All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two ...archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.
Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has ...called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.
Sensory stimuli drive the maturation and function of the mammalian nervous system in part through the activation of gene expression networks that regulate synapse development and plasticity. These ...networks have primarily been studied in mice, and it is not known whether there are species- or clade-specific activity-regulated genes that control features of brain development and function. Here we use transcriptional profiling of human fetal brain cultures to identify an activity-dependent secreted factor, Osteocrin (OSTN), that is induced by membrane depolarization of human but not mouse neurons. We find that OSTN has been repurposed in primates through the evolutionary acquisition of DNA regulatory elements that bind the activity-regulated transcription factor MEF2. In addition, we demonstrate that OSTN is expressed in primate neocortex and restricts activity-dependent dendritic growth in human neurons. These findings suggest that, in response to sensory input, OSTN regulates features of neuronal structure and function that are unique to primates.
It has been proposed that human embryonic stem cells could be used to provide an inexhaustible supply of differentiated cell types for the study of disease processes. Although methods for ...differentiating embryonic stem cells into specific cell types have become increasingly sophisticated, the utility of the resulting cells for modeling disease has not been determined. We have asked whether specific neuronal subtypes produced from human embryonic stem cells can be used to investigate the mechanisms leading to neural degeneration in amyotrophic lateral sclerosis (ALS). We show that human spinal motor neurons, but not interneurons, are selectively sensitive to the toxic effect of glial cells carrying an ALS-causing mutation in the SOD1 gene. Our findings demonstrate the relevance of these non-cell-autonomous effects to human motor neurons and more broadly demonstrate the utility of human embryonic stem cells for studying disease and identifying potential therapeutics.
Williams syndrome (WS), caused by a heterozygous microdeletion on chromosome 7q11.23, is a neurodevelopmental disorder characterized by hypersociability and neurocognitive abnormalities. Of the ...deleted genes, general transcription factor IIi (Gtf2i) has been linked to hypersociability in WS, although the underlying mechanisms are poorly understood. We show that selective deletion of Gtf2i in the excitatory neurons of the forebrain caused neuroanatomical defects, fine motor deficits, increased sociability and anxiety. Unexpectedly, 70% of the genes with significantly decreased messenger RNA levels in the mutant mouse cortex are involved in myelination, and mutant mice had reduced mature oligodendrocyte cell numbers, reduced myelin thickness and impaired axonal conductivity. Restoring myelination properties with clemastine or increasing axonal conductivity rescued the behavioral deficits. The frontal cortex from patients with WS similarly showed reduced myelin thickness, mature oligodendrocyte cell numbers and mRNA levels of myelination-related genes. Our study provides molecular and cellular evidence for myelination deficits in WS linked to neuronal deletion of Gtf2i.
We showed previously that high‐quality crystals of bacteriorhodopsin (bR) from Halobacterium salinarum can be obtained from bicelle‐forming DMPC/CHAPSO mixtures at 37°C. As many membrane proteins are ...not sufficiently stable for crystallization at this high temperature, we tested whether the bicelle method could be applied at a lower temperature. Here we show that bR can be crystallized at room temperature using two different bicelle‐forming compositions: DMPC/CHAPSO and DTPC/CHAPSO. The DTPC/CHAPSO crystals grown at room temperature are essentially identical to the previous, twinned crystals: space group P21 with unit cell dimensions of a = 44.7 Å, b = 108.7 Å, c = 55.8 Å, β = 113.6°. The room‐temperature DMPC/CHAPSO crystals are untwinned, however, and belong to space group C2221 with the following unit cell dimensions: a = 44.7 Å, b = 102.5 Å, c = 128.2 Å. The bR protein packs into almost identical layers in the two crystal forms, but the layers stack differently. The new untwinned crystal form yielded clear density for a previously unresolved CHAPSO molecule inserted between protein subunits within the layers. The ability to grow crystals at room temperature significantly expands the applicability of bicelle crystallization.
Neuronal activity-dependent gene expression is essential for brain development. Although transcriptional and epigenetic effects of neuronal activity have been explored in mice, such an investigation ...is lacking in humans. Because alterations in GABAergic neuronal circuits are implicated in neurological disorders, we conducted a comprehensive activity-dependent transcriptional and epigenetic profiling of human induced pluripotent stem cell-derived GABAergic neurons similar to those of the early developing striatum. We identified genes whose expression is inducible after membrane depolarization, some of which have specifically evolved in primates and/or are associated with neurological diseases, including schizophrenia and autism spectrum disorder (ASD). We define the genome-wide profile of human neuronal activity-dependent enhancers, promoters and the transcription factors CREB and CRTC1. We found significant heritability enrichment for ASD in the inducible promoters. Our results suggest that sequence variation within activity-inducible promoters of developing human forebrain GABAergic neurons contributes to ASD risk.
Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and ...whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS.
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•iPSC-derived motor neurons harboring SOD1 mutations exhibit cell survival deficits•Genetic correction rescues ALS-related phenotypes•RNA-seq reveals expression changes and mitochondrial and ER stress disturbances•Motor neurons exhibit inherent ER stress linked to electrical activity
Motor neurons differentiated from human-ALS-patient-derived iPSCs were used to define transcriptional and functional changes arising from SOD1 mutations, which could be reversed by genome engineering of the SOD1 locus.
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