CLEC16A-driven regulation of astrocytes

Brigham And Women'S Hospital, Boston MA

Investigators

Abstract

PROJECT SUMMARY Astrocytes are abundant cells of the central nervous system (CNS), with important roles in neurologic diseases such as multiple sclerosis (MS) and its model experimental autoimmune encephalomyelitis (EAE). Although mechanisms driving astrocyte pathogenic activities and subsets have been identified, less is known about the mechanisms that suppress them. In preliminary studies aimed to define astrocyte-intrinsic regulatory mechanisms we made the following observations: 1) A genome-wide CRISPR/Cas9 screen identified Clec16a, which has been linked to MS in genetic studies1,2, as a suppressor of NF-kB- and NLRP3 inflammasome-driven pathogenic astrocyte responses; 2) Genetic and pharmacologic studies established that mitophagy driven by CLEC16A expressed in astrocytes (CLEC16AAST) limits the accumulation of mitochondrial products that activate NF-kB and the NLRP3 inflammasome; 3) CLEC16AAST inactivation results in the worsening EAE and the expansion of pathogenic ACLY+ p300+ astrocytes that display increased NF-kB activation and IL-1b production; 4) CLEC16AAST inactivation boosts microglial pro-inflammatory responses in EAE; 5) A CNS-penetrant NLRP3 inhibitor abrogates the worsening of EAE induced by CLEC16AAST inactivation; and 6) CLEC16A and mitophagy are decreased in astrocytes in MS, while NLRP3 activation is upregulated. These are important findings because most MS-associated genes have so far been linked to the control of immune cells, but not astrocytes. In addition, mitophagy deficits in neurons, but not astrocytes, were previously linked to neurologic disorders. Finally, little is known about the role of the NLRP3 inflammasome in the control of astrocyte responses and subsets. Based on these findings, we hypothesize that CLEC16AAST limits NF-kB- and NLRP3 inflammasome-driven pathogenic astrocyte responses in CNS inflammation. Thus, we propose to study the regulation of astrocyte responses by this novel CLEC16A-NF-kB-NLRP3 inflammasome signaling axis. Specifically, we propose to: 1) Establish the effects of CLEC16AAST on pathologic astrocyte responses in EAE and MS; 2) Perform transcriptomic and epigenetic studies to define subsets of astrocytes and other cells controlled directly and indirectly by CLEC16AAST and the mechanisms involved; 3) Use genetic barcoding and cell-specific gene perturbation approaches to identify the effects of CLEC16AAST on astrocyte interactions with other cells and the mechanisms of cell-cell communication involved; 4) Map the CNS location of cell subsets and interactions controlled by CLEC16AST using in situ transcriptomics, immunofluorescence and single-cell RNA-seq datasets; and 5) Use CNS-penetrant NLRP3 inhibitors in combination with CLEC16AAST deficient mice and astrocyte-specific in vivo CRISPR/Cas9- driven gene perturbations to define the role of the NLRP3 inflammasome in the control of astrocyte responses and subsets, and its potential for the therapeutic management of astrocyte-driven CNS pathology. In summary, this project investigates the role of a novel CLEC16AAST-NF-kB-NLRP3 inflammasome signaling axis in the control of astrocyte pathogenic activities and subsets, and its potential as a therapeutic target for MS.