Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis.

Neurons are overproduced during cerebral cortical development. Neural progenitor cells (NPCs) divide rapidly and incur frequent DNA double-strand breaks (DSBs) throughout cortical neurogenesis. Although half of the neurons born during neurodevelopment die, many neurons with inaccurate DNA repair survive leading to brain somatic mosaicism. Recurrent DNA DSBs during neurodevelopment are associated with both gene expression level and gene length. We used imaging flow cytometry and a genome-wide DNA DSB capture approach to quantify and map DNA DSBs during human induced pluripotent stem cell (hiPSC)-based neurogenesis. Reduced p53 signaling was brought about by knockdown (p53KD); p53KD led to elevated DNA DSB burden in neurons that was associated with gene expression level but not gene length in neural progenitor cells (NPCs). Furthermore, DNA DSBs incurred from transcriptional, but not replicative, stress lead to p53 activation in neurotypical NPCs. In p53KD NPCs, DNA DSBs accumulate at transcription start sites of genes that are associated with neurological and psychiatric disorders. These findings add to a growing understanding of how neuronal genome dynamics are engaged by high transcriptional or replicative burden during neurodevelopment.

Loss of Coiled-Coil Protein Cep55 Impairs Neural Stem Cell Abscission and Results in p53-Dependent Apoptosis in Developing Cortex.

To build the brain, embryonic neural stem cells (NSCs) tightly regulate their cell divisions, undergoing a polarized form of cytokinesis that is poorly understood. Cytokinetic abscission is mediated by the midbody to sever the daughter cells at the apical membrane. In cell lines, the coiled-coil protein Cep55 was reported to be required for abscission. Mutations of Cep55 in humans cause a variety of cortical malformations. However, its role in the specialized divisions of NSCs is unclear. Here, we elucidate the roles of Cep55 in abscission and brain development. KO of Cep55 in mice causes abscission defects in neural and non-neural cell types, and postnatal lethality. The brain is disproportionately affected, with severe microcephaly at birth. Quantitative analyses of abscission in fixed and live cortical NSCs show that Cep55 acts to increase the speed and success rate of abscission, by facilitating ESCRT recruitment and timely microtubule disassembly. However, most NSCs complete abscission successfully in the absence of Cep55 Those that fail show a tissue-specific response: binucleate NSCs and neurons elevate p53, but binucleate fibroblasts do not. This leads to massive apoptosis in the brain, but not other tissues. Double KO of both p53 and Cep55 blocks apoptosis but only partially rescues Cep55-/- brain size. This may be because of the persistent NSC cell division defects and p53-independent premature cell cycle exit. This work adds to emerging evidence that abscission regulation and error tolerance vary by cell type and are especially crucial in neural stem cells as they build the brain.SIGNIFICANCE STATEMENT During brain growth, embryonic neural stem cells (NSCs) must divide many times. In the last step of cell division, the daughter cell severs its connection to the mother stem cell, a process called abscission. The protein Cep55 is thought to be essential for recruiting proteins to the mother-daughter cell connection to complete abscission. We find that Cep55 mutants have very small brains with disturbed structure, but almost normal size bodies. NSC abscission can occur, but it is slower than normal, and failures are increased. Furthermore, NSCs that do fail abscission activate a signal for programmed cell death, whereas non-neural cells do not. Blocking this signal only partly restores brain growth, showing that regulation of abscission is crucial for brain development.

Label-Free Quantification of Cell Cycle Synchronicity of Human Neural Progenitor Cells Based on Electrophysiology Phenotypes.

The ability to coax human-induced pluripotent stem cells (hiPSCs) into human neural progenitor cells (hNPCs) can lead to novel drug discovery and transplant therapy platforms for neurological diseases. Since hNPCs can form organoids that mimic brain development, there is emerging interest in their label-free characterization for controlling cell composition to optimize organoid formation in three-dimensional (3D) cultures. However, this requires the ability to quantify hNPCs in heterogeneous samples with subpopulations of similar phenotype. Using high-throughput (>6000 cells per condition), single-cell impedance cytometry, we present the utilization of electrophysiology for quantification of hNPC subpopulations that are altered in cell cycle synchronicity by camptothecin (CPT) exposure. Electrophysiology phenotypes are determined from impedance magnitude and phase metrics for distinguishing each cell cycle phase, as validated by flow cytometry, for a wide range of subpopulation proportions. Using multishell dielectric models for each cell cycle phase, electrophysiology alterations with CPT dose could be predicted. This label-free detection strategy can prevent loss of cell viability to speed the optimization of cellular compositions for organoid development.

Imaging Flow Cytometry Quantifies Neural Genome Dynamics.

