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Synaptic pruning is a fundamental process of neuronal circuit refinement in learning and memory. Accumulating evidence suggests that glia participates in sculpting the neuronal circuits through synapse engulfment. However, whether glial involvement in synaptic pruning has a role in memory formation remains elusive. Using newly developed phagocytosis reporter mice and three-dimensional ultrastructural characterization, we found that synaptic engulfment by cerebellar Bergmann glia (BG) frequently occurred upon cerebellum-dependent motor learning in mice. We observed increases in pre- and postsynaptic nibbling by BG along with a reduction in spine volume after learning. Pharmacological blockade of engulfment with Annexin V inhibited both the spine volume reduction and overnight improvement of motor adaptation. These results indicate that BG contribute to the refinement of the mature cerebellar cortical circuit through synaptic engulfment during motor learning.

Background: Previous studies have focused on the relationship between specific dietary factors (such as sodium intake or consumption of fruits and vegetables) and the development of hypertension. However, less is known about the role of overall dietary patterns (food intake, dietary behaviors, and cooking methods) in the development of hypertension. This study aims to address this gap in the literature by using unsupervised machine-learning techniques to identify dietary patterns associated with the incidence of hypertension. Methods: Data were obtained from Japanese participants enrolled in a prospective cohort study between August 2008 and August 2010. A total of 447 male participants were included in the analysis. Dimension reduction using Uniform Manifold Approximation and Projection (UMAP) and subsequent K-means clustering was used to derive dietary patterns. In addition, multivariable logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) to evaluate the association between dietary patterns and the incidence of hypertension. Results: We identified four dietary patterns: ‘Sweet Tooth,’ ‘Herbivorous,’ ‘Meat-based healthy,’ and ‘Seafood and Alcohol.’ Approximately 13.4% of the cohort study participants developed hypertension in the following two years. Compared with ‘Seafood and Alcohol’ as a reference, the protective dietary patterns for hypertension were ‘Herbivorous’ (OR = 0.39, 95% CI = 0.19–0.80, p = 0.013) and the ‘Meat-based healthy’ (OR = 0.37, 95% CI = 0.16–0.86, p = 0.022) after adjusting for potential confounding factors, including age, body mass index, smoking, education, physical activity, dyslipidemia, and diabetes. An age-matched sensitivity analysis confirmed this finding. Conclusions:From a methodological perspective, we successfully identified clear dietary patterns by clustering using the UMAP and K-means algorithms in an epidemiological dataset with a small sample size. The ‘Herbivorous’ and ‘Meat-based healthy’ dietary patterns were associated with a lower risk of hypertension in Japanese males than the ‘Seafood and Alcohol’ pattern. These findings provide helpful insights into hypertension-preventive interventions in Japanese males through dietary pattern regulation.

Environmental carbon dioxide (CO

The subcommissural organ (SCO) is a part of the circumventricular organs located in the dorsocaudal region of the third ventricle at the entrance of the aqueduct of Sylvius. The SCO comprises epithelial cells and produces high molecular weight glycoproteins, which are secreted into the third ventricle and become part of Reissner's fibre in the cerebrospinal fluid. Abnormal development of the SCO has been linked with congenital hydrocephalus, a condition characterized by excessive accumulation of cerebrospinal fluid in the brain. In the present study, we characterized the SCO cells in the adult mouse brain to gain insights into the possible role of this brain region. Immunohistochemical analyses revealed that expression of Pax6, a transcription factor essential for SCO differentiation during embryogenesis, is maintained in the SCO at postnatal stages from P0 to P84. SCO cells in the adult brain expressed known neural stem/progenitor cell (NSPC) markers, Sox2 and vimentin. The adult SCO cells also expressed proliferating marker PCNA, although expression of another proliferation marker Ki67, indicating a Gsub2/sub/M phase, was not detected. The SCO cells did not incorporate BrdU, a marker for DNA synthesis in the S phase. Therefore, the SCO cells have a potential for proliferation but are quiescent for cell division in the adult. The SCO cells also expressed GFAP, a marker for astrocytes or NSPCs, but not NeuN (for neurons). A few cells positive for Iba1 (microglia), Olig2 (for oligodendrocytes) and PDGFRα (oligodendrocyte progenitors) existed within or on the periphery of the SCO. These findings revealed that the SCO cells have a unique feature as secretory yet immature neuroepithelial cells in the adult mouse brain.

Infant crying is a communicative behavior impaired in neurodevelopmental disorders (NDDs). Because advanced paternal age is a risk factor for NDDs, we performed computational approaches to evaluate how paternal age affected vocal communication and body weight development in C57BL/6 mouse offspring from young and aged fathers. Analyses of ultrasonic vocalization (USV) consisting of syllables showed that advanced paternal age reduced the number and duration of syllables, altered the syllable composition, and caused lower body weight gain in pups. Pups born to young fathers had convergent vocal characteristics with a rich repertoire, whereas those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Additional analyses revealed that some pups from aged fathers displayed atypical USV trajectories. Thus, our study indicates that advanced paternal age has a significant effect on offspring's vocal development. Our computational analyses are effective in characterizing altered individual diversity.

