Discovery of a Novel Subset of Pro-Inflammatory Microglia in Amyotrophic Lateral Sclerosis
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CitationMifflin, Lauren. 2018. Discovery of a Novel Subset of Pro-Inflammatory Microglia in Amyotrophic Lateral Sclerosis. Master's thesis, Harvard Medical School.
AbstractMicroglia-derived inflammation or immune processes have been linked to a broad range of neurodegenerative and neuropsychiatric conditions, including multiple sclerosis, amyotrophic lateral sclerosis (ALS), Alzheimer’s Disease (AD), Parkinson’s Disease (PD), stroke, autism, and schizophrenia. In several of these human diseases, TNFα pro-inflammatory signaling has been identified as a pathological signature involved in mediating damage and cell death in the central nervous system (CNS). These deleterious effects have been shown to be primarily enacted through the TNF receptor 1 (TNFR1), which contains an intracellular death domain.
There are several downstream signaling molecules conserved between TNFα production through the NF-κB pathway and cell death pathways activated when TNFα binds to TNFR1. One such signaling molecule is RIP Kinase 1 (RIPK1), a kinase which, when genetically or therapeutically inactivated, can inhibit multiple forms of cell death and diminish TNFα-mediated inflammation. Small molecules can be highly effective kinase inhibitors, and necrostatin-1 (Nec-1s) is a unique RIPK1 inhibitor which can traverse the BBB, making it well suited for investigation in diseases with neuroinflammatory pathology.
Based on early success of RIPK1 inhibition genetically and pharmacologically in animal models of ALS and AD, a derivative of Nec-1s entered Phase I clinical trials in October 2016. However, it remains unclear in these diseases the extent to which RIPK1 inhibition is protecting from cell death in certain susceptible subtypes versus tuning the pro-inflammatory state of microglia and astrocytes. Advances in single-cell RNA sequencing (scRNASeq) allow simultaneous profiling of all CNS cells in disease models to more clearly elucidate the therapeutic benefit of RIPK1 inhibition to diminish TNFα-mediated inflammation.
In this project, we identify a novel subcluster of microglia expressing high levels of pro-inflammatory genes and upregulated in multiple mouse models of ALS using scRNASeq. These microglia are TNFα responsive and can be inhibited by Nec-1s treatment. Using multiplexed single molecule fluorescence in situ hybridization (smFISH), immunohistochemistry, and flow cytometric analysis, we confirm the presence of these novel microglia and expand our findings to additional mouse models of ALS. Collectively, this data provides a mechanistic rationale for the use of therapeutic RIPK1 inhibition in the treatment of ALS across multiple genetic and sporadic disease-causing variants.
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