Stem cell-derived brainstem mouse astrocytes obtain a neurotoxic phenotype in vitro upon neuroinflammation.

BACKGROUND: Astrocytes respond to injury and disease through a process known as reactive astrogliosis, of which inflammatory signaling is one subset. This inflammatory response is heterogeneous with respect to the inductive stimuli and the afflicted central nervous system region. This is of plausible importance in e.g. traumatic axonal injury (TAI), where lesions in the brainstem carries a particularly poor prognosis. In fact, astrogliotic forebrain astrocytes were recently suggested to cause neuronal death following axotomy. We therefore sought to assess if ventral brainstem- or rostroventral spinal astrocytes exert similar effects on motor neurons in vitro. METHODS: We derived brainstem/rostroventral spinal astrocyte-like cells (ES-astrocytes) and motor neurons using directed differentiation of mouse embryonic stem cells (ES). We activated the ES-astrocytes using the neurotoxicity-eliciting cytokines interleukin- (IL-) 1α and tumor necrosis factor-(TNF-)α and clinically relevant inflammatory mediators. In co-cultures with reactive ES-astrocytes and motor neurons, we assessed neurotoxic ES-astrocyte activity, similarly to what has previously been shown for other central nervous system (CNS) regions. RESULTS: We confirmed the brainstem/rostroventral ES-astrocyte identity using RNA-sequencing, immunocytochemistry, and by comparison with primary subventricular zone-astrocytes. Following cytokine stimulation, the c-Jun N-terminal kinase pathway down-stream product phosphorylated c-Jun was increased, thus demonstrating ES-astrocyte reactivity. These reactive ES-astrocytes conferred a contact-dependent neurotoxic effect upon co-culture with motor neurons. When exposed to IL-1ß and IL-6, two neuroinflammatory cytokines found in the cerebrospinal fluid and serum proteome following human severe traumatic brain injury (TBI), ES-astrocytes exerted similar effects on motor neurons. Activation of ES-astrocytes by these cytokines was associated with pathways relating to endoplasmic reticulum stress and altered regulation of MYC. CONCLUSIONS: Ventral brainstem and rostroventral spinal cord astrocytes differentiated from mouse ES can exert neurotoxic effects in vitro. This highlights how neuroinflammation following CNS lesions can exert region- and cell-specific effects. Our in vitro model system, which uniquely portrays astrocytes and neurons from one niche, allows for a detailed and translationally relevant model system for future studies on how to improve neuronal survival in particularly vulnerable CNS regions following e.g. TAI.

GPSeq reveals the radial organization of chromatin in the cell nucleus.

With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius. GPSeq relies on gradual restriction digestion of chromatin from the nuclear lamina toward the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100-kb resolution, which revealed radial patterns of genomic and epigenomic features and gene expression, as well as A and B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of whole-genome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations and found that they have distinctive radial arrangements in A and B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.