SorCS2 Is Important for Astrocytic Function in Neurovascular Signaling

Introduction: The receptor SorCS2 is involved in the trafficking of membrane receptors and transporters. It has been implicated in brain disorders and has previously been reported to be indispensable for ionotropic glutamatergic neurotransmission in the hippocampus. Aim: We aimed to study the role of SorCS2 in the control of astrocyte-neuron communication, critical for neurovascular coupling. Methods: Brain slices from P8 and 2-month-old wild-type and SorCS2 knockout (Sorcs2−/−) mice were immunostained for SorCS2, GFAP, AQP4, IB4, and CD31. Neurovascular coupling was assessed in vivo using laser speckle contrast imaging and ex vivo in live brain slices loaded with calcium-sensitive dye. Bulk and cell surface fraction proteomics was analyzed on freshly isolated and cultured astrocytes, respectively, and validated with Western blot and qPCR. Results: SorCS2 was strongly expressed in astrocytes, primarily in their endfeet, of P8 mice; however, it was sparsely repre-sented in 2-month-old mice. Sorcs2−/− mice demonstrated reduced neurovascular coupling associated with a reduced astrocytic calcium response to neuronal excitation. No differences in vascularization or endothelium-dependent relaxation ex vivo between the 2-month-old groups were observed. Proteomics suggested changes in glutamatergic signaling and suppressed calcium sign-aling in Sorcs2−/− brains from both P8 and 2-month-old mice. The increased abundance of glutamate metabotropic receptor 3 in Sorcs2−/− astrocytes was validated by PCR and Western blot. In cultured Sorcs2−/− astrocytes, AQP4 abundance was increased in the bulk lysate but reduced in the cell surface fraction, suggesting impaired trafficking

Spatial Transcriptomics and Proteomics Profiling after Ischemic Stroke Reperfusion:Insights into Vascular Alterations

BACKGROUND: More than half of patients with ischemic stroke experience futile reperfusion, increasing the risk of death and disabilities despite a successful recanalization. The reason behind this is debated, and we aim to investigate cerebrovascular changes toward a broader understanding of these conditions. We hypothesize that ischemic stroke reperfusion modifies the expression profile in the microvasculature in a spatial manner toward peri-infarct brain edema and circulatory failure. METHODS: We investigated the early (24-hour) changes in spatial gene expression in the brain parenchymal endothelial cells and mural cells following ischemia stroke reperfusion in 13- to 14-week-old C57BL/6JRj male mice (n=5). Ischemia was induced by occlusion of the middle cerebral artery for 60 minutes, and Nissl staining was used to validate infarct size. Spatial transcriptomics complemented by bulk proteomics was conducted in the peri-infarct cortex region and validated with immunohistochemical semiquantification of proteins of interest. To avoid individual biological variations, changes in the peri-infarct cortex region were expressed relatively to the matching contralateral hemisphere region. RESULTS: Ischemic stroke reperfusion impaired the blood-brain barrier integrity through junctional Cldn5 (claudin-5) downregulation, changes of the actin cytoskeleton adhesion, and high expression of the proinflammatory Il-6 (interleukin-6). Molecules important for extracellular Ca2+ influx and intracellular Ca2+ release, Cacna1e (R-type Ca2+ channels), Orai2, Ryr3, Itpr1, and Itpka (inositol-trisphosphate 3-kinase A), were markedly reduced. Furthermore, reduced Grm5 (glutamate receptor 5) associated with upregulated Nfatc3 and Stat3 implicates suppression of the contractile phenotype, suggesting reduced poststroke vascular resistance due to loss of mural cell tone. The complete spatial transcriptomics map over the ipsilateral and contralateral hemispheres is available online as a Web tool. CONCLUSIONS: Emphasizing the spatial molecular pattern behind blood-brain barrier disruption and loss of the vascular tone in the acute phase following ischemic stroke reperfusion suggests the gene expression contribution for a therapeutic target in ischemia-reperfusion abnormalities.</p