Task complexity could affect acquisition efficiency of motor skills and interlimb transfer; however, how task complexity affects interlimb transfer remains unclear. We hypothesized that left- and right-handed participants may have different interlimb transfer efficiency depending on the task complexity.Left-hand (n = 28) and right-hand (n = 28) dominant participants (age = 24.70 ± 4.02 years, male:female = 28:28) performed a finger sequence test with two levels of complexity (simple: one-digit with four fingers vs. complex: two-digit with five fingers) before and after ten trials of 2-min practice each on the same apparatus. The speed and task errors were measured and analyzed.Right-handed participants failed to improve performance on their right hand (non-trained hand) after contralateral left-hand practice in the simple finger sequence task. In contrast, the left-handed participants improved performance on non-trained hands both right and left after contralateral practices. In the complex task, however, both the left- and right-handed participants improved performance on non-trained hands by contralateral practices.Our results showed that task complexity of skilled practice gave different effects on interlimb transfer between right- and left-handed subjects. It appears that a certain level of appropriate complexity is necessary to detect inter-limb transfers in motor learning in right-handed subjects.

Despite recent advances in genome engineering technologies, traditional transgenic mice generated on a mixed genetic background of C57BL/6 and 129/Sv mice remain widely used in age-related hearing loss (AHL) research, since C57BL/6 mice exhibit early onset and progression of AHL due to a mutation in cadherin 23-encoding gene (Cdh23753G>A). In these transgenic mice, backcrossing for more than 10 generations results in replacement of the donor background (129/Sv) with that of the recipient (C57BL/6), so that approximately 99.9% of genes are C57BL/6-derived and are considered congenic. However, the regions flanking the target gene may still be of 129/Sv origin, creating a so-called "passenger gene problem" where the normal 129/Sv-derived Cdh23753G allele can travel with the target gene. In this study, we investigated the role of fatty acid-binding protein 7 (Fabp7), which is important for cellular uptake and intracellular trafficking of fatty acids in the cochlea, using traditional Fabp7 knockout (KO) mice on the C57BL/6 background. We found that Fabp7 KO mice showed delayed AHL progression and milder cochlear degeneration. However, the genotype of the Cdh23 region flanking Fabp7 was still that of 129/Sv origin (Cdh23753GG). Our findings reveal the potential risk of contamination for traditional transgenic mice generated on the C57BL/6 background.

Ependymal cells have an essential role in regulating the dynamics of the cerebrospinal fluid flow by the movement of their multiple cilia. Impaired generation or function of cilia could cause hydrocephalus due to the disordered dynamics of the cerebrospinal fluid flow. However, molecular bases regulating differentiation of the ependymal cells and their ciliogenesis have not been fully elucidated. We report here that bone morphogenetic proteins (BMPs), growth factors orchestrating tissue architecture throughout the body, inhibit ciliogenesis during ependymal cell differentiation in primary cell culture. Previous in vitro study has reported that ectopic expression of Smad6 and Smad7 promotes differentiation of embryonic stem cells into multi-ciliated ependymal-like cells. Since Smad6 and Smad7 have been known as the intracellular inhibitory factors of the BMP signaling pathway, the activation of the pathway could cause a deficit in ciliogenesis of ependymal cells. To examine whether activation of the pathway affects ciliogenesis, we investigated the effects of two BMPs, BMP2 and BMP4, on the ependymal differentiation of the primary cultured cells prepared from the neonatal mouse brain. Supplementation of BMP2 or BMP4 in culture media significantly reduced the number of cells with multiple cilia among the total cells, while most of the cells expressed FoxJ1, a master regulator of ciliogenesis. Activation of the pathway was confirmed by the phosphorylation of intracellular Smad1/5/8, downstream factors of the BMP receptors. These in vitro results suggest that inhibition of the BMP signaling pathway might be essential for ciliogenesis during the ependymal cell differentiation in vivo.

BACKGROUND Repressor element 1-silencing transcription factor (REST) is a master regulator that is highly expressed in multipotent stem cells to repress gene networks involving a wide range of biological processes. A recent study has suggested that REST might be involved in a misregulation of its target genes in the embryonic brain of offspring derived from aged fathers. However, detailed analyses of the REST function in spermatogenesis are lacking due to difficulty in the detection of REST protein in specific cell types. RESULTS To determine localization of REST, we generated an epitope tag knock-in (KI) mouse line with the C-terminus insertion of a podoplanin (PA)-tag at an endogenous Rest locus by the CRISPR/Cas9 system. Localization of the PA-tag was confirmed in neural stem cells marked with Pax6 in the embryonic brain. Moreover, PA-tagged REST was detected in undifferentiated and differentiating spermatogonia as well as Sertoli cells in both neonatal and adult testes. CONCLUSIONS We demonstrate that REST is expressed at the early step of spermatogenesis and suggest a possibility that REST may modulate the epigenetic state of male germline cells. Our KI mice may be useful for studying REST-associated molecular mechanisms of neurodevelopmental and age-related disorders.

