Asuka Uto

Ectopic Reelin Induces Neuronal Aggregation with a Normal Birthdate-Dependent “Inside-Out” Alignment in the Developing Neocortex

Neurons in the developing mammalian neocortex form the cortical plate (CP) in an “inside-out” manner; that is, earlier-born neurons form the deeper layers, whereas later-born neurons migrate past the existing layers and form the more superficial layers. Reelin, a glycoprotein secreted by Cajal–Retzius neurons in the marginal zone (MZ), is crucial for this “inside-out” layering, because the layers are inverted in the Reelin-deficient mouse, reeler (Relnrl ). Even though more than a decade has passed since the discovery of reelin, the biological effect of Reelin on individual migrating neurons remains unclear. In addition, although the MZ is missing in the reeler cortex, it is unknown whether Reelin directly regulates the development of the cell-body-sparse MZ. To address these issues, we expressed Reelin ectopically in the developing mouse cortex, and the results showed that Reelin caused the leading processes of migrating neurons to assemble in the Reelin-rich region, which in turn induced their cell bodies to form cellular aggregates around Reelin. Interestingly, the ectopic Reelin-rich region became cell-body-sparse and dendrite-rich, resembling the MZ, and the late-born neurons migrated past their predecessors toward the central Reelin-rich region within the aggregates, resulting in a birthdate-dependent “inside-out” alignment even ectopically. Reelin receptors and intracellular adaptor protein Dab1 were found to be necessary for formation of the aggregates. The above findings indicate that Reelin signaling is capable of inducing the formation of the dendrite-rich, cell-body-sparse MZ and a birthdate-dependent “inside-out” alignment of neurons independently of other factors/structures near the MZ.

Migration defects by DISC1 knockdown in C57BL/6, 129X1/SvJ, and ICR strains via in utero gene transfer and virus-mediated RNAi

Disrupted-in-Schizophrenia 1 (DISC1) is a promising genetic risk factor for major mental disorders. Many groups repeatedly reported a role for DISC1 in brain development in various strains of mice and rats by using RNA interference (RNAi) approach. Nonetheless, due to the complexity of its molecular disposition, such as many splice variants and a spontaneous deletion in a coding exon of the DISC1 gene in some mouse strains, there have been debates on the interpretation on these published data. Thus, in this study, we address this question by DISC1 knockdown via short-hairpin RNAs (shRNAs) against several distinct target sequences with more than one delivery methodologies into several mouse strains, including C57BL/6, ICR, and 129X1/SvJ. Here, we show that DISC1 knockdown by in utero electroporation of shRNA against exons 2, 6, and 10 consistently results in neuronal migration defects in the developing cerebral cortex, which are successfully rescued by co-expression of full-length DISC1. Furthermore, lentivirus-mediated shRNA also led to migration defects, which is consistent with two other methodologies already published, such as plasmid-mediated and retrovirus-mediated ones. The previous study by Songs group also reported that, in the adult hippocampus, the phenotype elicited by DISC1 knockdown with shRNA targeting exon 2 was consistently seen in both C57BL/6 and 129S6 mice. Taken together, we propose that some of DISC1 isoforms that are feasible to be knocked down by shRNAs to exon 2, 6, and 10 of the DISC1 gene play a key role for neuronal migration commonly in various mouse strains and rats.

Treatment of sarcopenia and glucose intolerance through mitochondrial activation by 5-aminolevulinic acid

Recently, sarcopenia has attracted attention as therapeutic target because it constitutes a risk factor for metabolic and cardiovascular diseases. We focused 5-aminolevulinic acid (ALA) which act as electron carriers in the mitochondrial electron transport system. The mice that received ALA for 8 weeks gained muscle strength and endurance, and exhibited increased muscle mass and mitochondrial amount. Administration of ALA to sarcopenia mice aged 100 weeks and chronic kidney disease (CKD) model mice also increased muscle mass and improved physical performance. Metabolome analysis revealed increased branched-chain amino acids (BCAAs) levels in the skeletal muscle of ALA-treated mice. Quantitative PCR analysis revealed decreased expression levels in branched-chain amino acid transaminases (BCATs) that degrade BCAAs and other muscle-degrading factors, and increased levels of mitochondria-activating factors. We also studied in cultured myocytes and obtained compatible results. ALA-treated mice tended to increase body weight, but reduced blood glucose level. These suggested that ALA treatment not only activated muscle mitochondria but also enhanced muscle mass through an increase in BCAAs contents, as to improve muscle strength, endurance and glucose tolerance in mice. In these ways, muscle mitochondrial activation with ALA is suggested to be useful for the treatment of sarcopenia and glucose intolerance.

