Lingyin Li

SUMMARY: Neuroinflammation drives neurodegeneration and cognitive decline in aging, but the signals that spread this inflammation across the CNS remain unclear. Here, we show in mice that advanced age and experimental autoimmune encephalomyelitis (EAE) both result in elevated cytosolic mtDNA in microglia, which stimulates the production and export of cGAMP from these cells. Meanwhile, ENPP1, the dominant cGAMP hydrolase in the brain and spinal cord, is downregulated in both EAE mice and MS patients, releasing the brake on extracellular cGAMP accumulation. In Enpp1 ^H362A mice, which selectively lack cGAMP hydrolysis activity, unrestrained extracellular cGAMP worsens EAE-associated paralysis and drives premature aging phenotypes including impaired motor coordination (rotarod, pole test) and reduced exploration (open field test) in a manner dependent on STING. Single-cell RNA sequencing pinpoints microglia, astrocytes, and inhibitory neurons as the principal cGAMP responders that activate STING-dependent interferon-stimulated gene induction in response to elevated extracellular cGAMP. Mechanistically, microglia import cGAMP through the LRRC8A:C channel, while astrocytes and neurons import cGAMP via the glutamate antiporter system xc-, reinforced by SLC1A2-mediated glutamate uptake in astrocytes and SLC38A1/2-fueled glutamine-to-glutamate synthesis in neurons. In isolated primary astrocytes and neurons, high intracellular glutamate and system xc--driven redox demands favor cGAMP uptake, raising the possibility that neurotransmitter cycling and redox burden shape cellular vulnerability to cGAMP immunotransmission in vivo. Together, these findings position ENPP1 as a protective brake on neuroinflammation in aging and MS, nominating cGAS inhibition and CNS ENPP1 enhancement as therapeutic strategies.

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has long been linked with metabolic diseases, with the common ENPP1 K173Q (historically K121Q) variant conferring increased risk for type 2 diabetes (T2D). However, the mechanistic basis of this association has remained unclear. Here, we demonstrate that the K173Q variant has decreased cGAMP hydrolysis activity, suggesting that this loss of enzymatic function could contribute to its pathogenesis. Using a cGAMP-hydrolysis-deficient knock-in mouse ( Enpp1 ^H362A ), we show that selective loss of this activity leads to a primary defect in energy expenditure and exacerbates high-fat diet (HFD)-induced weight gain and insulin resistance. An unbiased in vivo glucose-uptake screen reveals brown adipose tissue (BAT) as a focal site of metabolic impairment, characterized by profound extracellular cGAMP accumulation and a selective failure of insulin-stimulated glucose uptake. Mechanistically, we demonstrate that nutrient excess drives mitochondrial DNA leakage in brown adipocytes, triggering cGAMP production and export. Excess cGAMP directly propagates STING-dependent suppression of glucose uptake and lipogenesis in brown adipocytes. Additionally, when ENPP1-mediated clearance is compromised, extracellular cGAMP acts as a paracrine immunotransmitter that remodels the BAT microenvironment by recruiting and polarizing macrophages toward an M1-like phenotype. Together, our findings nominate the impaired ENPP1-dependent buffering of extracellular cGAMP as one mechanism by which ENPP1 variants influence metabolic homeostasis.

Sh-RNA knockdown sequences used in the study

A. Terminal Tumor volume of MOC2 WT/KO-Gal1 tumors in Rag2-/- IL2rg-/- mice. B. Gating strategy used for analyses of PMN-MDSCs and M-MDSCs in tumor and lung tissues. C. Unsupervised tSNE clustering of live cells in MOC2 WT/KO-Gal1 tumor showing the proportion of M-MDSC, PMN-MDSC, and CD8+ T cells. D. Visual representation of the spleens isolated from either naïve mice or mice bearing WT- or KO-Gal1 tumors. E. Comparison of MDSC enrichment scores between smaller T1-2 and larger T3-4 tumors in the HNSCC TCGA cohort. The student’s t-test was used to assess the significance of the difference between the two groups. F. Heat map showing gene expression clustering based on the MDSC enrichment score in the HNSCC TCGA cohort. Differentially expressed genes (DEGs) were defined by a false discovery rate smaller than 0.05 and fold change higher than 1.5. G. Representative images showing low and high staining Gal1 or LOX1 (OLR1, established marker of PMN-MDSCs) using immunohistochemistry (IHC). H. Table showing the distribution of tumors expressing high or low Gal1 and high or low LOX1 (PMN-MDSCs) in 81 patients with oral cavity tumors. Gal1 and LOX1 staining distribution was analyzed using a chi-square test and was not statistically significant.

Immunoblot showing knockdown of STING in MOC2 WT/KO cells.

A. Quantification of PMN-MDSCs (CD11b+Ly6Clo/intLy6G+) in the liver and kidneys of MOC2 WT- or KO-Gal1 tumor-bearing mice at matched tumor volumes in comparison with naïve mice. B. Gating strategy used to quantify Arginase (Arg1) and interleukin-10 (IL-10) expression on PMN-MDSCs isolated from the tumor or the lungs of naïve mice or mice with pre-metastatic niches (sub-gated on CD45+ live cells). C. Heat map showing differential gene expression from PMN-MDSCs isolated from tumor and lung tissues of MOC2 WT-Gal1 tumor-bearing mice compared with naïve neutrophils. D. STRING analysis showing two clusters of genes representing IL-17 and collagen synthesis pathways that are upregulated PMN-MDSCs isolated from pre-metastatic lung tissues. E. Reactome pathway enrichment analyses performed using RNA-SEQ data showing pathway represented by upregulated genes in PMN-MDSCs isolated from pre-metastatic lung tissues. F. Quantification of Trichrome staining intensity in mEERL WT- or KO-Gal1 tumor-bearing mice lungs, n=5/group. G. tSNE plot showing depletion of PMN-MDSCs in the blood following injection of anti-GR1 antibody in mice compared with isotype alone.

A. Gating strategy for CD8+ T cells in the spleen of mice treated with either MOC2 WT/KO-Gal1 CM. B. Quantitation of PMN-MDSCs and T cells in the spleen of mice bearing KO-Gal1 tumors injected with either WT- or KO-Gal1 CM for 3 weeks. C. Graph showing the 48-plex mouse cytokine Luminex assay results performed on the 10x concentrated condition media from MOC2 WT/KO-Gal1 tumor cells. The media was collected in 3 independent experiments and run as a single batch. D. Gating strategy for CXCR2 staining on PMN-MDSCs isolated from the tumor and pre-metastatic lungs.

A. Quantification of phospho-p65 (NF-kb) staining in MOC2 and mEERL WT/KO-Gal1 tumors (N=5 tumors, 3 random microscopic fields per tumor). B. Quantitation of STING mRNA levels in MOC2 and mEERL WT/KO-Gal1 cell lines. C. Immunoblot showing degradation kinetics of STING in LLC and FaDU WT/KD-Gal1 cells at 1, 2, 4h post cycloheximide treatment. D. Magnified images showing co-localization of STING and LAMP1 in MOC2 WT/KO-Gal1 cells. E. Representative images showing low and high IHC staining of STING in oral cavity cancer (Left) and the distribution of negative and positive STING staining in 83 oral cavity cancers on the TMA. F. Kaplan Meier curve showing progression-free survival (calculated as the time from the surgery date until death or progression) of patients based on Gal1 and STING expression in the tumor.