Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

Rapid eye movement sleep deprivation impairs neuronal plasticity and reduces hippocampal neuronal arborization in male albino rats: Noradrenaline is involved in the process.

Rapid eye movement sleep (REMS) favors brain development and memory, while it is decreased in neurodegenerative diseases. REMS deprivation (REMSD) affects several physiological processes including memory consolidation; however, its detailed mechanism(s) of action was unknown. REMS reduces, while REMSD elevates noradrenaline (NA) level in the brain; the latter induces several deficiencies and disorders, including changes in neuronal cytomorphology and apoptosis. Therefore, we proposed that REMS- and REMSD-associated modulation of NA level might affect neuronal plasticity and affect brain functions. Male albino rats were REMS deprived by flower-pot method for 6 days, and its effects were compared with home cage and large platform controls as well as post-REMSD recovered and REMS-deprived prazosin (α1-adrenoceptor antagonist)-treated rats. We observed that REMSD reduced CA1 and CA3 neuronal dendritic length, branching, arborization, and spine density, while length of active zone and expressions of pre- as well as post-synaptic proteins were increased as compared to controls; interestingly, prazosin prevented most of the effects in vivo. Studies on primary culture of neurons from chick embryo brain confirmed that NA at lower concentration(s) induced neuronal branching and arborization, while higher doses were destructive. The findings support our contention that REMSD adversely affects neuronal plasticity, branching, and synaptic scaffold, which explain the underlying cytoarchitectural basis of REMSD-associated patho-physio-behavioral changes. Consolidation of findings of this study along with that of our previous reports suggest that the neuronal disintegration could be due to either withdrawal of direct protective and proliferative role of low dose of NA or indirect effect of high dose of NA or both.

Noradrenergic ß-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na-K ATPase Subunit Expression in C6 Cells.

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na-K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na-K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca++ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on ß-adrenoceptor decreased Na-K ATPase activity and mRNA expression of the catalytic α2-Na-K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca++ played a critical role in such decreased α2-Na-K ATPase expression. In contrast, noradrenaline acting on ß-adrenoceptor up-regulated the expression of regulatory ß2-Na-K ATPase subunit, which although was cAMP and Ca++ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na-K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.

Rapid Eye Movement Sleep Deprivation Associated Increase in Na-K ATPase Activity in the Rat Brain is Due to Noradrenaline Induced α1-Adrenoceptor Mediated Increased α-Subunit of the Enzyme.

Rapid eye movement sleep (REMS) modulates Na-K ATPase activity and maintains brain excitability. REMS deprivation (REMSD)-associated increased Na-K ATPase activity is mediated by noradrenaline (NA) acting on α1-adrenoceptor (AR) in the brain. It was shown that NA-induced increased Na-K ATPase activity was due to allosteric modulation as well as increased turnover of the enzyme. Although the former has been studied in detail, our understanding on the latter was lacking, which we have studied. Male Wistar rats were REMS deprived for 4-days by classical flower-pot method; suitable control experiments were conducted. In another set, α1-AR antagonist prazosin (PRZ) was i.p. injected 48 h REMSD onward. At the end of experiments rats were sacrificed by cervical dislocation and brains were removed. Synaptosomes prepared from the brains were used to estimate Na-K ATPase activity as well as protein expressions of different isoforms of the enzyme subunits using western blot. REMSD significantly increased synaptosomal Na-K ATPase activity and that was due to differential increase in the expressions of α1-, α2- and α3-isoforms, but not that of ß1- and ß2-isoforms. PRZ reduced the REMSD-induced increased Na-K ATPase activity and protein expressions. We also observed that the increased Na-K ATPase subunit expression was not due to enhanced mRNA synthesis, which suggests the possibility of post-transcriptional regulation. Thus, the findings suggest that REMSD-associated increased Na-K ATPase activity is due to elevated level of α-subunit of the enzyme and that is induced by NA acting on α1-AR mediated mRNA-stabilization.

