitochondrial fission and fusion are important in developmental and physiological processes. Mitochondrial fragmentation occurs during apoptosis and necrosis, where the loss of the network facilitates cell death. However, under conditions of less severe stress, mitochondria can undergo hyperfusion, seemingly as a mechanism by which they can protect the cell from dying [4]. During nutrient starvation, which leads to the induction of macro-autophagy, mitochondria also undergo hyperfusion to prevent their encapsulation by autophagosomes. Hyperfusion is also important during the G1 to S transition of the cell cycle. Until now, the mechanisms regulating stress-induced mitochondrial hyperfusion have remained elusive.McBride and colleagues used an in vitro fusion assay, which consists of mixing mitochondria isolated from cell cultures expressing either an amino-terminal or a carboxy-terminal domain of luciferase, targeted to the mitochondrial matrix. Both luciferase domains contained a leucine zipper that leads to dimerization upon mitochondrial fusion thereby generating a functional enzyme. The authors found that in vitro, mitochondrial fusion was stimulated by oxidative stress including hydrogen peroxide treatment, whereas anti-oxidants inhibited the process. They then tested whether fusion might be responsive to reactive oxygen species or cellular oxidants. Glutathione (GSH), found in all parts of the cell, provides the main redox buffer for cells. GSH contains a free thiol group, and the formation of a disulphide bond between two GSH molecules gives rise to oxidized GSH (GSSG). GSH reductase recycles GSSG to GSH thus maintaining the cellular redox state. During oxidative stress, GSSG accumulates and can interact with other proteins, and induces either glutathionylation or the generation of a disulphide bond known as ‘disulphide switching''. McBride and colleagues found that GSSG stimulates mitochondrial fusion whereas GSH inhibits it. The fusion reaction could be stimulated by adding GSSG directly to isolated mitochondria whilst it was inhibited by the cysteine alkylating agent iodoacetate. This implies that mitochondrial proteins with free thiol groups are direct substrates of GSH-mediated oxidation and that this regulates mitochondrial fusion. Analysis of Mfn1 and Mfn2 revealed that GSSG treatment induces oligomer formation, which could be resolved on non-reducing SDS–PAGE as four distinct species of 160–220 kDa. Addition of a reductant led to the loss of the oligomeric species on SDS–PAGE, suggesting that the mitofusins might indeed form disulphide bonds. The presence of discrete oligomeric species on non-reducing gels suggests that intermolecular disulphides might occur and that the mitofusins interact with additional proteins. Interestingly, two proteins shown to regulate mitofusin activity directly, SLP2 and non-apoptotic Bax, were not modified by oxidant. Similarly, Drp1 was unaffected, pointing to a direct activation of fusion rather than a decrease in fission activity. However, it remains possible that fission might also be downregulated by oxidation, and inactivation, of mitochondrial fission receptors such as Fis1, Mff and MiD49/MiD51 or by a different post-translational modification of Drp1. The authors validated their work by incubating cultured cells with agents that cause an increase in the levels of GSSG. Such treatment induced mitochondrial hyperfusion as expected, but it also resulted in the increase of Mfn2 oligomers.This implies that mitochondrial proteins with free thiol groups are direct substrates of GSH-mediated oxidation and that this regulates mitochondrial fusionMitofusins contain a cytosolically exposed N-terminal GTPase domain followed by a heptad repeat (HR1), and two transmembrane regions that form a hairpin in the mitochondrial outer membrane allowing the exposure of another heptad repeat (HR2) to the cytosol. The heptad repeats are involved in membrane-tethering events, and structural analysis of a HR2 dimer revealed that it forms an antiparallel coiled-coil [5]. When the authors added non-hydrolysable GTP to their in vitro assay, the GSSG-mediated fusion was blocked. When mitochondria were diluted to reduce tethering between mitochondria, GSSG still induced mitofusin oligomerization and this depended on GTP hydrolysis. This suggests that stress-induced oligomerization occurs largely between mitofusin molecules on the surface of the same mitochondria—that is, in cis. At higher concentrations of mitochondria where tethering events are enhanced, GTP hydrolysis did not seem to be required to induce oligomerization of oxidized mitofusins between adjacent mitochondria—that is, in trans. To narrow down the cysteine residues that are oxidized in mitofusins, the authors performed site-directed mutagenesis of Mfn2 and introduced the mutants into Mfn2^−/− mouse embryonic fibroblasts. The authors found that mutation of Cys 684 in the hinge region of HR2 reduced the formation of oligomers and was less functional in rescuing mitochondrial fusion. The authors suggest that disulphide bond formation between this residue and cysteines in adjacent mitofusins might alter the coiled-coil interactions, thereby priming the proteins to activate fusion.…disulphide bond formation between […] adjacent mitofusins might alter the coiled-coil interactions, thereby priming the proteins to activate fusionWhy is hyperfusion needed? Mitochondrial fragmentation is an important aspect of cell death pathways, and hyperfusion might open a window of time to enable the cell to activate proteins involved in responding to cellular insults. However, cells can also block fusion during stress-induced apoptosis. It was found that stress-activated c-Jun N-terminal kinase phosphorylates Mfn2, leading to its ubiquitination and degradation [6]. How cells decide whether to turn on either survival mechanisms that activate mitofusins, or death mechanisms that inactivate them, requires additional studies.The work by McBride and colleagues points to the importance of intracellular redox conditions in regulating mitochondrial fusion. Changes in cellular GSH might also be important for regulating other members of the fission–fusion machinery. Ganglioside-induced differentiation associated protein 1 (GDAP1) functions in mitochondrial fission, and mutations in GDAP1 lead to the neurodegenerative disorder Charcot Marie-Tooth disease (CMT) 2A. GDAP1 contains GSH S-transferase domains, and loss of the protein has been found to decrease the levels of GSH [7]. Interestingly, mutations in Mfn2 lead to CMT4A, which is clinically almost indistinct from CMT2A. A study of a family with CMT revealed an asymptomatic mother with a mutation in GDAP1 and an asymptomatic father with a mutation in Mfn2. Their child who inherited both mutant alleles developed severe neuropathy [8]. The cumulative effects of inheriting both mutations points to an overlap in the function of these proteins. The new findings, pointing to the importance of GSH in mitochondrial fusion, warrant investigation into whether a closer connection between GDAP1 and mitofusins exists.? Open in a separate windowFigure 1Model for mitochondrial fusion induced by oxidative stress. Oxidation of cysteines in mitofusins induces oligomer formation in cis, probably through the formation of one or more disulphide bonds, which might cause a conformational change in the heptad repeat (HR) regions aiding in tethering to mitofusins in trans to enhance membrane fusion.