👤 Kazutsune Yamagata

The melanocortin receptor 4 (MC4R) plays a key role in the CNS regulation of metabolism. In addition to its role within the hypothalamus, other brain areas, including the dorsal raphe nucleus (DRN), express MC4R. However, the identity and role of these neurons in metabolism regulation are not fully understood. We performed studies to address these questions. We generated Mc4r-cre;Vgat-FlpO and Mc4r-cre;Vglut2-FlpO mice to determine the contribution of these MC4R neuronal populations in DRN. We then chemogenetically activated or inhibited the GABAergic and glutamatergic populations of MC4R. Finally, we selectively deleted MC4R from these two neuronal populations and studied the impact on whole-body metabolism. We found that about 60% of DRN MC4R neurons are GABAergic (Vgat), while only about 20% are glutamatergic (Vglut2). Most of the projections onto DRN neurons originated from the arcuate nucleus (ARC)-POMC neurons, and only a small input from the nucleus of the solitary tract (NTS)-POMC neurons was identified. Significant projections of DRN MC4R/Vgat neurons were observed in the paraventricular nucleus of the hypothalamus (PVN). Chemogenetic activation or inhibition of MC4R/Vgat neurons increased or inhibited food intake, respectively. No effects were observed when the same approach was used in MC4R/Vglut2 neurons. Furthermore, only chemogenetic manipulation of the MC4R/Vgat neurons affected anxiety-like behavior, which was associated with changes in serotonin staining in the DRN. Finally, MC4R-selective deletion in Vgat but not Vglut2 neurons affected whole-body metabolism. These findings suggest that DRN MC4R/Vgat neurons receiving projections from the ARC POMC neurons and projecting to the hypothalamic PVN play a role in metabolism regulation. In addition, this same DRN neuronal subpopulation affects anxiety-like behavior by modulating DRN serotonin neurons. Show less

We aimed to determine the similarities and differences in the roles of genic and regulatory copy number variations (CNVs) in bipolar disorder (BD), schizophrenia (SCZ), and autism spectrum disorder (ASD). Based on high-resolution CNV data from 8708 Japanese samples, we performed to our knowledge the largest cross-disorder analysis of genic and regulatory CNVs in BD, SCZ, and ASD. In genic CNVs, we found an increased burden of smaller (<100 kb) exonic deletions in BD, which contrasted with the highest burden of larger (>500 kb) exonic CNVs in SCZ/ASD. Pathogenic CNVs linked to neurodevelopmental disorders were significantly associated with the risk for each disorder, but BD and SCZ/ASD differed in terms of the effect size (smaller in BD) and subtype distribution of CNVs linked to neurodevelopmental disorders. We identified 3 synaptic genes (DLG2, PCDH15, and ASTN2) as risk factors for BD. Whereas gene set analysis showed that BD-associated pathways were restricted to chromatin biology, SCZ and ASD involved more extensive and similar pathways. Nevertheless, a correlation analysis of gene set results indicated weak but significant pathway similarities between BD and SCZ or ASD (r = 0.25-0.31). In SCZ and ASD, but not BD, CNVs were significantly enriched in enhancers and promoters in brain tissue. BD and SCZ/ASD differ in terms of CNV burden, characteristics of CNVs linked to neurodevelopmental disorders, and regulatory CNVs. On the other hand, they have shared molecular mechanisms, including chromatin biology. The BD risk genes identified here could provide insight into the pathogenesis of BD. Show less

Mesenchymal stem cells (MSC) can differentiate into chondrocytes. Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed during chondrogenic differentiation and can be produced by MSC. EBI3 is also a subunit of interleukin (IL)-27 and IL-35, and it accumulates in the endoplasmic reticulum (ER) when its partners, such as IL-27 p28 and IL-35 p35, are insufficient. ER stress induced by protein accumulation is responsible for chondrogenic differentiation. However, the role of EBI3 and its relevance to the ER stress in chondrogenic differentiation of MSC have never been addressed. Here, we demonstrate that EBI3 protein is expressed in the early stage of chondrogenic differentiation of MSC. Additionally, knockdown, overexpression, or induction of EBI3 through IL-1β inhibits chondrogenesis. We show that EBI3 localizes and accumulates in the ER of MSC after overexpression or induction by IL-1β and TNF-α, whereas ER stress inhibitor 4-phenylbutyric acid decreases its accumulation in MSC. Moreover, EBI3 modulates ER stress sensor inositol-requiring enzyme 1 α (IRE1α) after induced by IL-1β, and MSC-like cells coexpress EBI3 and IRE1α in rheumatoid arthritis (RA) synovial tissue. Altogether, these data demonstrate that intracellular EBI3 commits to chondrogenic differentiation by regulating ER stress sensor IRE1α. Show less

