Calsyntenins (CLSTNs) are important synaptic molecules whose molecular functions are not fully understood. Although mutations in calsyntenin (CLSTN) genes have been associated with psychiatric ...disorders in humans, their function is still unclear. One of the reasons why the function of CLSTNs in the nervous system has not been clarified is the functional redundancy among the three paralogs. Therefore, to investigate the functions of mammalian CLSTNs, we generated triple knockout (TKO) mice lacking all CLSTN paralogs and examined their behavior. The mutant mice tended to freeze in novel environments and exhibited hypersensitivity to stress. Consistent with this, glucose levels under stress were significantly higher in the mutant mice than in the wild-type controls. In particular, phenotypes such as decreased motivation, which had not been reported in single Clstn KO mice, were newly discovered. The TKO mice generated in this study represent an important mouse model for clarifying the function of CLSTN in the future.
22q11.2 deletion syndrome (22q11.2DS) is a disorder caused by the segmental deletion of human chromosome 22. This chromosomal deletion is known as high genetic risk factors for various psychiatric ...disorders. The different deletion types are identified in 22q11.2DS patients, including the most common 3.0-Mb deletion, and the less-frequent 1.5-Mb and 1.4-Mb deletions. In previous animal studies of psychiatric disorders associated with 22q11.2DS mainly focused on the 1.5-Mb deletion and model mice mimicking the human 1.5-Mb deletion have been established with diverse genetic backgrounds, which resulted in the contradictory phenotypes. On the other hand, the contribution of the genes in 1.4-Mb region to psychiatric disorders is poorly understood. In this study, we generated two mouse lines that reproduced the 1.4-Mb and 1.5-Mb deletions of 22q11.2DS Del(1.4 Mb)/+ and Del(1.5 Mb)/+ on the pure C57BL/6N genetic background. These mutant mice were analyzed comprehensively by behavioral tests, such as measurement of locomotor activity, sociability, prepulse inhibition and fear-conditioning memory. Del(1.4 Mb)/+ mice displayed decreased locomotor activity, but no abnormalities were observed in all other behavioral tests. Del(1.5 Mb)/+ mice showed reduction of prepulse inhibition and impairment of contextual- and cued-dependent fear memory, which is consistent with previous reports. Furthermore, apparently intact social recognition in Del(1.4 Mb)/+ and Del(1.5 Mb)/+ mice suggests that the impaired social recognition observed in Del(3.0 Mb)/+ mice mimicking the human 3.0-Mb deletion requires mutations both in 1.4-Mb and 1.5 Mb regions. Our previous study has shown that Del(3.0 Mb)/+ mice presented disturbance of behavioral circadian rhythm. Therefore, we further evaluated sleep/wakefulness cycles in Del(3.0 Mb)/+ mice by electroencephalogram (EEG) and electromyogram (EMG) recording. EEG/EMG analysis revealed the disturbed wakefulness and non-rapid eye moving sleep (NREMS) cycles in Del(3.0 Mb)/+ mice, suggesting that Del(3.0 Mb)/+ mice may be unable to maintain their wakefulness. Together, our mouse models deepen our understanding of genetic contributions to schizophrenic phenotypes related to 22q11.2DS.
Lysosome-associated membrane protein 5 (LAMP5) is a mammalian ortholog of the Caenorhabditis elegans protein, UNC-46, which functions as a sorting factor to localize the vesicular GABA transporter ...UNC-47 to synaptic vesicles. In the mouse forebrain, LAMP5 is expressed in a subpopulation of GABAergic neurons in the olfactory bulb and the striato-nigral system, where it is required for fine-tuning of GABAergic synaptic transmission. Here we focus on the prominent expression of LAMP5 in the brainstem and spinal cord and suggest a role for LAMP5 in these brain regions. LAMP5 was highly expressed in several brainstem nuclei involved with auditory processing including the cochlear nuclei, the superior olivary complex, nuclei of the lateral lemniscus and grey matter in the spinal cord. It was localized exclusively in inhibitory synaptic terminals, as has been reported in the forebrain. In the absence of LAMP5, localization of the vesicular inhibitory amino acid transporter (VIAAT) was unaltered in the lateral superior olive and the ventral cochlear nuclei, arguing against a conserved role for LAMP5 in trafficking VIAAT. Lamp5 knockout mice showed no overt behavioral abnormality but an increased startle response to auditory and tactile stimuli. In addition, LAMP5 deficiency led to a larger intensity-dependent increase of wave I, II and V peak amplitude of auditory brainstem response. Our results indicate that LAMP5 plays a pivotal role in sensorimotor processing in the brainstem and spinal cord.
Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by a CTG repeat expansion in the 3′‐UTR of dystrophia myotonica‐protein kinase. Aberrant regulation of alternative splicing is a ...characteristic feature of DM. Dozens of genes have been found to be abnormally spliced; however, few reported splicing abnormalities explain the phenotypes of DM1 patients. Thus, we hypothesized that other, unknown abnormal splicing events exist. Here, by using exon array, we identified aberrant inclusion of myomesin 1 (MYOM1) exon 17a as a novel splicing abnormality in DM1 muscle. A cellular splicing assay with a MYOM1 minigene revealed that not only MBNL1‐3 but also CELF1 and 2 decreased the inclusion of MYOM1 exon 17a in HEK293T cells. Expression of expanded CUG repeat impeded MBNL1 activity but did not affect CELF1 activity on the splicing of MYOM1 minigene. Our results suggest that the downregulation of MBNL proteins should lead to the abnormal splicing of MYOM1 exon 17a in DM1 muscle.
