Tip60-mediated H2A.Z acetylation promotes neuronal fate specification and bivalent gene activation.

Cell lineage specification is accomplished by a concerted action of chromatin remodeling and tissue-specific transcription factors. However, the mechanisms that induce and maintain appropriate lineage-specific gene expression remain elusive. Here, we used an unbiased proteomics approach to characterize chromatin regulators that mediate the induction of neuronal cell fate. We found that Tip60 acetyltransferase is essential to establish neuronal cell identity partly via acetylation of the histone variant H2A.Z. Despite its tight correlation with gene expression and active chromatin, loss of H2A.Z acetylation had little effect on chromatin accessibility or transcription. Instead, loss of Tip60 and acetyl-H2A.Z interfered with H3K4me3 deposition and activation of a unique subset of silent, lineage-restricted genes characterized by a bivalent chromatin configuration at their promoters. Altogether, our results illuminate the mechanisms underlying bivalent chromatin activation and reveal that H2A.Z acetylation regulates neuronal fate specification by establishing epigenetic competence for bivalent gene activation and cell lineage transition.

HIV-1 infection of genetically engineered iPSC-derived central nervous system-engrafted microglia in a humanized mouse model.

IMPORTANCE: Our mouse model is a powerful tool for investigating the genetic mechanisms governing central nervous system (CNS) human immunodeficiency virus type-1 (HIV-1) infection and latency in the CNS at a single-cell level. A major advantage of our model is that it uses induced pluripotent stem cell-derived microglia, which enables human genetics, including gene function and therapeutic gene manipulation, to be explored in vivo, which is more challenging to study with current hematopoietic stem cell-based models for neuroHIV. Our transgenic tracing of xenografted human cells will provide a quantitative medium to develop new molecular and epigenetic strategies for reducing the HIV-1 latent reservoir and to test the impact of therapeutic inflammation-targeting drug interventions on CNS HIV-1 latency.

An induced pluripotent stem cell line carrying a silencing-resistant calcium reporter allele.

Calcium indicators are sensitive tools to image neural activity. However, their use in human induced pluripotent stem cells (iPSC)-derived neurons is limited by silencing of the transgene. We generated the iPSC line MSE2336A carrying heterozygous insertion in the safe-harbor locus AAVS1 of the ultrasensitive protein calcium sensor (GCaMP6) under the control of CAG promoter and UCOE to maintain robust transgene expression in differentiated cells. The iPSC exhibited normal cell morphology, expression of pluripotency markers, genome integrity, and the ability to differentiate into the three primary germ layers. This line provides a powerful model to study activity in human neurons.

Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.

Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.

Neuroligin-4 Regulates Excitatory Synaptic Transmission in Human Neurons.

The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.

Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling.

Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 (FMR1) gene, but how FMR1 inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional FMR1 mutations, we show here that loss of FMR1 function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. FMR1 inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the FMR1 gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by FMR1 mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.