Failure of multiple sclerosis (MS) lesions to resolve in the months after they form leads to smouldering demyelination and axon degeneration, identifiable in vivo as paramagnetic rim lesions on MRI. [1][2][3] To define mechanisms underlying this disabling, progressive neurodegenerative state, 4-6 and to foster development of new therapeutics, we used MRI-informed single-nucleus RNA sequencing to profile the edge of demyelinated white matter (WM)

Oligodendrocyte precursor cells (OPCs) are abundant in the adult central nervous system, and have the capacity to regenerate oligodendrocytes and myelin. However, in inflammatory diseases such as multiple sclerosis (MS) remyelination is often incomplete. To investigate how neuroinflammation influences OPCs, we perform in vivo fate-tracing in an inflammatory demyelinating mouse model. Here we report that OPC differentiation is inhibited by both effector T cells and IFNγ overexpression by astrocytes. IFNγ also reduces the absolute number of OPCs and alters remaining OPCs by inducing the immunoproteasome and MHC class I. In vitro, OPCs exposed to IFNγ cross-present antigen to cytotoxic CD8 T cells, resulting in OPC death. In human demyelinated MS brain lesions, but not normal appearing white matter, oligodendroglia exhibit enhanced expression of the immunoproteasome subunit PSMB8. Therefore, OPCs may be co-opted by the immune system in MS to perpetuate the autoimmune response, suggesting that inhibiting immune activation of OPCs may facilitate remyelination.

Summary Paralytic polio once afflicted almost half a million children each year. The attenuated oral polio vaccine (OPV) has enabled world-wide vaccination efforts, which resulted in nearly complete control of the disease. However, poliovirus eradication is hampered globally by epidemics of vaccine-derived polio. Here, we describe a combined theoretical and experimental strategy that describes the molecular events leading from OPV to virulent strains. We discover that similar evolutionary events occur in most epidemics. The mutations and the evolutionary trajectories driving these epidemics are replicated using a simple cell-based experimental setup where the rate of evolution is intentionally accelerated. Furthermore, mutations accumulating during epidemics increase the replication fitness of the virus in cell culture and increase virulence in an animal model. Our study uncovers the evolutionary strategies by which vaccine strains become pathogenic, and provides a powerful framework for rational design of safer vaccine strains and for forecasting virulence of viruses.

While neuroinflammation is an evolving concept and the cells involved and their functions are being defined, microglia are understood to be a key cellular mediator of brain injury and repair. The ability to measure microglial activity specifically and noninvasively would be a boon to the study of neuroinflammation, which is involved in a wide variety of neuropsychiatric disorders including traumatic brain injury, demyelinating disease, Alzheimer’s disease (AD), and Parkinson’s disease, among others. We have developed [11C]CPPC [5-cyano-N-(4-(4-[11C]methylpiperazin-1-yl)-2-(piperidin-1-yl)phenyl)furan-2-carboxamide], a positron-emitting, high-affinity ligand that is specific for the macrophage colony-stimulating factor 1 receptor (CSF1R), the expression of which is essentially restricted to microglia within brain. [11C]CPPC demonstrates high and specific brain uptake in a murine and nonhuman primate lipopolysaccharide model of neuroinflammation. It also shows specific and elevated uptake in a murine model of AD, experimental allergic encephalomyelitis murine model of demyelination and in postmortem brain tissue of patients with AD. Radiation dosimetry in mice indicated [11C]CPPC to be safe for future human studies. [11C]CPPC can be synthesized in sufficient radiochemical yield, purity, and specific radioactivity and possesses binding specificity in relevant models that indicate potential for human PET imaging of CSF1R and the microglial component of neuroinflammation.

c RNA polymerase II (Pol II) and the pausing complex, NELF and DSIF, are detected near the transcription start site (TSS) of many active and silent genes. Active transcription starts when the pause release factor P-TEFb is recruited to initiate productive elongation. However, the mechanism of P-TEFb recruitment and regulation of NELF/DSIF during transcription is not fully understood. We investigated this question in interferon (IFN)-stimulated transcription, focusing on BRD4, a BET family protein that interacts with P-TEFb. Besides P-TEFb, BRD4 binds to acetylated histones through the bromodomain. We found that BRD4 and P-TEFb, although not present prior to IFN treatment, were robustly recruited to IFN-stimulated genes (ISGs) after stimulation. Likewise, NELF and DSIF prior to stimulation were hardly detectable on ISGs, which were strongly recruited after IFN treatment. A shRNA-based knockdown assay of NELF revealed that it negatively regulates the passage of Pol II and DSIF across the ISGs during elongation, reducing total ISG transcript output. Analyses with a BRD4 small-molecule inhibitor showed that IFN-induced recruitment of P-TEFb and NELF/DSIF was under the control of BRD4. We suggest a model where BRD4 coordinates both positive and negative regulation of ISG elongation.

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Recent studies of the histone H3.3 variant indicate that it is incorporated into nucleosomal chromatin in association with active gene expression (1-4). Although other H3 variants, H3.1 and H3.2, are synthesized predominantly in S phase and are deposited onto newly replicated DNA, H3.3 is synthesized throughout the cell cycle, independent of DNA replication. Replication-independent incorporation of H3.3 is mediated by the HIRA complex, through a mechanism distinct from that of replication-dependent deposition of H3.1 mediated by the CAF1 complex (5). In Drosophila as well as in mammalian cells, H3.3 is enriched in nucleosomes carrying post-translational modification patterns characteristic of active transcription (6, 7). On the other hand, H3K9 dimethylation, typically seen in transcriptionally repressed chromatin, is scarce in H3.3. Histone H3.3 is accumulated in the transcriptionally active ribosomal DNA array in the Drosophila nucleus (8). A genome-wide analysis of H3.3 distribution patterns showed that H3.3 is distributed predominantly over regions of active genes and is enriched in the promoter regions coinciding with methylated Lys-4 in H3 and abundant RNA polymerase II binding (9). These studies led to a proposition that H3.3 marks active chromatin and may be involved in the epigenetic maintenance of chromatin status (3, 10 -12). Evidence supporting the role of H3.3 in the inheritance of activated gene status was recently presented by nuclear transplantation experiments in Xenopus (13).It has been shown that H3.3 replacement is triggered upon transcriptional activation of the HSP 70 genes in Drosophila (14). In that study, H3.3 deposition began within minutes of heat shock stimulation. This induced deposition coinciding with chromosomal puffs, providing an immediate link between transcription and H3.3 enrichment. Further supporting transcription-coupled H3.3 deposition, Janicki et al. (15) Despite these studies, there are questions that have remained uncertain regarding H3.3 incorporation. For example, it is unclear whether transcriptional activation is a prerequisite of H3.3 deposition. Also unclear is the stability of transcriptioninduced H3.3 deposition as well as the spatial patterns of H3.3 incorporation within induced genes. In addition, the biological significance of transcription-coupled H3.3 enrichment has remained elusive.With respect to the sites of H3.3 enrichment within a gene, widely varied results are reported for vertebrate cells, ranging from promoter-biased H3.3 incorporation to broader distribution patterns that include coding regions (16,17). A study by Jin and Felsenfeld (18) on chicken erythroid cells concluded that there is no straightforward correlation between gene expression and H3.3 enrichment.In this report, we have studied interferon (IFN)-dependent transcription as a model to address signal-induced H3.3

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