•Opalin expression shows a region-specific reduction in aged mouse brains.•The molecular size of Opalin becomes larger with aging.•The increasing molecular weight of Opalin with aging due to ...hypersialylation.
Opalin/Tmem10 is a myelin-associated sialylglycoprotein that is specific to only the mammalian central nervous system. However, little is known about the properties or function of this protein. Here, we analyzed the expression and glycosylation patterns of Opalin in the postnatal mouse brain. Immunolocalization patterns of Opalin were similar to those of myelin basic protein in juvenile and adolescent mice. On the other hand, in the adult mouse brain, decreasing immunoreactivity for Opalin was observed in the hindbrain region especially in the cerebellar white matter compared with the corpus callosum in the forebrain. In addition, Opalin showed increasing molecular size with mouse aging. This age-dependent increase in Opalin molecular weight was mainly owing to hypersialylation of O-glycans. These results indicate that the regional redistribution and the degree of sialylation of Opalin protein are age-dependently regulated in mouse brains.
► PMA regulates endogenous SERCA1 splicing in HEK293 cells. ► PMA’s effects were completely abolished by the PKC inhibitor Ro 31-8220. ► PKCβII and PKCθ are the specific PKC isozymes regulating ...SERCA1 splicing.
Myotonic dystrophy type 1 (DM1) is a multi-systemic disease with no established treatment to date. Small, cell-permeable molecules hold the potential to treat DM1. In this study, we investigated the association between protein kinase C (PKC) signaling and splicing of sarcoplasmic reticulum Ca2+-ATPase1 (SERCA1). Our aim was to clarify the mechanisms underlying the regulation of alternative splicing, in order to explore new therapeutic strategies for DM1. By assessing the splicing pattern of the endogenous SERCA1 gene in HEK293 cells, we found that treatment with phorbol 12-myristate 13-acetate (PMA) regulated SERCA1 splicing. Interestingly, treatment with PMA for 48h normalized SERCA1 splicing, while treatment for 1.5h promoted aberrant splicing. These two responses showed dose dependency and were completely abolished by the PKC inhibitor Ro 31-8220. Furthermore, repression of PKCβII and PKCθ by RNAi mimicked prolonged PMA treatment. These results indicate that PKC signaling is involved in the splicing of SERCA1 and provide new evidence for a link between alternative splicing and PKC signaling.
Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of ...disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.
Thermogenic brown and beige adipocytes counteract obesity by enhancing energy dissipation via uncoupling protein-1 (Ucp1). However, the effect of genetic variation on these cells, a major source of ...disease susceptibility, has been less well studied. Here we examined beige adipocytes from obesity-prone C57BL/6J (B6) and obesity-resistant 129X1/SvJ (129) mouse strains and identified a cis-regulatory variant rs47238345 that is responsible for differential Ucp1 expression. The alternative T allele of rs47238345 at the Ucp1 -12kb enhancer in 129 facilitates the allele-specific binding of nuclear factor I-A (NFIA) to mediate allele-specific enhancer-promoter interaction and Ucp1 transcription. Furthermore, CRISPR-Cas9/Cpf1-mediated single nucleotide polymorphism (SNP) editing of rs47238345 resulted in increased Ucp1 expression. We also identified Lim homeobox protein 8 (Lhx8), whose expression is higher in 129 than in B6, as a trans-acting regulator of Ucp1 in mice and humans. These results demonstrate the cis- and trans-acting effects of genetic variation on Ucp1 expression that underlie phenotypic diversity.
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•NFIA in adipocytes determines Ucp1 expression between obesity-prone and -resistant mouse strains•Allele-specific binding of NFIA at the Ucp1 -12kb enhancer mediates differential Ucp1 expression•Editing of a SNP at the Ucp1 -12kb enhancer is sufficient to increase Ucp1 in obesity-prone strain
Physiology; Molecular physiology; Molecular biology; Epigenetics
Pain is one of the most frequent non-motor symptoms of Parkinson’s disease (PD). Neuropathic pain is highly prevalent in PD and negatively affects the quality of life of patients with PD. However, ...there is currently no evidence-based treatment for its control. Safinamide, a monoamine oxidase (MAO)-B inhibitor with a sodium channel inhibitory effect, showed improvement in PD-related pain in several clinical trials. However, it is unclear for which of the various types of pain in PD safinamide is effective. The aim of the present study was to examine the effect of safinamide on neuropathic pain in a rat model of chronic constriction injury (CCI). Pain was evaluated on postoperative days 14 and 21 using von Frey or weight-bearing tests. Male CCI model rats showed a decreased paw withdrawal threshold and a weight-bearing deficit on postoperative days 14 and 21. Single oral administration of safinamide (15, 30, 45 or 70 mg/kg) dose-dependently improved neuropathic pain in both pain assessments on day 14. Subsequently, the 15 and 45 mg/kg dose groups were administered safinamide orally once daily until day 21. With repeated administration, the effect of safinamide on pain was enhanced. The present findings show that safinamide improves neuropathic pain in male CCI model rats. Further animal model research and pathological and molecular pharmacological investigations are warranted.
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