S1P Lyase Deficiency in the Brain Promotes Astrogliosis and NLRP3 Inflammasome Activation via Purinergic Signaling.

Astrocytes are critical players in brain health and disease. Brain pathologies and lesions are usually accompanied by astroglial alterations known as reactive astrogliosis. Sphingosine 1-phosphate lyase (SGPL1) catalysis, the final step in sphingolipid catabolism, irreversibly cleaves its substrate sphingosine 1-phosphate (S1P). We have shown that neural ablation of SGPL1 causes accumulation of S1P and hence neuronal damage, cognitive deficits, as well as microglial activation. Moreover, the S1P/S1P-receptor signaling axis enhances ATP production in SGPL1-deficient astrocytes. Using immunohistochemical methods as well as RNA Seq and CUT&Tag we show how S1P signaling causes activation of the astrocytic purinoreceptor P2Y1 (P2Y1R). With specific pharmacological agonists and antagonists, we uncover the P2Y1R as the key player in S1P-induced astrogliosis, and DDX3X mediated the activation of the NLRP3 inflammasome, including caspase-1 and henceforward generation of interleukin-1ß (IL-1ß) and of other proinflammatory cytokines. Our results provide a novel route connecting S1P metabolism and signaling with astrogliosis and the activation of the NLRP3 inflammasome, a central player in neuroinflammation, known to be crucial for the pathogenesis of numerous brain illnesses. Thus, our study opens the door for new therapeutic strategies surrounding S1P metabolism and signaling in the brain.

Sphingosine Kinases at the Intersection of Pro-Inflammatory LPS and Anti-Inflammatory Endocannabinoid Signaling in BV2 Mouse Microglia Cells.

Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and cis-vaccenic acid ethanolamide, palmitoylethanolamide, and docosahexaenoyl ethanolamide. Furthermore, treatment with JWH133, a selective agonist of the eCB-binding cannabinoid 2 (CB2) receptor, mimicked the anti-inflammatory effects of URB597. Interestingly, LPS induced transcription of both SphK1 and SphK2, and the selective inhibitors of SphK1 (SLP7111228) and SphK2 (SLM6031434) strongly reduced LPS-induced TNFα and IL-1ß production. Thus, the two SphKs were pro-inflammatory in BV2 cells in a non-redundant manner. Most importantly, the inhibition of FAAH by URB597, as well as the activation of CB2 by JWH133, prevented LPS-stimulated transcription of SphK1 and SphK2. These results present SphK1 and SphK2 at the intersection of pro-inflammatory LPS and anti-inflammatory eCB signaling, and suggest the further development of inhibitors of FAAH or SphKs for the treatment of neuroinflammatory diseases.