Background Golimumab (GLM) has been reported to have lower immunogenicity than do other TNF inhibitors used for treating rheumatoid arthritis (RA). We previously found a prolonged effect of and improvement similar to that associated with infliximab (IFX) after switching to subcutaneous GLM (GLM-SC) for control of RA activity or adverse events. Thus, this study aimed to evaluate the continued maintenance of treatment efficacy and safety for > 2 years by switching to GLM-SC in RA patients with low disease activity or in remission after previous treatment with another tumor necrosis factor (TNF) inhibitor. Methods Thirty-two patients treated with etanercept or infliximab were switched to GLM-SC and maintained low disease activity. The patients were divided into two groups (GLMq4w and GLMq8w) through discussion with each patient, considering their general condition and convenience. The groups included patients with low disease activity or in remission who switched to 50-mg GLM therapy at 4-week and 8-week intervals, respectively. Results The mean DAS28-ESR and DAS-CRP values in the GLMq4w group (17 patients) and GLMq8w group (15 patients) were maintained from baseline throughout the 104-week treatment period. Two patients from the GLMq4w group showed disease flaring to moderate disease activity. No serious adverse events occurred, and the treatment continuation rate at 104 weeks was 100% in both groups. After > 2 years of treatment, three patients in the GLMq8w group and one patient in the GLMq4w group discontinued GLM treatment due to relapse or complications. The 5-year survival rates were 88.2% and 75.5% in the GLMq4w and GLMq8w groups, respectively. The average treatment duration was 5.0 (2.0–7.5) years. Conclusion Administration of GLM-SC at 4-week and 8-week intervals after switching from TNF inhibitors showed sustained long-term efficacy and acceptable safety in RA patients with low disease activity.

Objective Dietary intervention is a practical prevention strategy for age-related hearing loss (AHL). Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) may be effective in prevention of AHL due to their anti-inflammatory and tissue-protective functions. Age-related changes in the hearing function of wild-type and Fat-1 transgenic mice derived from the C57BL/6N strain, which can convert omega-6 PUFAs to n-3 PUFAs and consequently produce enriched endogenous n-3 PUFAs, were investigated to test the efficacy of n-3 PUFAs for AHL prevention. Results At 2 months, the baseline auditory brainstem response (ABR) thresholds were the same in Fat-1 and wild-type mice at 8–16 kHz but were significantly higher in Fat-1 mice at 4 and 32 kHz. In contrast, the ABR thresholds of Fat-1 mice were significantly lower at 10 months. Moreover, the ABR thresholds of Fat-1 mice at low-middle frequencies were significantly lower at 13 months (12 kHz). Body weights were significantly reduced in Fat-1 mice at 13 months, but not at 2, 10, and 16–17 months. In conclusion, enriched endogenous n-3 PUFAs produced due to the expression of the Fat-1 transgene partially alleviated AHL in male C57BL/6N mice.

Pax6 is an important transcription factor expressed in several discrete domains of the developing central nervous system. It has been reported that Pax6 is involved in the specification of subtypes of hindbrain motor neurons. Pax6 homozygous mutant (rSey2/rSey2) rats die soon after birth, probably due to impaired respiratory movement. To determine whether the respiratory center in the medulla functions normally, we analyzed the histological and neurophysiological properties of the medulla and spinal cord in fetal rats with this mutation. First, the medulla of rSey2/rSey2 at embryonic (E) 21.5-E22.5 tended to be smaller than those from heterozygous mutant (rSey2/+) and wild-type (+/+) littermates. Through immunohistochemical analysis, we confirmed normal distribution of Phox2b-expressing cells in the parafacial respiratory group (pFRG) of rSey2/rSey2 rats. Expression of neurokinin-1 receptor (NK-1R) was weak and dispersed in rSey2/rSey2 rats. In addition, rSey2/rSey2 rats have a defect of the hypoglossal nerve root. Electrophysiological analysis using brainstem-spinal cord preparations (E21.5-E22.5) revealed that rSey2/rSey2 rats showed larger fluctuation of the amplitude of inspiratory activity monitored from the fourth cervical root although there was no significant difference in the respiratory rate among rSey2/rSey2, rSey2/+, and +/+ littermates. The response of respiratory rhythm to high CO2 was similar among all genotypes. Optical recordings of neuronal activity revealed that the activity of the pFRG tended to be weaker and inspiratory activity appeared in more scattered areas in the caudal ventral medulla in the rSey2/rSey2 rats. These results suggest that the basal activity of the respiratory system was preserved with mild impairment of the inspiratory activity in the rSey2/rSey2 rats and that the Pax6 gene is involved in the functional development of the neuronal system producing effective inspiratory motor outputs for survival.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal‐aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole‐genome target DNA methylome analyses of sperm from aged mice reveal more hypo‐methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de‐methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo‐methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes., Paternal aging affects health and disease of the next generation, but the causative mechanisms are largely unknown. This study identifies hypo‐methylated sites enriched in REST/NRSF motifs in sperm DNA as epigenetic key features for inheritance.