Full-length isoform transcriptome of the developing human brain provides further insights into autism.

Alternative splicing plays an important role in brain development, but its global contribution to human neurodevelopmental diseases (NDDs) requires further investigation. Here we examine the relationships between splicing isoform expression in the brain and de novo loss-of-function mutations from individuals with NDDs. We analyze the full-length isoform transcriptome of the developing human brain and observe differentially expressed isoforms and isoform co-expression modules undetectable by gene-level analyses. These isoforms are enriched in loss-of-function mutations and microexons, are co-expressed with a unique set of partners, and have higher prenatal expression. We experimentally test the effect of splice-site mutations and demonstrate exon skipping in five NDD risk genes, including SCN2A, DYRK1A, and BTRC. Our results suggest that the splice site mutation in BTRC reduces translational efficiency, likely affecting Wnt signaling through impaired degradation of ß-catenin. We propose that functional effects of mutations should be investigated at the isoform- rather than gene-level resolution.

Mechanism of noradrenaline-induced α1-adrenoceptor mediated regulation of Na-K ATPase subunit expression in Neuro-2a cells.

Rapid eye movement sleep (REMS) plays an important role in maintaining brain excitability by regulating noradrenaline (NA) level and Na-K ATPase activity. We showed earlier that REMS deprivation (REMSD) associated elevated NA increased neuronal, while decreased glial Na-K ATPase activity. However, our knowledge was insufficient on how the REMSD-associated effect is sustained particularly under chronic condition. Using Neuro-2a cells as a model, we investigated the molecular mechanism of NA-induced increase in mRNA expression of Na-K ATPase subunit and the enzyme activity. The cells were treated with NA in the presence or absence of either α1- or ß-adrenoceptor (AR) antagonists, Ca++-channel blocker or SERCA-inhibitor, and PKA or PKC inhibitor. We observed that NA acting on α1-AR increased Na-K ATPase activity and mRNA expression of the catalytic α1- and α3-Na-K ATPase subunits in the Neuro-2a cells. Further, PLC and PKC mediated modulation of intracellular Ca++ played a critical role in inducing the mRNA expression. On the other hand NA, acting on ß-AR up-regulated expression of the regulatory ß1-subunit of Na-K ATPase. The involvement of SP1 as well as phospho-CREB transcription factors in the NA-mediated increased expression of various subunit isoforms was established. The results of this study along with that of earlier reports support our proposed working model of NA-induced increase in mRNA expression of specific Na-K ATPase subunit leading to increased Na-K ATPase activity. The findings help us understand the molecular mechanism of NA-induced increased brain excitability, for example, upon REMSD including under chronic condition.

Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain.

Rapid eye movement sleep (REMS) deprivation (REMSD) has been reported to elevate neurotransmitter level in the brain; however, intracellular mechanism of its increased release was not studied. Phosphorylation of synapsinI, a synaptic vesicle-associated protein, is involved in the regulation of neurotransmitter release. In this study, rats were REMS deprived by classical flowerpot method; free moving control (FMC), large platform control (LPC) and recovery control (REC) was carried out. In another set REMS deprived rats were intraperitoneally (i.p.) injected with α1-adrenoceptor antagonist, prazosin (PRZ). Effects of REMSD on Na-K ATPase activity and on the total synapsinI as well as phosphorylated synapsinI levels were estimated in synaptosomes prepared from whole brain. It was observed that REMSD significantly increased synaptosomal Na-K ATPase activity, which was prevented by PRZ. Western blotting of the same samples by anti-synapsinI and anti-synapsinI-phosphoSer603 showed that REMSD increased both the total as well as phospho-form of synapsinI as compared to respective levels in FMC and LPC samples. These findings suggest a functional link between REMSD and synaptic vesicular mobilization at the presynaptic terminal, a process that is essential for neurotransmitter release. The findings help explaining the intracellular mechanism of elevated neurotransmitter release associated to REMSD.