Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

MITOL regulates phosphatidic acid-binding activity of RMDN3/PTPIP51.

The transfer of phospholipids from the endoplasmic reticulum (ER) to mitochondria via the mitochondria-ER contact site (MERCS) is essential for maintaining mitochondrial function and integrity. Here, we identified RMDN3/PTPIP51, possessing phosphatidic acid (PA)-transfer activity, as a neighbouring protein of the mitochondrial E3 ubiquitin ligase MITOL/MARCH5 by proximity-dependent biotin labelling using APEX2. We found that MITOL interacts with and ubiquitinates RMDN3. Mutational analysis identified lysine residue 89 in RMDN3 as a site of ubiquitination by MITOL. Loss of MITOL or the substitution of lysine 89 to arginine in RMDN3 significantly reduced the PA-binding activity of RMDN3, suggesting that MITOL regulates the transport of PA to mitochondria by activating RMDN3. Our findings imply that ubiquitin signalling regulates phospholipid transport at the MERCS.

Oscillation of Cdc20-APC/C-mediated CAMDI stability is critical for cortical neuron migration.

Radial migration during cortical development is required for formation of the six-layered structure of the mammalian cortex. Defective migration of neurons is linked to several developmental disorders such as autism and schizophrenia. A unique swollen structure called the dilation is formed in migrating neurons and is required for movement of the centrosome and nucleus. However, the detailed molecular mechanism by which this dilation forms is unclear. We report that CAMDI, a gene whose deletion is associated with psychiatric behavior, is degraded by cell division cycle protein 20 (Cdc20)-anaphase-promoting complex/cyclosome (APC/C) cell-cycle machinery after centrosome migration into the dilation in mouse brain development. We also show that CAMDI is restabilized in the dilation until the centrosome enters the dilation, at which point it is once again immediately destabilized. CAMDI degradation is carried out by binding to Cdc20-APC/C via the destruction box degron of CAMDI. CAMDI destruction box mutant overexpression inhibits dilation formation and neuronal cell migration via maintaining the stabilized state of CAMDI. These results indicate that CAMDI is a substrate of the Cdc20-APC/C system and that the oscillatory regulation of CAMDI protein correlates with dilation formation for proper cortical migration.

Forebrain-specific deficiency of the GTPase CRAG/Centaurin-γ3 leads to immature dentate gyri and hyperactivity in mice.

Mouse models of various neuropsychiatric disorders, such as schizophrenia, often display an immature dentate gyrus, characterized by increased numbers of immature neurons and neuronal progenitors and a dearth of mature neurons. We previously demonstrated that the CRMP5-associated GTPase (CRAG), a short splice variant of Centaurin-γ3/AGAP3, is highly expressed in the dentate gyrus. CRAG promotes cell survival and antioxidant defense by inducing the activation of serum response factors at promyelocytic leukemia protein bodies, which are nuclear stress-responsive domains, during neuronal development. However, the physiological role of CRAG in neuronal development remains unknown. Here, we analyzed the role of CRAG using dorsal forebrain-specific CRAG/Centaurin-γ3 knockout mice. The mice revealed maturational abnormality of the hippocampal granule cells, including increased doublecortin-positive immature neurons and decreased calbindin-positive mature neurons, a typical phenotype of immature dentate gyri. Furthermore, the mice displayed hyperactivity in the open-field test, a common measure of exploratory behavior, suggesting that these mice may serve as a novel model for neuropsychiatric disorder associated with hyperactivity. Thus, we conclude that CRAG is required for the maturation of neurons in the dentate gyrus, raising the possibility that its deficiency might promote the development of psychiatric disorders in humans.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

MITOL promotes cell survival by degrading Parkin during mitophagy.

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-ß oligomer generation.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aß fibrils, but not the spontaneous formation of Aß fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aß oligomers. Consistent with this, MITOL-deficient mice with Aß etiology exhibited worsening cognitive decline depending on Aß oligomers rather than Aß plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aß form, oligomers or plaques, responsible for disease development.

MITOL dysfunction causes dwarfism with anterior pituitary hypoplasia.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients.

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.

Critical role of CRAG, a splicing variant of centaurin-γ3/AGAP3, in ELK1-dependent SRF activation at PML bodies.

