Novel method for collecting hippocampal interstitial fluid extracellular vesicles (EVISF ) reveals sex-dependent changes in microglial EV proteome in response to Aß pathology.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. The internal cargo of EVs is protected from degradation, making EVs attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF ) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labelling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g., APPswe, PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with Aß deposition. Genotype, age, and Aß deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

Cerebrovascular responses to insulin in rats.

Effects of insulin on cerebral arteries have never been examined. Therefore, we determined cerebrovascular actions of insulin in rats. Both PCR and immunoblot studies identified insulin receptor expression in cerebral arteries and in cultured cerebral microvascular endothelial cells (CMVECs). Diameter measurements (% change) of isolated rat cerebral arteries showed a biphasic dose response to insulin with an initial vasoconstriction at 0.1 ng/mL (-9.7%+/-1.6%), followed by vasodilation at 1 to 100 ng/mL (31.9%+/-1.4%). Insulin also increased cortical blood flow in vivo (30%+/-8% at 120 ng/mL) when applied topically. Removal of reactive oxygen species (ROS) abolished the vasoconstriction to insulin. Endothelial denudation, inhibition of K(+) channels, and nitric oxide (NO) synthase, all diminished insulin-induced vasodilation. Inhibition of cytochrome P450 enhanced vasodilation in endothelium-intact arteries, but promoted vasoconstriction after endothelial denudation. Inhibition of cyclooxygenase abolished vasoconstriction and enhanced vasodilation to insulin in all arteries. Inhibition of endothelin type A receptors enhanced vasodilation, whereas endothelin type B receptor blockade diminished vasodilation. Insulin treatment in vitro increased Akt phosphorylation in cerebral arteries and CMVECs. Fluorescence studies of CMVECs showed that insulin increased intracellular calcium and enhanced the generation of NO and ROS. Thus, cerebrovascular responses to insulin were mediated by complex mechanisms originating in both the endothelium and smooth muscle.