Cell stress in cortical organoids impairs molecular subtype specification.

Cortical organoids are self-organizing three-dimensional cultures that model features of the developing human cerebral cortex1,2. However, the fidelity of organoid models remains unclear3-5. Here we analyse the transcriptomes of individual primary human cortical cells from different developmental periods and cortical areas. We find that cortical development is characterized by progenitor maturation trajectories, the emergence of diverse cell subtypes and areal specification of newborn neurons. By contrast, organoids contain broad cell classes, but do not recapitulate distinct cellular subtype identities and appropriate progenitor maturation. Although the molecular signatures of cortical areas emerge in organoid neurons, they are not spatially segregated. Organoids also ectopically activate cellular stress pathways, which impairs cell-type specification. However, organoid stress and subtype defects are alleviated by transplantation into the mouse cortex. Together, these datasets and analytical tools provide a framework for evaluating and improving the accuracy of cortical organoids as models of human brain development.

Selective Kv1.3 channel blocker as therapeutic for obesity and insulin resistance.

Obesity is an epidemic, calling for innovative and reliable pharmacological strategies. Here, we show that ShK-186, a selective and potent blocker of the voltage-gated Kv1.3 channel, counteracts the negative effects of increased caloric intake in mice fed a diet rich in fat and fructose. ShK-186 reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. These changes mimic the effects of Kv1.3 gene deletion. ShK-186 did not alter weight gain in mice on a chow diet, suggesting that the obesity-inducing diet enhances sensitivity to Kv1.3 blockade. Several mechanisms may contribute to the therapeutic benefits of ShK-186. ShK-186 therapy activated brown adipose tissue as evidenced by a doubling of glucose uptake, and increased ß-oxidation of fatty acids, glycolysis, fatty acid synthesis, and uncoupling protein 1 expression. Activation of brown adipose tissue manifested as augmented oxygen consumption and energy expenditure, with no change in caloric intake, locomotor activity, or thyroid hormone levels. The obesity diet induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. This action on the liver may underlie the differential effectiveness of ShK-186 in mice fed a chow vs. an obesity diet. Our results highlight the potential use of Kv1.3 blockers for the treatment of obesity and insulin resistance.

Intracellular trafficking of the KV1.3 potassium channel is regulated by the prodomain of a matrix metalloprotease.

Matrix metalloproteases (MMPs) are endopeptidases that regulate diverse biological processes. Synthesized as zymogens, MMPs become active after removal of their prodomains. Much is known about the metalloprotease activity of these enzymes, but noncanonical functions are poorly defined, and functions of the prodomains have been largely ignored. Here we report a novel metalloprotease-independent, channel-modulating function for the prodomain of MMP23 (MMP23-PD). Whole-cell patch clamping and confocal microscopy, coupled with deletion analysis, demonstrate that MMP23-PD suppresses the voltage-gated potassium channel KV1.3, but not the closely related KV1.2 channel, by trapping the channel intracellularly. Studies with KV1.2-1.3 chimeras suggest that MMP23-PD requires the presence of the KV1.3 region from the S5 trans-membrane segment to the C terminus to modulate KV1.3 channel function. NMR studies of MMP23-PD reveal a single, kinked trans-membrane α-helix, joined by a short linker to a juxtamembrane α-helix, which is associated with the surface of the membrane and protected from exchange with the solvent. The topological similarity of MMP23-PD to KCNE1, KCNE2, and KCNE4 proteins that trap KV1.3, KV1.4, KV3.3, and KV3.4 channels early in the secretory pathway suggests a shared mechanism of channel regulation. MMP23 and KV1.3 expression is enhanced and overlapping in colorectal cancers where the interaction of the two proteins could affect cell function.

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience.

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks.

Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes.

Intracellular calcium dysregulation in autism spectrum disorder: An analysis of converging organelle signaling pathways.

Autism spectrum disorder (ASD) is a group of complex, neurological disorders that affect early cognitive, social, and verbal development. Our understanding of ASD has vastly improved with advances in genomic sequencing technology and genetic models that have identified >800 loci with variants that increase susceptibility to ASD. Although these findings have confirmed its high heritability, the underlying mechanisms by which these genes produce the ASD phenotypes have not been defined. Current efforts have begun to "functionalize" many of these variants and envisage how these susceptibility factors converge at key biochemical and biophysical pathways. In this review, we discuss recent work on intracellular calcium signaling in ASD, including our own work, which begins to suggest it as a compelling candidate mechanism in the pathophysiology of autism and a potential therapeutic target. We consider how known variants in the calcium signaling genomic architecture of ASD may exert their deleterious effects along pathways particularly involving organelle dysfunction including the endoplasmic reticulum (ER), a major calcium store, and the mitochondria, a major calcium ion buffer, and theorize how many of these pathways intersect.

High-throughput screen detects calcium signaling dysfunction in typical sporadic autism spectrum disorder.

Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental disorders without any defined uniting pathophysiology. Ca2+ signaling is emerging as a potential node in the genetic architecture of the disorder. We previously reported decreased inositol trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum in several rare monogenic syndromes highly comorbid with autism - fragile X and tuberous sclerosis types 1 and 2 syndromes. We now extend those findings to a cohort of subjects with sporadic ASD without any known mutations. We developed and applied a high throughput Fluorometric Imaging Plate Reader (FLIPR) assay to monitor agonist-evoked Ca2+ signals in human primary skin fibroblasts. Our results indicate that IP3 -mediated Ca2+ release from the endoplasmic reticulum in response to activation of purinergic receptors is significantly depressed in subjects with sporadic as well as rare syndromic forms of ASD. We propose that deficits in IP3-mediated Ca2+ signaling represent a convergent hub function shared across the spectrum of autistic disorders - whether caused by rare highly penetrant mutations or sporadic forms - and holds promise as a biomarker for diagnosis and novel drug discovery.

Channelopathy pathogenesis in autism spectrum disorders.

Autism spectrum disorder (ASD) is a syndrome that affects normal brain development and is characterized by impaired social interaction as well as verbal and non-verbal communication and by repetitive, stereotypic behavior. ASD is a complex disorder arising from a combination of multiple genetic and environmental factors that are independent from racial, ethnic and socioeconomical status. The high heritability of ASD suggests a strong genetic basis for the disorder. Furthermore, a mounting body of evidence implies a role of various ion channel gene defects (channelopathies) in the pathogenesis of autism. Indeed, recent genome-wide association, and whole exome- and whole-genome resequencing studies linked polymorphisms and rare variants in calcium, sodium and potassium channels and their subunits with susceptibility to ASD, much as they do with bipolar disorder, schizophrenia and other neuropsychiatric disorders. Moreover, animal models with these genetic variations recapitulate endophenotypes considered to be correlates of autistic behavior seen in patients. An ion flux across the membrane regulates a variety of cell functions, from generation of action potentials to gene expression and cell morphology, thus it is not surprising that channelopathies have profound effects on brain functions. In the present work, we summarize existing evidence for the role of ion channel gene defects in the pathogenesis of autism with a focus on calcium signaling and its downstream effects.