Background Some rheumatoid arthritis (RA) patients initially respond to treatment with infliximab (IFX), but subsequently their responsiveness decreases. Objectives Our objective was to evaluate the efficacy and safety of switching from IFX to subcutaneous golimumab (GLM-SC) in RA patients. Methods Thirty-three patients who had been treated for a mean 4.4 years with IFX (3–6 mg/kg/8 weeks) were switched to GLM-SC to control disease activity or adverse events. The patients with low disease activity (LDA) or remission were divided into two groups: the LDA group and the LDA every 8 weeks (q8w) group, which included patients with LDA or remission who switched to GLM therapy with 50 mg at 4- and 8-week intervals, respectively. The moderate disease activity (MDA) group included patients with MDA who switched to GLM therapy with 50 mg at 4-week intervals. Effects of the IFX to GLM-SC switch were evaluated at weeks 12, 24, and 52 after switching. Results The mean disease activity score 28-ESR and -C-reactive protein values in the LDA and LDAq8w groups were maintained from baseline throughout the 52-week treatment period. The mean disease activity score 28 values at 12, 24, and 52 weeks in the MDA group were improved significantly compared with baseline. Treatment discontinuations due to adverse events occurred in one patient in the MDA group, and no serious adverse events occurred during the observation period in the LDA group or the LDAq8w group. The GLM continuation rates at 52 weeks were 100% in the LDA and LDAq8w groups and 83.3% in the MDA group. Thus, GLM-SC treatment regimens were effective in controlling disease activity and improving the clinical response related to adverse events caused by IFX. Conclusion The clinical efficacy of GLM-SC was sustained or improved in patients who switched from IFX without serious safety concerns.

The study was conducted to evaluate continued maintenance of the efficacy and safety of therapy by switching to subcutaneous golimumab (GLM-SC) in rheumatoid arthritis patients with low disease activity or remission who previously received a tumor necrosis factor (TNF) inhibitor. Thirty patients who had been treated with etanercept or infliximab were switched to GLM-SC in maintaining disease activity at a low level. The patients were divided into two groups through discussion with each patient, considering general condition and convenience: the low disease activity (LDA) group and the LDAq8w group, which included patients with low disease activity or remission who switched to 50 mg GLM therapy at 4- and 8-week intervals, respectively. The effects of the TNF inhibitors to GLM-SC switch were evaluated at 12, 24, and 52 weeks after switching. The mean DAS28-ESR and DAS-CRP values in the LDA groups (16 patients) and LDAq8w groups (14 patients) were maintained from baseline throughout the 52-week treatment period. DAS28-ESR remission (93.8 and 92.3%) rates were also maintained through week 52 from the baseline remission rate (75.0 and 78.6%) in the LDA and LDAq8w groups, respectively. Thus, both GLM-SC treatment regimens were effective in maintaining the clinical response achieved with LDA secondary to TNF inhibitors. No serious adverse events occurred, and the continuation rate at 52 weeks was 100% in both groups. Therapeutic efficacy is adequately maintained in most patients switching from TNF inhibitor to GLM-SC (50 mg/4-8 weeks). Patients receiving TNF inhibitor can seamlessly switch to GLM-SC without serious safety concerns.

Fragile X mental retardation protein (FMRP) is encoded byFMR1gene that is responsible for Fragile X Syndrome (FXS) showing intellectual disability and autism spectrum disorder. FMRP is an RNA binding protein highly expressed in the brain. Although several target genes for FMRP have been identified, limited studies have suggested the role of FMRP in corticogenesis. Here we performed RNA immunoprecipitation sequencing against the murine embryonic neocortex, and identified 124 genes as potential FMRP mRNA targets. We found 48 of these genes as overlapped with autism-related genes, which were categorized in four functional groups: “transcriptional regulation”, “regulation of actin cytoskeleton”, “ubiquitin-mediated proteolysis” and “calcium signaling pathway”. Four of these genes showed significant difference in expression in the cortical primordium ofFmr1-KO mice;Huwe1andKat6aincreased, whileKmt2candApcdecreased. Although the change in expression of these four genes was relatively small, these subtle changes due to dysregulated transcription could collectively contribute to impaired corticogenesis to cause phenotypes of FXS. Investigating the transcriptional control of FMRP on its mRNA targets may provide new insight to understand neurodevelopmental pathogenesis of FXS.