Neuronal ceroid lipofuscinoses (NCLs; CLN) are mainly autosomal recessive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigments in neuronal and other cells. Symptoms include visual disabilities, motor decline, and epilepsy. Causative genes are CLN1, CLN2, CLN3, CLN5, CLN6, CLN7, CLN8, CLN10, CLN11, CLN12, CLN13, and CLN14. We present the fourth Japanese case with a CLN6 mutation. At 3 years of age, our patient became clumsy and fell down easily. He developed focal seizures with impaired consciousness and was started on carbamazepine. He showed ataxic walking and dysarthria with increased deep tendon reflexes. Interictal electroencephalogram revealed slow waves in the left temporal and occipital areas. Brain magnetic resonance imaging showed cerebellar atrophy and ventriculomegaly. In optical coherence tomography (OCT), the inner layer of the retina was thick and highly reflective. Exome sequencing revealed a known homozygous mutation, C.794₉₇₆del, p. (Ser265del) in CLN6. A total of 130 cases of NCL with CLN6 mutations have been reported globally, of which only four were from Japan including the current patient. The deletion of serine at position 265 has been reported in six cases. Ser265 is located in a region of short repeated sequences that is susceptible to mutation. Clinical trials of gene therapy using adeno-associated virus serotype 9 have started for NCL6, making early diagnosis crucial. OCT examination might be helpful in achieving a diagnosis. Show less

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation. Show less

Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future subject since a deletion mutation in PIK3C3 was detected in a patient with specific learning disorders (SLD). Show less

The mammalian Class III phosphoinositide 3-kinase (PIK3C3, also known as mammalian vacuolar protein sorting 34 homologue, Vps34) is a regulator of vesicular trafficking, autophagy, and nutrient sensing. In this study, we generated a specific antibody against PIK3C3, and carried out expression and morphological analyses of PIK3C3 during mouse brain development. In Western blotting, PIK3C3 was detected throughout the developmental process with higher expression in the early embryonic stage. In immunohistochemical analyses with embryonic day 16 mouse brain, PIK3C3 was detected strongly in the axon of cortical neurons. While PIK3C3 was distributed at the soma, nucleus, axon, and dendrites in primary cultured mouse hippocampal neurons at 3 days in vitro (div), it was also found in a punctate distribution with partial colocalization with synaptic marker, synaptophysin, at 21 div. The obtained results indicate that PIK3C3 is expressed and may have a physiological role in central nervous system during corticogenesis. Show less

The CALM-AF10 fusion gene, which results from a t(10;11) translocation, is found in a variety of hematopoietic malignancies. Certain HOXA cluster genes and MEIS1 genes are upregulated in patients and mouse models that express CALM-AF10. Wild-type clathrin assembly lymphoid myeloid leukemia protein (CALM) primarily localizes in a diffuse pattern within the cytoplasm, whereas AF10 localizes in the nucleus; however, it is not clear where CALM-AF10 acts to induce leukemia. To investigate the influence of localization on leukemogenesis involving CALM-AF10, we determined the nuclear export signal (NES) within CALM that is necessary and sufficient for cytoplasmic localization of CALM-AF10. Mutations in the NES eliminated the capacity of CALM-AF10 to immortalize murine bone-marrow cells in vitro and to promote development of acute myeloid leukemia in mouse models. Furthermore, a fusion of AF10 with the minimal NES can immortalize bone-marrow cells and induce leukemia in mice. These results suggest that during leukemogenesis, CALM-AF10 plays its critical roles in the cytoplasm. Show less

To know the involvement of glycosaminoglycans (GAGs) in the metastasis of mouse FBJ osteosarcoma cells, N(α)-lauroyl-O-(β-D-xylopyranosyl)-L-serinamide (Xyl-Ser-C12), which initiates elongation of GAG chains using the glycan biosynthesis system in cells, was administered to FBJ cells with different metastatic capacities. Production of glycosylated products derived from Xyl-Ser-C12, especially heparan sulfate (HS) GAG-type oligosaccharides such as GalNAc-GlcA-GlcNAc-GlcA-Gal-Gal-Xyl-Ser-C12, was indicated in poorly metastatic FBJ-S1 cells more than in highly metastatic FBJ-LL cells by LC-MS. The results of RT-PCR revealed that HS synthases, Ext1 and Ext2, were expressed in FBJ-S1 cells more than in FBJ-LL cells. Furthermore, siRNA against Ext1 suppressed the expression of HS and enhanced the motility of FBJ-S1 cells. In addition, the expression of heparanase (HPSE) was enhanced in Ext-1-knockdown FBJ-S1 cells, and responsible for the increase in cell motility caused by the down-regulation of Ext1 expression. Our data provide the first evidence that Ext1 regulates the expression of HPSE and also indicated that levels of Ext1 and HPSE influenced the motility of FBJ cells. Show less

Two boys from separate families presented with hereditary multiple exostoses (EXT) and autism associated with mental retardation. Their fathers both expressed a clinical phenotype of hereditary multiple exostoses milder than those of the patients and without the associated mental disorder. The EXT1 and EXT2 genes from lymphocytes of the affected individuals were analyzed by using denaturing high-performance liquid chromatography and direct sequencing. A novel deletion mutation, 1742delTGT-G in exon 9 of EXT1, causing a frameshift was detected in one boy and his father. Another novel deletion mutation, 2093delTT in exon 11 of EXT1, causing transcription termination was detected in the other affected boy and his father. EXT1 is expressed in the brain, and both EXT1 and EXT2 proteins are associated with glycosyltransferase activities required for the biosynthesis of heparan sulfate, which also has activity in the brain. The coincidental association of mental disorders in the boys was not completely excluded. However, these results suggest the involvement of EXT1 in the development of mental disorders, including mental retardation and autism. Show less