Ghrelin treatment improves physical decline in sarcopenia model mice through muscular enhancement and mitochondrial activation

Chronic kidney disease (CKD) impairs physical performance in humans, which leads to a risk of all-cause mortality. In our previous study, we demonstrated that a reduction in muscle mitochondria rather than muscle mass was a major cause of physical decline in 5/6 nephrectomized CKD model mice. Because ghrelin administration has been reported to enhance oxygen utilization in skeletal muscle, we examined the usefulness of ghrelin for a recovery of physical decline in 5/6 nephrectomized C57Bl/6 mice, focusing on the epigenetic modification of peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. The mice were intraperitoneally administered acylated ghrelin (0.1 nmol/gBW; three times per week) for a month. Muscle strength and exercise endurance were measured by using a dynamometer and treadmill, respectively. Mitochondrial DNA copy number was determined by quantitative PCR. The methylation levels of the cytosine residue at 260 base pairs upstream of the translation initiation point (C-260) of PGC-1α, which has been demonstrated to decrease the expression, was evaluated by methylation-specific PCR and bisulfite genomic sequencing methods after the ghrelin administration. Ghrelin administration improved both muscle strength and exercise endurance in the mice and was associated with an increase in muscle mass and muscle mitochondrial content. Ghrelin administration decreased the methylation ratio of C-260 of PGC-1α in the skeletal muscle and increased the expression. Therefore, ghrelin administration effectively reduced the physical decline in 5/6 nephrectomized mice and was accompanied with an increased mitochondrial content through de-methylation of the promoter region of PGC-1α in the muscle.

TRANSIENT SALT LOADING CAUSES PERSISTENT HYPERTENSION THROUGH EPIGENETIC MODIFICATION OF THE RENAL ARTERIOLES

Objective: We have previously reported that the medial hypertrophy of the renal arterioles induced by transient salt loading causes sustained elevation of blood pressure in spontaneous hypertensive rat (SHR) even after the salt loading had completed [Oguchi et al. Hypertension 2014]. Epigenetic modification of gene expression has attracted attention as a possible mechanism for sustained biological effects and onset of hypertension. The present study investigated the significance of histone acetylation in each segment of the kidney in the induction of hypertension after transient salt loading. Design and method: C57bl6 mice were implanted deoxycorticosterone acetate (DOCA) pellets and given drinking water containing 1% NaCl for 2 weeks to induce hypertension. We evaluated blood pressure, histological findings and gene expressions of the kidney during and after the transient salt loading. The degree of histone acetylation was assessed by immunostaining of acetylated H3 and H4 in each segment of the kidney including renal arterioles, segmental arteries, glomeruli and tubules. Gene expressions were examined in each segment of the kidney collected by laser capture microdissection (LCM). Results: Transient salt loading caused elevation in blood pressure during and even after stopping salt loading associated with persistent medial hypertrophy of renal arterioles. In the media of renal arterioles, histone acetylation was enhanced during salt loading, and the enhanced histone acetylation persisted even after stopping salt loading. The gene expression of MMPs in the renal arterioles collected by LCM increased during salt loading, and did not decline even after stopping salt loading. On the other hand, in the segmental arteries, neither hyper-acetylation nor hyper-expression of MMPs was observed. Also, in the tubules, enhanced histone acetylation by the salt loading returned to the initial level after the completion. Conclusions: The persistent medial hypertrophy along with focally sustained histone hyper-acetylation and elevation in MMPs expressions in the renal arterioles were suggested to cause sustained elevation of blood pressure after transient salt loading. The focal epigenetic modification in the kidney due to environmental factors would participate in the onset of persistent hypertension.