CRMP-5-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, is predominantly expressed in the developing brain. We previously demonstrated that CRAG, but not centaurin-γ3, translocates to the nucleus and activates the serum response factor (SRF)-c-Fos pathway in cultured neuronal cells. However, the physiological relevance of CRAG in vivo is unknown. Here, we found that CRAG/centaurin-γ3-knockout mice showed intensively suppressed kainic acid-induced c-fos expression in the hippocampus. Analyses of molecular mechanisms underlying CRAG-mediated SRF activation revealed that CRAG has an essential role in GTPase activity, interacts with ELK1 (a co-activator of SRF), and activates SRF in an ELK1-dependent manner. Furthermore, CRAG and ELK1 interact with promyelocytic leukaemia bodies through SUMO-interacting motifs, which is required for SRF activation. These results suggest that CRAG plays a critical role in ELK1-dependent SRF-c-fos activation at promyelocytic leukaemia bodies in the developing brain.

CAMDI interacts with the human memory-associated protein KIBRA and regulates AMPAR cell surface expression and cognition.

Little is known about the molecular mechanisms of cognitive deficits in psychiatric disorders. CAMDI is a psychiatric disorder-related factor, the deficiency of which in mice results in delayed neuronal migration and psychiatrically abnormal behaviors. Here, we found that CAMDI-deficient mice exhibited impaired recognition memory and spatial reference memory. Knockdown of CAMDI in hippocampal neurons increased the amount of internalized alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) and attenuated the chemical long-term potentiation (LTP)-dependent cell surface expression of AMPAR. KIBRA was identified as a novel CAMDI-binding protein that retains AMPAR in the cytosol after internalization. KIBRA inhibited CAMDI-dependent Rab11 activation, thereby attenuating AMPAR cell surface expression. These results suggest that CAMDI regulates AMPAR cell surface expression during LTP. CAMDI dysfunction may partly explain the mechanism underlying cognitive deficits in psychiatric diseases.

MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

MITOL deletion in the brain impairs mitochondrial structure and ER tethering leading to oxidative stress.

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.

Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor.

The DISC1-interacting protein CAMDI has been suggested to promote radial migration through centrosome regulation. However, its physiological relevance is unclear. Here, we report the generation and characterization of CAMDI-deficient mice. CAMDI-deficient mice exhibit delayed radial migration with aberrant neural circuit formation and psychiatric behaviors including hyperactivity, repetitive behavior, and social abnormality typically observed in autism spectrum disorder patients. Analyses of direct targets of CAMDI identify HDAC6 whose α-tubulin deacetylase activity is inhibited by CAMDI at the centrosome. CAMDI deficiency increases HDAC6 activity, leading to unstable centrosomes with reduced γ-tubulin and acetylated α-tubulin levels. Most importantly, psychiatric behaviors as well as delayed migration are significantly rescued by treatment with Tubastatin A, a specific inhibitor of HDAC6. Our findings indicate that HDAC6 hyperactivation by CAMDI deletion causes psychiatric behaviors, at least in part, through delayed radial migration due to impaired centrosomes.

Regulation of B cell differentiation by the ubiquitin-binding protein TAX1BP1.

Tax1-binding protein 1 (TAX1BP1) is a ubiquitin-binding protein that restricts nuclear factor-κB (NF-κB) activation and facilitates the termination of aberrant inflammation. However, its roles in B-cell activation and differentiation are poorly understood. To evaluate the function of TAX1BP1 in B cells, we established TAX1BP1-deficient DT40 B cells that are hyper-responsive to CD40-induced extracellular signal-regulated kinase (ERK) activation signaling, exhibit prolonged and exaggerated ERK phosphorylation and show enhanced B lymphocyte-induced maturation protein 1 (Blimp-1; a transcription factor inducing plasma cell differentiation) expression that is ERK-dependent. Furthermore, TAX1BP1-deficient cells exhibit significantly decreased surface IgM expression and increased IgM secretion. Moreover, TAX1BP1-deficient mice display reduced germinal center formation and antigen-specific antibody production. These findings show that TAX1BP1 restricts ERK activation and Blimp-1 expression and regulates germinal center formation.

Roles of mitochondrial ubiquitin ligase MITOL/MARCH5 in mitochondrial dynamics and diseases.

Accumulating evidence indicate physiological significance of mitochondrial dynamics such as mitochondrial fusion and division, the dynamic movement of mitochondria along microtubules and interaction of mitochondria with the endoplasmic reticulum. A disruption in mitochondrial dynamics leads to a functional deterioration of mitochondria, resulting in a variety of diseases including neurodegenerative disorders. We previously identified a mitochondrial ubiquitin ligase MITOL/MARCH5, which belongs to the membrane-associated RING-CH E3 ubiquitin ligase (MARCH) family (also called MARCH5). MITOL plays an important role in the regulation of mitochondrial dynamics including mitochondrial morphology, transport and interaction with ER, at least in part, through the ubiquitinations of mitochondrial fission factor Drp1, microtubule-associated protein 1B and mitofusin2, respectively. This review focuses on recent findings that show how MITOL regulates mitochondrial dynamics and which suggest physiological disorders resulting from a failure in such regulation.

MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Mitochondrial ubiquitin ligase MITOL blocks S-nitrosylated MAP1B-light chain 1-mediated mitochondrial dysfunction and neuronal cell death.

Nitric oxide (NO) is implicated in neuronal cell survival. However, excessive NO production mediates neuronal cell death, in part via mitochondrial dysfunction. Here, we report that the mitochondrial ubiquitin ligase, MITOL, protects neuronal cells from mitochondrial damage caused by accumulation of S-nitrosylated microtubule-associated protein 1B-light chain 1 (LC1). S-nitrosylation of LC1 induces a conformational change that serves both to activate LC1 and to promote its ubiquination by MITOL, indicating that microtubule stabilization by LC1 is regulated through its interaction with MITOL. Excessive NO production can inhibit MITOL, and MITOL inhibition resulted in accumulation of S-nitrosylated LC1 following stimulation of NO production by calcimycin and N-methyl-D-aspartate. LC1 accumulation under these conditions resulted in mitochondrial dysfunction and neuronal cell death. Thus, the balance between LC1 activation by S-nitrosylation and down-regulation by MITOL is critical for neuronal cell survival. Our findings may contribute significantly to an understanding of the mechanisms of neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction.

CRMP5-associated GTPase (CRAG) protein protects neuronal cells against cytotoxicity of expanded polyglutamine protein partially via c-Fos-dependent activator protein-1 activation.

We previously demonstrated that CRAM (CRMP5)-associated GTPase (CRAG), a short splicing variant of centaurin-γ3/AGAP3, facilitated degradation of expanded polyglutamine protein (polyQ) via the nuclear ubiquitin-proteasome pathway. Taking advantage of this feature, we also showed that lentivirus-mediated CRAG expression in the Purkinje cells of mice expressing polyQ resulted in clearance of the polyQ aggregates and rescue from ataxia. However, the molecular basis of the function of CRAG in cell survival against polyQ remains unclear. Here we report that CRAG, but not centaurin-γ3, induces transcriptional activation of c-Fos-dependent activator protein-1 (AP-1) via serum response factor (SRF). Mutation analysis indicated that the nuclear localization signal and both the N- and C-terminal regions of CRAG are critical for SRF-dependent c-Fos activation. CRAG knockdown by siRNA or expression of a dominant negative mutant of CRAG significantly attenuated the c-Fos activation triggered by either polyQ or the proteasome inhibitor MG132. Importantly, c-Fos expression partially rescued the enhanced cytotoxicity of CRAG knockdown in polyQ-expressing or MG132-treated cells. Finally, we suggest the possible involvement of CRAG in the sulfiredoxin-mediated antioxidant pathway via AP-1. Taken together, these results demonstrated that CRAG enhances the cell survival signal against the accumulation of unfolded proteins, including polyQ, through not only proteasome activation, but also the activation of c-Fos-dependent AP-1.

A mitochondrial ubiquitin ligase MITOL controls cell toxicity of polyglutamine-expanded protein.

Expansion of a polyglutamine tract in ataxin-3 (polyQ) causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation. Several lines of evidence demonstrate that polyQ also accumulates in mitochondria and causes mitochondrial dysfunction. To uncover the mechanism of mitochondrial quality-control via the ubiquitin-proteasome pathway, we investigated whether MITOL, a novel mitochondrial ubiquitin ligase localized in the mitochondrial outer membrane, is involved in the degradation of pathogenic ataxin-3 in mitochondria. In this study, we used N-terminal-truncated pathogenic ataxin-3 with a 71-glutamine repeat (ΔNAT-3Q71) and found that MITOL promoted ΔNAT-3Q71 degradation via the ubiquitin-proteasome pathway and attenuated mitochondrial accumulation of ΔNAT-3Q71. Conversely, MITOL knockdown induced an accumulation of detergent-insoluble ΔNAT-3Q71 with large aggregate formation, resulting in cytochrome c release and subsequent cell death. Thus, MITOL plays a protective role against polyQ toxicity, and thereby may be a potential target for therapy in polyQ diseases. Our findings indicate a protein quality-control mechanism at the mitochondrial outer membrane via a MITOL-mediated ubiquitin-proteasome pathway.