Human epidemiological studies have shown that paternal aging as one of the risk factors for neurodevelopmental disorders, such as autism, in offspring. A recent study has suggested that factors other than de novo mutations due to aging can influence the biology of offspring. Here, we focused on epigenetic alterations in sperm that can influence developmental programs in offspring. In this study, we qualitatively and semiquantitatively evaluated histone modification patterns in male germline cells throughout spermatogenesis based on immunostaining of testes taken from young (3 months old) and aged (12 months old) mice. Although localization patterns were not obviously changed between young and aged testes, some histone modification showed differences in their intensity. Among histone modifications that repress gene expression, histone H3 lysine 9 trimethylation (H3K9me3) was decreased in the male germline cells of the aged testis, while H3K27me2/3 was increased. The intensity of H3K27 acetylation (ac), an active mark, was lower/higher depending on the stages in the aged testis. Interestingly, H3K27ac was detected on the putative sex chromosomes of round spermatids, while other chromosomes were occupied by a repressive mark, H3K27me3. Among other histone modifications that activate gene expression, H3K4me2 was drastically decreased in the male germline cells of the aged testis. In contrast, H3K79me3 was increased in M-phase spermatocytes, where it accumulates on the sex chromosomes. Therefore, aging induced alterations in the amount of histone modifications and in the differences of patterns for each modification. Moreover, histone modifications on the sex chromosomes and on other chromosomes seems to be differentially regulated by aging. These findings will help elucidate the epigenetic mechanisms underlying the influence of paternal aging on offspring development.

SummaryInfant crying is an innate communicative behavior that is frequently impaired in certain neurodevelopmental disorders (NDDs). Since advanced paternal age is a reported risk factor for NDDs in offspring, we evaluated the impact of a father’s age on early vocal development in C57BL/6J mice. We recorded and applied a unique combination of computational analyses to ultrasonic vocalizations (USVs) emitted by mouse pups sired by young and aged fathers. Our data showed that advanced paternal age reduced the number and duration of USVs, and altered the syllable composition in pups. Moreover, pups born to young fathers showed convergent vocal characteristics with a rich repertoire during postnatal development, while those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Principal component analysis in conjunction with clustering analysis demonstrated that pups from aged fathers deviated from typical trajectories of vocal development, which were considered as atypical individuals. Thus, our study indicates that advanced paternal age has a significant effect on offspring’s early vocal development. It is suggested that the trajectories of vocal development could be a useful marker of the NDD-like phenotype associated with the advanced paternal age. In addition, our comprehensive computational analysis described here is an effective approach to characterize the altered individual diversity relevant to neurodevelopmental disorders.One Sentence SummaryAdvanced paternal age affects vocal development in early postnatal mice, with more pups showing atypical developmental trajectories.

Advanced paternal age has deleterious effects on mental health of next generation. Using a mouse model, we have confirmed that offspring derived from aged fathers showed impairments in behavior and abnormalities in the brain structure and activity. Comprehensive target DNA methylome analyses revealed in aged sperm more hypo-methylated genomic regions, in which REST/NRSF binding motif was enriched. Gene set enrichment analyses also identified enrichment of “REST/NRSF target genes”, in addition to “Late-fetal genes” and autism spectrum disorder-related “SFARI genes”, in up-regulated genes of developing brains from aged father. Indeed, gene sets near hypo-methylated genomic regions with REST/NRSF binding motif were also enriched in up-regulated genes of developing brains. Taken altogether, DNA hypo-methylation due to paternal aging in sperm will induce leaky expression of REST/NRSF target genes in the developing brain, thereby causing neuronal abnormalities and subsequent behavioral alteration in offspring.

In the mammalian olfactory epithelium (OE), olfactory receptor neurons (ORNs) are continuously regenerated throughout the animal's lifetime. Horizontal basal cells (HBCs) in the OE express the epithelial marker keratin 5 (K5) and the stem cell marker Pax6 and are considered relatively quiescent tissue stem cells in the OE. Pax6 is a key regulator of several developmental processes in the central nervous system and in sensory organs. Although Pax6 is expressed in the OE, its precise role remains unknown, particularly with respect to stem cell-like HBCs. To investigate the function of Pax6 in the developmental and regenerative processes in the OE, we generated conditional Pax6-knockout mice carrying a loxP-floxed Pax6 gene. Homozygous Pax6-floxed mice were crossed with K5-Cre transgenic mice to generate HBC-specific Pax6-knockout (Pax6-cKO) mice. We confirmed that the deletion of Pax6 expression in HBCs was sufficiently achieved in zone 1 of the OE in Pax6-cKO mice 3 days after methimazole-induced severe damage. In this condition, regeneration of the OE was dramatically impaired; both OE thickness and the number of ORNs were significantly decreased in the regenerated OE of Pax6-cKO mice. These results suggest that Pax6 expression is essential for HBCs to differentiate into neuronal cells during the regeneration process following severe injury.

