Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

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.

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.

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.

Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial division.

Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria-ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIß [PI(4)KIIIß], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P-containing vesicles at mitochondria-ER contact sites may trigger final events leading to mitochondrial scission.

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.

The novel heart-specific RING finger protein 207 is involved in energy metabolism in cardiomyocytes.

A failing heart shows severe energy insufficiency, and it is presumed that this energy shortage plays a critical role in the development of cardiac dysfunction. However, little is known about the mechanisms that cause energy metabolic alterations in the failing heart. Here, we show that the novel RING-finger protein 207 (RNF207), which is specifically expressed in the heart, plays a role in cardiac energy metabolism. Depletion of RNF207 in neonatal rat cardiomyocytes (NRCs) leads to a reduced cellular concentration of adenosine triphosphate (ATP) and mitochondrial dysfunction. Consistent with this result, we observed here that the expression of RNF207 was significantly reduced in mice with common cardiac diseases including heart failure. Intriguingly, proteomic approaches revealed that RNF207 interacts with the voltage-dependent anion channel (VDAC), which is considered to be a key regulator of mitochondria function, as an RNF207-interacting protein. Our findings indicate that RNF207 is involved in ATP production by cardiomyocytes, suggesting that RNF207 plays an important role in the development of heart failure.

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.

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.