Somatic mosaicism is a common consequence of normal development. DNA repair is simply not perfect, and each cell's genome incurs continuous DNA damage as a consequence of transcription, replication, and other cell biological stressors. Brain somatic mosaicism is particularly noteworthy because the vast majority of an individual's neurons are with that individual for life and neural circuits give rise directly to behavioral phenotypes. Brain somatic mosaicism, now revealed and tractable due to advances in single cell 'omic approaches, has emerged as an intriguing and unexplored aspect of neuronal diversity. Furthermore, the study of DNA damage during early neurodevelopment, when the rate of mutagenesis is high, is the perfect starting point to understand the origins of brain mosaicism. Flow cytometry is a highly efficient technique to study cell cycle and intracellular proteins of interest, particularly those related to DNA damage, but it lacks the high resolution of microscopy to examine the localization of these proteins. In this study, we outline a novel single-cell approach to quantify DNA double-strand break (DNA DSB) dynamics during early human neurodevelopment by applying imaging flow cytometry (IFC) to human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) undergoing neurogenesis. We establish an increase of DNA DSBs by quantifying γH2AX foci in mildly stressed NPCs using various single-cell approaches in addition to IFC including fluorescent microscopy, conventional flow cytometry, and measuring DNA DSBs with the comet assay. We demonstrate the dose-dependent sensitive detection of γH2AX foci through IFC and reveal the dynamics of DNA DSBs in proliferating and differentiating neural cells in early neurogenesis. © 2019 International Society for Advancement of Cytometry.

The effect of rho kinase inhibition on morphological and electrophysiological maturity in iPSC-derived neurons.

Induced pluripotent stem cell (iPSC)-derived neurons permit the study of neurogenesis and neurological disease in a human setting. However, the electrophysiological properties of iPSC-derived neurons are consistent with those observed in immature cortical neurons, including a high membrane resistance depolarized resting membrane potential and immature firing properties, limiting their use in modeling neuronal activity in adult cells. Based on the proven association between inhibiting rho kinase (ROCK) and increased neurite complexity, we seek to determine if short-term ROCK inhibition during the first 1-2 weeks of differentiation would increase morphological complexity and electrophysiological maturity after several weeks of differentiation. While inhibiting ROCK resulted in increased neurite formation after 24 h, this effect did not persist at 3 and 6 weeks of age. Additionally, there was no effect of ROCK inhibition on electrophysiological properties at 2-3, 6, or 12 weeks of age, despite an increase in evoked and spontaneous firing and a more hyperpolarized resting membrane potential over time. These results indicate that while there is a clear effect of time on electrophysiological maturity, ROCK inhibition did not accelerate maturity.

Single-cell analysis of diversity in human stem cell-derived neurons.

Neural stem and progenitor cells produce one of the most remarkable organs in nature, the human brain. Among neural stem cell progeny, post-mitotic neurons are likewise remarkably diverse. Single-cell transcriptomic approaches are now cataloging a long-sought-after molecular taxonomy of neuronal diversity in the brain. Contemporary single-cell omic classifications of neuronal diversity build from electrophysiological approaches that for decades have measured and cataloged diverse biophysical properties of single neurons. With the widespread application of human pluripotent stem cell-based models of neurogenesis to investigate disease pathology and to develop new drugs, a high-resolution understanding of neuronal diversity in vivo is essential to benchmark the state of in vitro models of human neurological disease.

Dysregulation of Prefrontal Cortex-Mediated Slow-Evolving Limbic Dynamics Drives Stress-Induced Emotional Pathology.

Circuits distributed across cortico-limbic brain regions compose the networks that mediate emotional behavior. The prefrontal cortex (PFC) regulates ultraslow (<1 Hz) dynamics across these networks, and PFC dysfunction is implicated in stress-related illnesses including major depressive disorder (MDD). To uncover the mechanism whereby stress-induced changes in PFC circuitry alter emotional networks to yield pathology, we used a multi-disciplinary approach including in vivo recordings in mice and chronic social defeat stress. Our network model, inferred using machine learning, linked stress-induced behavioral pathology to the capacity of PFC to synchronize amygdala and VTA activity. Direct stimulation of PFC-amygdala circuitry with DREADDs normalized PFC-dependent limbic synchrony in stress-susceptible animals and restored normal behavior. In addition to providing insights into MDD mechanisms, our findings demonstrate an interdisciplinary approach that can be used to identify the large-scale network changes that underlie complex emotional pathologies and the specific network nodes that can be used to develop targeted interventions.

Prefrontal cortex reactivity underlies trait vulnerability to chronic social defeat stress.

Psychological stress contributes to the onset and exacerbation of nearly all neuropsychiatric disorders. Individual differences in stress-regulatory circuits can therefore dramatically affect vulnerability to these illnesses. Here we identify neural circuit mechanisms underlying individual differences in vulnerability to stress using a murine model of chronic social defeat stress. In chronically stressed mice, we find that the degree of prefrontal cortex (PFC) control of amygdala activity predicts stress susceptibility in individual mice. Critically, we also find that individual differences in PFC activation (that is, reactivity) during exposure to an aggressor mouse predict the emergence stress-induced behavioural deficits in stress-naïve mice. Finally, we show that naturally occurring differences in PFC reactivity directly correspond to the intrinsic firing rate of PFC neurons. This demonstrates that naturally occurring differences in PFC function underlie individual differences in vulnerability to stress, raising the hypothesis that PFC modulation may prevent stress-induced psychiatric disorders.

Gαz regulates BDNF-induction of axon growth in cortical neurons.

The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles--predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation--suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.