CLEC16A in astrocytes promotes mitophagy and limits pathology in a multiple sclerosis mouse model [Single Cell Dataset]

Astrocytes promote neuroinflammation and neurodegeneration in multiple sclerosis (MS) through cell-intrinsic activities and their ability to recruit and activate other cell types. Here, in a genome-wide CRISPR-based forward genetic screen investigating regulators of astrocyte proinflammatory responses, we identified the C-type lectin domain-containing 16A gene (CLEC16A), linked to MS susceptibility, as a suppressor of nuclear factor-?B (NF-?B) signaling. Gene and small-molecule perturbation studies in mouse primary and human embryonic stem cell-derived astrocytes in combination with mitochodrial, metabolic and multiomic analyses established that CLEC16A promotes mitophagy, limiting mitochondrial dysfunction and the accumulation of mitochondrial products that activate NF-?B, the NLRP3 inflammasome and gasdermin D. Astrocyte-specific Clec16a inactivation increased NF-?B, NLRP3 and gasdermin D activation in vivo, worsening experimental autoimmune encephalomyelitis, a mouse model of MS. Moreover, we detected disrupted mitophagic capacity and gasdermin D activation in astrocytes in samples from individuals with MS. These findings identify CLEC16A as a suppressor of astrocyte pathological responses and a candidate therapeutic target in MS. Scheduled for publication in Nature Neuroscience DOI: 10.1038/s41593-025-01875-9 Overall design: For single cell RNA-sequencing, CNS cells from n=3 Control EAE and n=3 CLEC16AGFAP EAE (Day 23) were hashed before FACS sorting using multiplex 3' Cell Plex Set Kit A (#100261, 10x Genomics). After sorting and washing, astrocytes from 6 samples were pooled and loaded into one well of a 10X Chromium Next GEM Chip G (10X Genomics). Single-cell libraries were further generated using the Single Cell 3' Reagent Kit v3.1 chemistry (10x Genomics) using Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (PN-1000128) according to the manufacturer's protocol. The generated scRNA-seq libraries were sequenced by NOVA-Seq S1 at the Broad Institute.