A MASS IMAGING TECHNIQUE REVEALED A RENO-PROTECTIVE EFFECT OF THE XANTHINE OXIDASE INHIBITOR FEBUXOSTAT IN THE ISCHEMIC KIDNEY BY PROMOTING ATP RECOVERY IN THE CORTEX

Objective: The kidney has different energy metabolism depending on the region. However, the distribution of phosphorylated adenosine (ATP, ADP and AMP) and their alteration after transient ischemia have not been known due to the technical difficulties. Design and method: Imaging mass spectrometry (IMS) with metabolome analysis is a novel technique to quantify the small metabolites in the tissues. We performed the IMS analysis in the ischemic kidney after transient ischemia by renal artery clipping. Results: In the normal kidney, ATP was significantly rich in both the cortex and outer medulla. After transient ischemia, ATP in the cortex degraded and the energy charge value decreased within a minute. ATP in the inner medulla did not decrease within a minute and needed 10 minutes to start decreasing. After the 10 minutes of ischemia, total adenylates decreased in the cortex, although the decrease in energy charge value was homogeneous in the kidney. During the 24 hours reperfusion after 10 minutes ischemia, restoration of total adenylates in the cortex was not sufficient. Febuxostat is a xanthine oxidase inhibitor which might promote reuse of hypoxanthine as a progenitor of adenylates and therefore might improve the restoration of total adenylates and ATP after transient ischemia. The administration of febuxostat in accordance with the reperfusion period supported the restoration of ATP level in the cortex and improved renal function which was impaired by transient ischemia. Conclusions: In these ways, IMS revealed the region-specific alteration of phosphorylated adenosine the ischemic kidney and the novel effect of febuxostat on the restoration of total adenylates and ATP in the cortex after transient ischemia.

HYPERTENSION-RELATED PROTEIN DEACETYLASE SIRT3 AFFECTS GLUCOSE METABOLISM THROUGH REGULATION OF GLUCAGON-LIKE PEPTIDE 1 IN MICE

Objective: Sirt3 is a NAD-dependent protein deacetylase that is presumably related to the onset of hypertension. Recent reports have shown that Sirt3-deficient knockout (KO) mice develop pulmonary hypertension, high-fat diet induced obesity and metabolic syndrome-like phenotype. However, the precise mechanism by which they cause glucose intolerance has not known. To elucidate the regulatory mechanism of glucose metabolism through Sirt3, the metabolic parameters in the Sirt3-KO mice were investigated. Design and method: Blood glucose levels in the Sirt3-KO mice after glucose loading were examined, associated with blood glucagon-like peptide-1 (GLP-1) levels and dipeptidyl peptidase-4 (DPP-4) activity. Gene expressions which were associated with GLP-1 and DPP-4 were quantified by qPCR. The expression levels of sirtuins in various organs in wild-type mice and, the histone acetylation in the Sirt3-KO mice. The levels of histone acetylation in various organs of the KO mice were estimated by immunostaining of acetylated histone H3 (H3Ac) and H4 (H4Ac). Results: The Sirt3-KO mice fed with a standard or a high-fat diet were subjected to an oral glucose tolerance test (OGTT), together with an intraperitoneal glucose tolerance test (ipGTT). OGTT revealed an increase in the blood glucose levels in the KO mice, although ipGTT did not show the increase after the glucose loading. Blood GLP-1 and insulin levels which were examined before and 15 minutes after oral glucose loading were found to decrease in the KO mice. The plasma DPP-4 activity increased significantly in the Sirt3-KO mice. qPCR analysis revealed a predominance of Sirt3 expression in the small intestine, colon, kidney, and liver in the wild-type mice. The immunostaining of H3Ac and H4Ac showed enhanced intensity in the glandular and stromal cells in the small intestine and colon, and in the renal tubular cells and the vascular endothelial cells in the KO mice. Conclusions: These results suggest that Sirt3 deficiency impairs the incretin effect by a suppression of blood GLP-1 levels and causes glucose intolerance. Enhanced acetylation of histone H3 and H4 in the Sirt3-KO mice might relate to the increase in the DPP-4 activity, which lead to the decrease in the GLP-1 levels.