Fabp7 gene encodes a brain-specific fatty acid-binding protein that is widely used as a marker for neural stem cells. Here, we report that the activity of rat Fabp7 promoter was regulated directly by a transcription factor, Pax6. Deletion analyses identified an essential region (-837 to -64 from transcription start site) in the rat Fabp7 promoter. This region controls promoter activity in rat embryos and in the mouse cultured cell line MEB5. Over-expressing wild-type Pax6 or a dominant-negative Pax6 mutant enhanced and suppressed, respectively, the promoter activity. Pax6 can bind the region directly, although the region contains no clear binding motif for Pax6. The rat Fabp7 promoter also contains conserved binding sites for Pbx/POU (-384 to -377) and CBF1 (-270 to -262). However, specific deletion of the sites showed no significant reduction in the promoter activity, although a gel mobility shift assay confirmed that CBF1 binds the conserved sequence. Taken together, these results suggest that the rat Fabp7 promoter is mainly regulated by Pax6. The Pax6-dependent regulation of the rat Fabp7 expression might have an evolutionary aspect between rat and mouse; the former may need to efficiently use fatty acids to make the brain bigger than the latter.

Background Paternal age has deleterious effects on the transmission of phenotypes to offspring, including higher prevalence of major psychiatric and neurodevelopmental disorders. Methods Here, we examined transgenerational transmission of selected somatic and cognitive phenotypes in mice and explored a possible epigenetic mechanism. Results F1 offspring of aged fathers had lower body weight and showed impairment in pup's vocalization, sensorimotor gating and spatial learning. From comprehensive methylome analyses, we identified in aged sperm 16 hypermethylated and 96 hypomethylated genomic loci. Motif analysis identified 19 potential REST/NRSF-binding sites in hypomethylated regions, which was confirmed as 37 motifs with actual binding of REST in the ChIP-seq database using ES-derived neural stem cells. Since we also found reduction of thickness in the neocortex, especially in the deep layer, we analyzed expression of potential REST-target genes. Discussion All of the data consistently support the scenario that hypomethylation in sperm due to paternal age may eventually result in ectopic binding of REST repressor to its target genes, downregulating their expression during brain development, thereby inducing behavioral abnormalities in the offspring.

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP 2 ), although the structural rearrangements occurring on PIP 2 binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP 2 binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence 121 KRWRK 125 , which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP 2 from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP 2 facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP 2 -binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP 2 mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP 2 interacting site and conditions that deplete PIP 2 or restrict access of TRPV4 to PIP 2 —in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.

OBJECTIVE TRPM2 is a Ca2+-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5′-diphosphoribose, which has been suggested to be involved in intracellular Ca2+ increase in immunocytes and pancreatic β-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. RESEARCH DESIGN AND METHODS Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca2+ in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets. RESULTS Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated β-cells, smaller intracellular Ca2+ increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups. CONCLUSIONS These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.

The “eyeless” animal C. elegans possesses the sense of light and engages in phototaxis behavior mediated by photoreceptor cells. However, the molecular and cellular mechanisms underlying phototransduction in C. elegans remain largely unclear. By recording the photoreceptor neuron ASJ in wild-type and various mutant worms, here we show that phototransduction in ASJ is a G protein-mediated process and requires membrane-associated guanylate cyclases but not typical cGMP-cleaving phosphodiesterases (PDEs). In addition, we find that C. elegans phototransduction requires LITE-1, a candidate photoreceptor protein known to be a member of the invertebrate taste receptor family. Genetic, pharmacological and electrophysiological data suggest a model whereby LITE-1 transduces light signals in ASJ through G-protein signaling, which leads to up-regulation of the second messenger cGMP followed by opening of cGMP-sensitive CNG channels and thereby stimulation of photoreceptor cells. Our results identify a phototransduction cascade in C. elegans and implicate the function of a “taste receptor" in phototransduction.

Charcot-Marie-Tooth disease type 2C (CMT2C) is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. All genes in this region were sequenced and heterozygous missense mutations were identified in the TRPV4 gene at positions c.805C>T and c.806G>A, causing the amino acid substitutions R269C and R269H. TRPV4 is a well known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of peripheral nerves. The CMT2C mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this striking phenotypic variability may be due to differential effects on regulatory protein-protein interactions.

Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH-related sensory disorders whose mechanisms are largely unknown.

Background and purpose Transient receptor potential melastatin 2 (TRPM2) is a non-selective Ca(2+)-permeable cation channel and is known to be activated by adenosine 5'-diphosphoribose (ADP-ribose) and hydrogen peroxide. TRPM2 current responses are reported to be drastically potentiated by the combination of each of these ligands with heat. Furthermore, the combination of cyclic ADP-ribose with heat also activates TRPM2. Although flufenamic acid, antifungal agents (miconazole and clotrimazole), and a phospholipase A(2) inhibitor (N-(p-amylcinnamoyl)anthranilic acid) inhibit TRPM2, their inhibition was either gradual or irreversible. Experimental approach To facilitate future research on TRPM2, we screened several compounds to investigate their potential to activate or inhibit the TRPM2 channels using the patch-clamp technique in HEK293 cells, transfected with human TRPM2. Key results 2-aminoethoxydiphenyl borate (2-APB) exhibited a rapid and reversible inhibition of TRPM2 channels that had been activated by its ADP-ribose or cADP-ribose and heat in a dose-dependent manner (IC(50) about 1 microM). 2-APB also inhibited heat-evoked insulin release from pancreatic islets, isolated from rats. Conclusions and implications 2-APB proved to be a powerful and effective tool for studying the function of TRPM2.

