Abstract
Background: The actin cytoskeleton regulates many important cellular processes in the brain, including cell division and proliferation, migration, and differentiation. The interactions of actin microfilaments with plaque proteins zonula occludens (ZO)-1, -2, -3 are required ensuring the formation of a polarized brain endothelium which ultimately drives development of blood-brain barrier (BBB). In this study, we carried out an extensive evaluation of proteins involved in the structure and function of mouse brain cortical microvessels (MVs) using a discovery-based quantitative proteomic approach. Methods: Cortical MVs were isolated from equal number of age-matched, male and female, young (4–6 months), middle-aged (12–14 months), and old (20–21 months) mice obtained from Jackson Laboratory [Tg(Thy1-EGFP)MJrs/J] and bred in a C57B16J background (n = 6/group). The presence of end-arterioles, capillaries, and venules in MVs was confirmed by light microscopy and by alkaline phosphate staining. Proteomics analysis was performed using liquid chromatography/mass spectrometry. Results: We quantified 4746, 4216, and 4579 differentially expressed proteins by proteomics in brain cortical MVs of young, middle-aged, and old mice, respectively. We found that several actin cytoskeletal-related alpha actinin-1, -2, -3 (ACTN1/2/4), actin-related protein -2, -3, -3B (ACTR2/3/3B), actin-related protein 2/3 complex subunit -1A, -2, -4, -5 (ARPC1A/2/4/5), and [F-actin]-monooxygenase MICAL3 proteins were significantly downregulated in mice cortical MVs with aging. Under physiological conditions, cerebral endothelial cells are connected by tight junctions (TJs) and adherens junctions (AJs). The TJ and AJ proteins are anchored to the actin cytoskeleton by multiple accessory proteins, ZO-1, ZO-2 and ZO-3. Interestingly, we observed that several TJ, AJ, and their signaling (PKC-α/γ/ε, RHOA, RAB3 and YES1) proteins were significantly downregulated in mice cortical MVs with aging. Moreover, we previously documented that oxidative stress during aging leads to adverse protein profile changes of brain cortical MVs that affect mRNA/protein stability and ATP synthesis capacity in mice. Conclusions: We suggest that increased oxidative stress during aging leads to protein expression profile changes of brain cortical MVs that affect ATP synthesis capacity, which leads to actin cytoskeletal dysregulation, and accelerating to endothelial and BBB dysfunction in the brain microvasculature with aging. Funding: AG075988, HL148836, AG063345, AG074489, NS114286, AG047296, HL141143, HL168568, and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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