Background: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, and the resulting cyclic dinucleotide, 2′3-cGAMP, binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS-STING pathway in human neurodegenerative diseases. Methods: Post-mortem CNS tissue from donors with multiple sclerosis (n = 4), Alzheimer’s disease (n = 6), Parkinson’s disease (n = 3), amyotrophic lateral sclerosis (n = 3) and non-neurodegenerative controls (n = 11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-β, α-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1–400 μM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarker interferon-β release and changes in ICAM-1 integrin expression. Results: In neurodegenerative brain diseases, elevated STING protein was observed mainly in brain endothelial cells and neurons, compared to non-neurodegenerative control tissues where STING protein staining was weaker. Interestingly, a higher STING presence was associated with toxic protein aggregates (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander’s coefficient; p < 0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS and cell surface ICAM. In addition, a dose response in the secretion of interferon-β was observed, but it failed to reach statistical significance. Conclusions: The histological evidence shows that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data, this suggests that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage, resulting in downstream neuroinflammation; hence, this pathway may be a target for future STING therapeutics.

Background: Some neurodegenerative diseases have an element of neuroinflammation that is triggered by viral nucleic acids, resulting in the generation of type I interferons. In the cGAS-STING pathway, microbial and host-derived DNA bind and activate the DNA sensor cGAS, the resulting cyclic dinucleotide, 2’3-cGAMP binds to a critical adaptor protein, stimulator of interferon genes (STING), which leads to activation of downstream pathway components. However, there is limited work demonstrating the activation of the cGAS- STING pathway in human neurodegenerative diseases. Methods: Post-mortem CNS tissue from donors with multiple sclerosis (n=4), Alzheimer's diseases (n=6) and Parkinson's disease (n=3), amyotrophic lateral sclerosis (n=3) and non-neurodegenerative controls (n=11) were screened by immunohistochemistry for STING and relevant protein aggregates (e.g., amyloid-b, a-synuclein, TDP-43). Human brain endothelial cells were cultured and stimulated with the STING agonist palmitic acid (1-400µM) and assessed for mitochondrial stress (release of mitochondrial DNA into cytosol, increased oxygen consumption), and downstream regulator factors, TBK-1/pIRF3 and inflammatory biomarkers interferon-β release and changes ICAM-1 integrin expression. Results: In neurodegenerative brain, elevated STING protein was observed mainly in brain endothelial cells and neurons compared to non-neurodegenerative control tissues where STING protein staining was weaker in comparison. Interestingly, higher STING presence was associated with toxic protein aggregates. (e.g., in neurons). Similarly high STING protein levels were observed within acute demyelinating lesions in multiple sclerosis subjects. To understand non-microbial/metabolic stress activation of the cGAS-STING pathway, brain endothelial cells were treated with palmitic acid. This evoked mitochondrial respiratory stress up to a ~2.5-fold increase in cellular oxygen consumption. Palmitic acid induced a statistically significant increase in cytosolic DNA leakage from endothelial cell mitochondria (Mander’s coefficient; P <0.05) and a significant increase in TBK-1, phosphorylated transcription factor IFN regulatory factor 3, cGAS, cell surface ICAM. In addition, a dose response in secretion of interferon-b was observed but failed to reach statistical significance. Conclusions: The histological evidence show that the common cGAS-STING pathway appears to be activated in endothelial and neural cells in all four neurodegenerative diseases examined. Together with the in vitro data suggest that the STING pathway might be activated via perturbation of mitochondrial stress and DNA leakage resulting in downstream neuroinflammation hence this pathway may be a target for future STING therapeutics.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood–brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood–brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.