The body size of Caenorhabditis elegans is thought to be controlled by sensory inputs because many mutants with sensory cilium structure defects exhibit small body size. The EGL-4 cGMP-dependent protein kinase acts in sensory neurons to reduce body size when animals fail to perceive sensory signals. In addition to body size control, EGL-4 regulates various other behavioral and developmental pathways, including those involved in the regulation of egg laying and chemotaxis behavior. Here we have identified gcy-12, which encodes a receptor-type guanylyl cyclase, as a gene involved in the sensory regulation of body size. Analyses with GFP fusion constructs showed that gcy-12 is expressed in several sensory neurons and localizes to sensory cilia. Genetic analyses indicated that GCY-12 acts upstream of EGL-4 in body size control but does not affect other EGL-4 functions. Our studies indicate that the function of the GCY-12 guanylyl cyclase is to provide cGMP to the EGL-4 cGMP-dependent kinase only for limited tasks including body size regulation. We also found that the PDE-2 cyclic nucleotide phosphodiesterase negatively regulates EGL-4 in controlling body size. Thus, the cGMP level is precisely controlled by GCY-12 and PDE-2 to determine body size through EGL-4, and the defects in the sensory cilium structure may disturb the balanced control of the cGMP level. The large number of guanylyl cyclases encoded in the C. elegans genome suggests that EGL-4 exerts pleiotropic effects by partnering with different guanylyl cyclases for different downstream functions.

Thermotaxis (TTX) is one of the sophisticated behaviors in the nematode Caenorhabditis elegans. Although the mechanisms of thermotaxis have been deduced from different studies, they are controversial. Previous studies proposed a behavioral model where thermotaxis is regulated by the counterbalance between two opposite driving forces, while recent studies proposed stochastic models. In this study, we analyzed thermotaxis by a novel quantitative population TTX assay using a gentle linear thermal gradient. Analysis of thermotaxis in wild type animals revealed a clear thermal preference to a cultivation temperature with regard to the distribution of animals and the TTX mean expressing temperature preference. A time course assay revealed that the behavioral response to a preferred temperature was initially suppressed for at least 15 min in the animals cultivated at 23 degrees C, but not in those cultivated at 17 degrees C. Our result provides a possible explanation for the inconsistency between the various studies on thermotaxis and is consistent with the early behavioral model, where thermotaxis is regulated by the counterbalance between two driving forces.

The brain is rich in lipid and fatty molecules. In this review article, we focus on fatty acid binding proteins (Fabps) that bind to fatty acids such as arachidonic acid and docosahexianoic acid and transfer these lipid ligands within the cytoplasm. Among Fabp family molecules, Fabp3, Fabp5, and Fabp7 are specifically localized in neural stem/progenitor cells, neurons and glia in a cell-type specific manner. Quantitative trait locus analysis has revealed that Fabp7 is related with performance of prepulse inhibition (PPI) that is used as an endophenotype of psychiatric diseases such as schizophrenia. Fabp5 and Fabp7 play important roles on neurogenesis and differentially regulate acoustic startle response and PPI. However, other behavior performances including spatial memory, anxiety-like behavior, and diurnal changes in general activity were not different in mice deficient for Fabp7 or Fabp5. Considering the importance of fatty acids in neurogenesis, we would like to emphasize that lipid nutrition and its dynamism via Fabps play significant roles in mental conditions. This might provide a good example of how nutritional environment can affect psychiatric conditions at the molecular level.

The rpoB gene for the beta subunit of rice chloroplast RNA polymerase was found to be highly expressed in unexpanded immature leaves that contained proplastids, indicating the specific expression of rpoB at an early stage of chloroplast development. A putative transcription start site (tss) was identified, but the 5' upstream region of the tss had no sequences resembling typical --35 and --10 elements. A palindromic sequence and high AT-content were recognized.

We have treated three patients with avascular osteonecrosis using vascularized scapular bone graft. To predict blood perfusion in both the diseased femoral head and the transferred bone, all the hips were followed up using dynamic magnetic resonance (MR) scans, performed 1 and 7 months after surgery. In the present cases, it was shown that conventional enhanced MR imaging sometimes depicts increased intensity in bone marrow without blood perfusion due to the leakage of gadolinium-DTPA (Gd-DTPA) from the capillaries surrounding the avascular tissue. It was found that Gd-DTPA remaining in the dead bone marrow resulted in a false-positive image. By contrast, the dynamic MR scan evaluated only those images taken before the leakage. This is one of the advantages of the dynamic study, which reflects actual blood flow in the bone. The fast rise in the time-intensity curve following bolus injection of Gd-DTPA indicates that there is fast blood perfusion in the bone. The dynamic MR scan has demonstrated that there is little blood perfusion in the diseased bone 1 month after the operation and that vascular ingrowth from the transferred bone flap proceeds gradually between 1 and 7 months after surgery. These findings indicate that the dynamic MR scan is very useful in demonstrating vascular ingrowth after surgery in avascular necrosis of the femoral head and can be a reliable monitoring technique for anastomotic patency of the vascularized bone flap.

