BACKGROUND Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ≥200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.)
Aging is a major risk factor for the progression of neurodegenerative diseases, including Huntington disease (HD). Reduced neuronal IGF1 or Irs2 signaling have been shown to extend life span in mice. To determine whether Irs2 signaling modulates neurodegeneration in HD, we genetically modulated Irs2 concentrations in the R6/2 mouse model of HD. Increasing Irs2 levels in the brains of R6/2 mice significantly reduced life span and increased neuronal oxidative stress and mitochondrial dysfunction. In contrast, reducing Irs2 levels throughout the body (except in β cells, where Irs2 expression is needed to prevent diabetes onset; R6/2•Irs2 +/-•Irs2 βtg mice) improved motor performance and extended life span. The slower progression of HD-like symptoms was associated with increased nuclear localization of the transcription factor FoxO1 and increased expression of FoxO1-dependent genes that promote autophagy, mitochondrial function, and resistance to oxidative stress. Mitochondrial function improved and the number of autophagosomes increased in R6/2•Irs2 +/-•Irs2 βtg mice, whereas aggregate formation and oxidative stress decreased. Thus, our study suggests that Irs2 signaling can modulate HD progression. Since we found the expression of Irs2 to be normal in grade II HD patients, our results suggest that decreasing IRS2 signaling could be part of a therapeutic approach to slow the progression of HD.
The cellular distribution of GABAC receptor rho1 and rho2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum rho1 and rho2 mRNAs were expressed in Purkinje cells and basket-like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the rho2 signal was consistently stronger than rho1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both rho1 and rho2 mRNAs showed somatodendritic localization, extending out for >100 microm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of rho1 and rho2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.
Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing beta-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single beta-cells. The insulin signal in the embryonic beta-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in beta-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of beta-cells.
SUMMARY Irs2-mediated insulin/IGF1 signaling in the CNS modulates energy balance and glucose homeostasis; however, the site for Irs2 function is unknown. The hormone leptin mediates energy balance by acting on leptin receptor (LepR-b)-expressing neurons. To determine whether LepR-b neurons mediate the metabolic actions of Irs2 in the brain, we utilized Leprcre together with Irs2L/L to ablate Irs2 expression in LepR-b neurons (LeprΔIrs2). LeprΔIrs2-mice developed obesity, glucose intolerance, and insulin resistance. Leptin action was not altered in young LeprΔIrs2-mice, although insulin-stimulated FoxO1 nuclear exclusion was reduced in LeprΔIrs2-mice. Indeed, deletion of Foxo1 from LepR-b neurons in LeprΔIrs2-mice normalized energy balance, glucose homeostasis, and arcuate nucleus gene expression in LeprΔIrs•2ΔFoxo1-mice. Thus, Irs2 signaling in LepR-b neurons plays a crucial role in metabolic sensing and regulation. While not required for leptin action, Irs2 suppresses FoxO1 signaling in LepR-b neurons to promote energy balance and metabolism.
The aim of this study was to define when an insulin-positive cell becomes functional in vivo and starts to exocytose insulin in a regulated nutrient-dependent manner. Insulin-positive cells appear in embryonic life (midgestation) and complete their maturation, presumably around birth. In order to work with embryonic and newborn endocrine pancreas, we used organotypic slices. The mouse embryonic pancreas slices show high basal insulin release that is not further elevated by high glucose levels. Despite the presence of functional voltage-activated ion channels, the cells are not electrically active in the presence of secretagogues. At birth, the high basal insulin release drops and, after postnatal day 2, the insulin-positive cells show both adult-like bursting electrical activity and hormone release induced by high glucose levels. These properties allowed us to define them as beta cells. Despite the apparent stability of the transcription factor profile reported in insulin-positive cells during late-embryonic life, functional beta cells appear only 2 days after birth.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.