Sex‐Dependent Differences of Mitochondria‐Related Proteins in Rat Brain Microvessels Revealed by Mass Spectrometry

Abstract

Background Mitochondrial responses to experimental strokes differ in large cerebral arteries of male and female rats. However, it is unclear whether sex‐dependent differences extend to the cerebral microcirculation. Therefore, it is important to study the cerebral microcirculation since recent evidence suggests this vascular segment is a major participant in the development of neurological diseases such as cognitive impairment, vascular dementia, and Alzheimer’s disease. We performed an unbiased quantitative discovery‐based proteomic experiment to help elucidate the sex‐dependent differential expression of mitochondria‐related proteins in cerebral microvessels (MVs) for the first time. Methods MVs were isolated from young, age‐matched, male and female, Sprague‐Dawley rats. The population and the quality of the isolated MVs (< 70 μm diameter) were confirmed by light microscopy. MVs were then used for a 3 vs. 3 quantitative multiplexed experiment utilizing tandem mass tags coupled with liquid chromatography‐mass spectrometry (LC‐MS). MS data and bioinformatic analyses were performed using Proteome Discoverer version 2.2. and Ingenuity Pathway Analysis (IPA), respectfully. Results The LC‐MS proteomic analysis identified 42 mitochondria‐related proteins with significant (p < 0.05) male and female differences. Twenty‐four proteins were more abundant in MVs of females and 18 were more prevalent in male MVs. In general, males displayed more mitochondria destructive proteins, e.g., microtubule‐associated proteins 1A/1B light chain 3B and mitochondria‐eating protein. Overexpression of neuronal pentraxin‐1 in male MVs may influence the translocation of proapoptotic proteins to mitochondria. Increased expression of monocarboxylate transporter 1 and its chaperone protein, basigin (CD147), in male MVs reflects inhibited glycolysis, impaired oxidative phosphorylation, and reduced mitochondrial anaplerotic reactions. On the other hand, females showed more inner and outer membrane proteins (several ATP synthase subunits), anti‐ROS (peroxiredoxin‐2), and proteins involved in use of alternative fuels via fatty acid beta‐oxidation (indicated by the induction of 2,4‐dienoyl‐CoA reductase, and Δ3.5‐Δ2,4‐dienoyl‐CoA isomerase). Bioinformatic analysis showed the top 5 canonical pathways that exhibit sex‐based differences are mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, TCA cycle, and eIF2 signaling. Conclusion Female rat MVs have more anti‐inflammatory/pro‐healing proteins associated with mitochondrial activity than the male MVs. Female MV mitochondria are also more stable and versatile than male MV mitochondria. Finally, our findings reveal significant sexual dimorphism in MV mitochondrial metabolic protein profiles, all of which may contribute to the phenotypic basis for many sex‐dependent cerebrovascular pathologies Support or Funding Information HL‐093554, AHA17SDG33410366, NS094834, U54GM 104940.