Toshifumi Fukuda

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.

Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells

The RET receptor tyrosine kinase plays a critical role in the development of the enteric nervous system (ENS) and the kidney. Upon glial-cell-line-derived neurotrophic factor (GDNF) stimulation, RET can activate a variety of intracellular signals, including the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K)/AKT, and RAC1/JUN NH2-terminal kinase (JNK) pathways. We recently demonstrated that the RAC1/JNK pathway is regulated by serine phosphorylation at the juxtamembrane region of RET in a cAMP-dependent manner. To determine the importance of cAMP-dependent modification of the RET signal in vivo, we generated mutant mice in which serine residue 697, a putative protein kinase A (PKA) phosphorylation site, was replaced with alanine (designated S697A mice). Homozygous S697A mutant mice lacked the ENS in the distal colon, resulting from a migration defect of enteric neural crest cells (ENCCs). In vitro organ culture showed an impaired chemoattractant response of the mutant ENCCs to GDNF. JNK activation by GDNF but not ERK, AKT and SRC activation was markedly reduced in neurons derived from the mutant mice. The JNK inhibitor SP600125 and the PKA inhibitor KT5720 suppressed migration of the ENCCs in cultured guts from wild-type mice to comparable degrees. Thus, these findings indicated that cAMP-dependent modification of RET function regulates the JNK signaling responsible for proper migration of the ENCCs in the developing gut.

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin-proteasome pathway.

Distinct regulation of mitochondrial localization and stability of two human Sirt5 isoforms

Seven human Sir2 homologues (sirtuin) have been identified to date. In this study, we clarified the mechanism of subcellular localization of two SIRT5 isoforms (i.e., SIRT5iso1 and SIRT5iso2) encoded by the human SIRT5 gene and whose C‐termini slightly differ from each other. Although both isoforms contain cleavable mitochondrial targeting signals at their N‐termini, we found that the cleaved SIRT5iso2 was localized mainly in mitochondria, whereas the cleaved SIRT5iso1 was localized in both mitochondria and cytoplasm. SIRT5ΔC, which is composed of only the common domain, showed the same mitochondrial localization as that of SIRT5iso2. These results suggest that the cytoplasmic localization of cleaved SIRT5iso1 is dependent on the SIRT5iso1‐specific C‐terminus. Further analysis showed that the C‐terminus of SIRT5iso2, which is rich in hydrophobic amino acid residues, functions as a mitochondrial membrane insertion signal. In addition, a de novo protein synthesis inhibition experiment using cycloheximide showed that the SIRT5iso1‐specific C‐terminus is necessary for maintaining the stability of SIRT5iso1. Moreover, genome sequence analysis from each organism examined indicated that SIRT5iso2 is a primate‐specific isoform. Taken together, these results indicate that human SIRT5 potentially controls various primate‐specific functions via two isoforms with different intracellular localizations or stabilities.

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.

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.

CAMDI, a Novel Disrupted in Schizophrenia 1 (DISC1)-binding Protein, Is Required for Radial Migration

Centrosomes play a crucial role in the directed migration of developing neurons. However, the underlying mechanism is poorly understood. This study has identified a novel disrupted in schizophrenia 1 (DISC1)-interacting protein, named CAMDI after coiled-coil protein associated with myosin II and DISC1, which translocates to the centrosome in a DISC1-dependent manner. Knockdown of CAMDI by shRNA revealed severely impaired radial migration with disoriented centrosomes. A yeast two-hybrid screen identified myosin II as a binding protein of CAMDI. CAMDI interacts preferentially with phosphomyosin II and induces an accumulation of phosphomyosin II at the centrosome in a DISC1-dependent manner. Interestingly, one single nucleotide polymorphism of the CAMDI gene (R828W) is identified, and its gene product was found to reduce the binding ability to phosphomyosin II. Furthermore, mice with overexpression of R828W in neurons exhibit an impaired radial migration. Our findings indicate that CAMDI is required for radial migration probably through DISC1 and myosin II-mediated centrosome positioning during neuronal development.

Phospholipase Cδ3 Regulates RhoA/Rho Kinase Signaling and Neurite Outgrowth

Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLCδ3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLCδ3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLCδ3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLCδ3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLCδ3KD cells. We also found that exogenous expression of PLCδ3 down-regulated RhoA protein, and constitutively active PLCδ3 promotes the RhoA down-regulation more significantly than PLCδ3 upon differentiation. These results indicate that PLCδ3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.

Activation of c-Jun amino-terminal kinase by GDNF induces G2/M cell cycle delay linked with actin reorganization.

It is well known that the cell cycle is controlled by several cyclin/cyclin‐dependent kinase (Cdk) complexes whose expression and phosphorylation states vary with orderly periodicity. During the cell cycle, activity of the cyclin/Cdk complexes can be regulated directly or indirectly by a number of molecules, including protein kinases and phosphatases, p53, and Cdk inhibitors. Here, we show that the addition of glial cell line‐derived neurotrophic factor (GDNF) induced G2/M cell cycle delay in human SK‐N‐MC neuroectodermal tumor cells that express RET tyrosine kinase, accompanying actin reorganization. Cell cycle delay at G2/M was characterized by accelerated and prolonged Cdc2 phosphorylation and stabilization of cyclin B1 and Wee1 kinase expression. Interestingly, we found that phosphorylation and/or expression of Cdc2, cyclinB1, and Wee1 was controlled by the Rac1/c‐Jun NH2‐terminal kinase (JNK) pathway. Immunohistochemical analysis suggested that the G2/M cell cycle delay may be necessary to prevent the mitotic progression of SK‐N‐MC cells with perturbed actin cytoskeletons.

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.