TRPV2 is a member of the superfamily of the Transient Receptor Potential (TRP) ion channels. These channels are assembled into homotetramers and allow cations across the membrane in response to physico-chemical stimuli such as heat, pressure, osmotic changes, etc. TRPV2 is an orphan receptor, since no specific endogenous ligand has been identified yet. To better understand the role of TRPV2 and to go further into its function, sequence analysis of orthologs for TRPV2 has been performed in order to define common and differential architectural regions. Preliminary biophysical characterization such as thermal stability, and secondary structure composition analysis has been carried out on the different TRPV2 orthologs to identify key structural points in the TRPV2 topology.

A trimethoxysilyl group, which reacts easily with water, was introduced into poly(vinyl chloride) (PVC) in homogeneous and heterogeneous reactions. The polymers obtained were crosslinked in the presence of water, and crosslinking was accelerated in the presence of acid or base.

A short pulse response for surface waves involved in the backscattering by dielectric spheres is considered in the time domain. Since the surface-wave contributions for the cw backscattering are known, pulse returns for the surface waves are obtained from the cw solutions by Fourier synthesis. Large savings in computation time for a Fourier series were realized with the use of the fast Fourier transform algorithm. The return positions in the short pulse response can be approximately estimated from a scattering model for surface waves depicted by Van de Hulst. With the propagation constant in the dielectric sphere rather than in free space for a wave traveling along the sphere surface, a better agreement between the predicted and calculated positions for the short pulse returns was obtained. Significant returns of surface waves for the backscattering come from the surface waves that have made the maximum number of shortcuts possible through the sphere.

Diffracted field contributions to backscattering of an electromagnetic plane wave by a spherical particle are calculated. The diffracted fields give rise to surface waves in the shadow region and can be evaluated by finding surface wave poles and computing their residues. In order to compute the residues the valid range of the Schobe and Debye asymptotic expansion formulas for the Hankel function is examined. With these asymptotic formulas numerical values of the surface wave complex poles are tabulated. Curves for backscattering cross section due to the first six surface waves are presented as a function of the size parameter kappaa between 5 and 60 for absorbing spheres of refractive index m = 1.61-i0.0025 as well as nonabsorbing spheres with m = 1.60.

Homeothermic animals, including humans, live by adapting to changes in ambient temperature. Numerous studies have demonstrated cold exposure (at approximately 5 degrees C) improves glucose tolerance despite reducing insulin secretion and increasing energy expenditure. To determine the effects of a small reduction in ambient temperature on energy metabolism, we compared two groups of mice; one exposed to a cool environment (20 degrees C) and the other maintained in a near-thermoneutral environment (25 degrees C) for 10 days. Both glucose-induced insulin secretion and glucose response were significantly impaired in mice exposed to a cool environment. In the cool temperature-exposed mice, skin temperatures were reduced, and plasma norepinephrine levels were increased, suggesting that impairment of insulin secretion was facilitated by induction of sympathetic nervous activity due to skin cooling. In addition, expression of GLUT4 mRNA was increased significantly in inguinal subcutaneous adipose tissue (IWAT) but not in epididymal or brown adipose tissue or skeletal muscle in these mice. Moreover, expression of Dok1, a molecule linked to activation of insulin receptors in adipocyte hypertrophy, and Cd36, a molecule related to NEFA uptake, were also increased at mRNA and/or protein levels only in IWAT of the cool temperature-exposed mice. Fatty acid synthesis was also facilitated, and fat weights were increased only in IWAT from mice kept at 20 degrees C. These results suggest that a small reduction in ambient temperature can affect glucose homeostasis through regulation of insulin secretion and preferentially enhances fat storage in IWAT. These adaptations can be interpreted as preparation for a further reduction in ambient temperature.

Ligand-gated ion channels are important in sensory and synaptic transduction. The PKD1L3–PKD2L1 channel complex is a sour taste receptor candidate that is activated by acids. Here, we report that the proton-activated PKD1L3–PKD2L1 ion channels have the unique ability to be activated after the removal of an acid stimulus. We refer to this property as the off-response (previously described as a delayed response). Electrophysiological analyses show that acid-induced responses are observed only after the removal of an acid solution at less than pH 3.0. A small increase in pH is sufficient for PKD1L3–PKD2L1 channel activation, after exposure to an acid at pH 2.5. These results indicate that this channel is a new type of ion channel—designated as an ‘off-channel'—which is activated during stimulus application but not gated open until the removal of the stimulus. The off-response property of PKD1L3–PKD2L1 channels might explain the physiological phenomena occurring during sour taste sensation.