MPS 07-06 5-AMINOLEVULINIC ACID, A MATERIAL FOR MITOCHONDRIAL ELECTRON CARRIER, IMPROVES EXERCISE ENDURANCE THROUGH BOTH MUSCLE ENHANCEMENT AND ACTIVATION OF MUSCLE MITOCHONDRIA IN SARCOPENIC MICE.

Objective: Sarcopenia affects not only physical performance, but also increases the risk for cardiovascular diseases. Aminolevulinic acid (ALA) is a raw material for the mitochondrial electron carriers, heme and cytochrome C. In the present study, physical performance of ALA-treated mice (0.03%-ALA/ chow weight) was examined as to elucidate the usefulness of ALA for the treatment of sarcopenia. Design and Method: Alteration in physical performance and muscle mass were examined in ALA-treated mice. Harvested skeletal muscle was examined for expression of genes related to mitochondrial amount and function. Results: Muscle mass and mitochondrial amount were increased in the skeletal muscle of ALA-treated mice. Expressions of the mitochondrial activation genes, PGC1a and UCP3 were up-regulated. Decreased muscle strength, exercise endurance and glucose tolerance of aging mice and 5/6 nephrectomized mice, which were sarcopenic with reduced amount of muscle mitochondria, were improved by the ALA-feeding for 8weeks. In the experiments using cultured myocytes treated with ALA, expressions of PGC1a and UCP3, and mitochondrial amount were increased, associated with augmented oxygen utilization. Suppression of electron transport by antimycin A attenuated the increases in oxygen utilization, PGC1a expression and mitochondrial amount in ALA-treated myocytes. Conclusions: ALA improved exercise endurance and glucose intolerance of sarcopenic mice through increases in muscle mass, muscle PGC1a expression and mitochondrial amount. These results indicated the usefulness of ALA for improvement of physical performance and glucose tolerance in sarcopenic patients through both muscle enhancement and activation of muscle mitochondria.

PS 16-03 EFFECTS OF 72 HOURS FASTING AND REFEEDING ON FAT OXIDATION AND WEIGHT SUPPRESSION

Objective: Intermittent fasting has been shown to have several health benefits including reduced blood pressure, increased insulin sensitivity, stress resistance and reduced morbidity. However, the mechanism for the beneficial effects has not been well investigated. Design and method: Body weight and body temperature of c57BL/6 mice were examined when they were maintained on the intermittent fasting (IF) of 72 hours fasting and 96 hours refeeding in a week for 4 weeks, and subsequently administered a high fat diet (HFD) for 4 weeks. After the IF and HFD administration, glucose tolerance test and respiratory gas analysis were performed. Expressions of genes responsible for fat oxidation and lipolysis in the skeletal muscle and white adipose tissue were examined by PCR analysis. Results: There was no significant difference in body weight and body temperature between the adlib eating and IF groups before HFD administration. After HFD administration, the body weight in IF group was significantly lower and the body temperature became higher when compared to adlib eating group. Glucose tolerance test after HFD showed a decrease in glucose level in IF group. In the PCR analysis after HFD, expressions of genes responsible for fat oxidation, heat production and lipolysis (UCP1, DiO2, AGTL and HSL) were increased in the skeletal muscle and white adipose tissue. Expressions of hormone receptors responsible for fat oxidation (GHSR and AdipoR1) were also increased. Conclusions: The operation of IF resulted in suppression of body weight gain after HFD administration. From the results of PCR analysis, promotions of fat oxidation, lipolysis and heat production in the skeletal muscle and white adipose tissue in IF group were suggested to be resulted in suppression of body weight gain. Furthermore, the alteration in hormone responses might be involved in these beneficial effects of the IF.