| Project Detail |
Background and rationale: Viral infections and autoimmune diseases of the central nervous system (CNS) frequently result in long-lasting neurological deficits, which correlate best with the extent of neuronal alterations. These alterations manifest in various forms, but often include damage along neuronal processes affecting neuronal signal transmission. However, the underlying mechanisms that lead to lasting neuronal dysfunction in chronic inflammatory CNS diseases are only partially known. Overall objective: This project aims to gain insights into how the transcription factor regulatory networks and associated epigenetic remodeling lead to cellular dyshomeostasis and neuronal dysfunction in chronic neuroinflammatory diseases.Specific aims: Taking advantage of an experimental model of CNS neuroinflammation, we aim to elucidate the regulatory network operating in disease-associated phagocytes (aim 1) and address if neuronal long-lasting epigenetic alterations associated with persistent neurological impairments can be reverted (aim 2). We further aim to investigate epigenetic remodeling events in the gray matter of multiple sclerosis (MS) patients (aim 3).Methods to be used: For aims 1 and 2, we will harness a mouse viral encephalitis experimental model established in our laboratory. In aim 1, we will apply a computational method for simultaneous gene regulatory network reconstruction and cell-state identification (SCENIC) from single-cell RNA-sequencing (RNA-Seq) in microglia and monocyte-derived macrophages over the course of the disease. Predicted DNA regulatory elements and candidate transcription factors (TFs) will be corroborated by genome-wide chromatin accessibility using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq). The implication of identified TFs for phagocyte activation and their association with neuronal alterations in various disease stages will be corroborated by targeted gene ablation specifically in microglia or monocyte-derived inflammatory macrophages using conditional knock-out mouse lines of tamoxifen-inducible phagocyte-specific genes. In aim 2, we will harness transgenic mice with CRISPR-based systems in combination with a guide RNA expressing cassette delivered via adeno-associated virus (AAV), which allows temporally controlled and neuronal-restricted in vivo epigenome editing of long-lasting epigenetically remodeled neuronal gene loci revealed by ATAC-Seq and RNA-Seq. For aim 3, we will perform fluorescence-activated nuclear sorting (FANS) on human postmortem cortex tissue from multiple sclerosis (MS) and matched control cases to isolate neuronal and non-neuronal subsets that will be profiled with DNA methylation microarray (Infinium MethylationEPIC BeadChip Array, Illumina) and chromatin immunoprecipitation and sequencing technology (ChIP-Seq) and RNA-Seq. In aims 1 to 3, our analysis will be complemented by quantitative multiplexed immunohistochemistry and in situ hybridization approaches. Expected results: This project will provide new insights into cell-intrinsic and intercellular effects underlying long-lasting neuronal damage. Specifically, we will gain an understanding of the regulatory network underpinning aberrant phagocyte activation and intrinsic neuronal alterations associated with long-lasting synaptic pathology in neuroinflammatory conditions. Furthermore, our project will discern the relationship between epigenetic and transcriptomic alterations occurring in the different cellular subsets of the gray matter of MS patients. Impact: A better understanding of the molecular mechanisms associated with cellular dyshomeostasis and the resulting long-lasting neuronal changes may inform us about new, potentially actionable therapeutic targets to alleviate chronic clinical disability in neuroinflammatory diseases in the future. |
