**Background:** Aging is associated with cognitive decline and dementias like Alzheimer's disease, linked to functional and structural alterations of the brain microvasculature. Mitochondrial dysfunction, including loss of mitochondria and reduced efficiency, is thought to play a major role. The mitochondria-targeted tetrapeptide SS-31 (elamipretide) has shown benefits in aging and disease, but its effects on the brain microvascular proteome had not been examined. This study tested whether targeting mitochondria with SS-31 attenuates aging-associated changes in the brain microvascular proteome.
**Methods:** Aged male C57Bl6/J mice (>18 months, n=10) were randomly assigned to receive daily intraperitoneal injections of SS-31 (10 mg/kg, n=5) or vehicle saline (n=5) for 14 days. Cerebral blood flow (CBF) was measured using laser speckle imaging at baseline, day 3, day 7, and day 14. After treatment, cortical microvessels (MVs, <70 µm, including end arterioles, capillaries, and venules) were isolated via gradient centrifugation and size-filtering. Protein extracts were analyzed using Orbitrap Eclipse Tribrid mass spectrometry with tandem mass tag 10-plex isobaric labeling. MaxQuant was used for protein identification (FDR <1%), and data were normalized using variance stabilizing normalization (VSN). Differential expression was assessed using limma with empirical Bayes smoothing (p<0.05 considered significant). Pathway analysis was performed using Ingenuity Pathway Analysis and EGSEA.
**Key Results:** CBF was similar between groups at all time points (vehicle: 11.7±13.9, 8.6±24.7, -2.1±13.0; SS-31: 2.5±11.9, 8.7±10.3, -5.9±7.2 percent change on days 3, 7, 14 respectively; p>0.05). Proteomics identified 6267 total proteins, of which 12% were mitochondria-associated. Of these, 107 were significantly differentially expressed (48 downregulated, 59 upregulated) in SS-31 vs vehicle. Key findings include: (1) OXPHOS: 13 proteins significantly changed, including upregulation of Complex I subunits (Ndufa5, Ndufa8, Ndufb8, Ndufs5), Complex II (Sdhc), Complex III (Cyc1, Uqcrb, Bcs1l), Complex IV (Cox7a2l, Higd1a), and cytochrome c (Cycs); downregulation of Cox4i2 and Atp5c1. (2) Metabolism: 59 proteins changed, including upregulation of glycolysis enzymes (Hk1, Gpi, Pfkp, Aldoa/c, Tpi, GAPDH, Pgk1, Pgam1, Eno1/2, Pkm) and TCA cycle proteins (Acly, Idh3g, Sdhc); downregulation of fatty acid oxidation proteins (Acad10, Acadl, Acadm, Acads, Acadvl, Cpt1a, Decr1, Hadha, Hadhb). (3) Antioxidant defense: 15 of 51 detoxification proteins changed, including upregulation of Sod1, Prdx2, Prdx5, Prdx6, Msra, Oxr1, Txnrd1, Hagh, Nit1; downregulation of Mgst1 and Prxl2a. (4) Mitochondrial dynamics: 9 proteins changed, including upregulation of fission proteins Dnm1l, Spire1, and mitophagy proteins Park7 and Pgam5. Top canonical pathways for the global dataset included synaptogenesis signaling (p=1.12E-36), spliceosomal cycle (p=1.91E-24), and androgen signaling (p=3.49E-24). For the mitochondrial dataset, top pathways included mitochondrial dysfunction (p=1.34E-15), sirtuin signaling (p=3.46E-12), and oxidative phosphorylation (p=6.90E-11). Upstream regulators for the global dataset included MAPT (p=1.33×10^-43), CST5 (p=5.47×10^-34), and APP (p=1.11×10^-27).
**Clinical Implications:** This study demonstrates that SS-31 treatment in aged mice induces widespread proteomic changes in brain microvessels, affecting not only mitochondrial proteins but also glycolysis, antioxidant defense, and cellular metabolism. These changes may underlie the previously reported improvements in neurovascular coupling and cognitive function with SS-31. The findings suggest that mitochondrial dysfunction is a key driver of aging-related cerebrovascular pathology and that targeting mitochondria with agents like SS-31 could be a therapeutic strategy for preventing or reversing vascular contributions to cognitive decline and dementia. The lack of change in baseline CBF indicates that proteomic effects are direct rather than secondary to improved blood flow. Limitations include use of only male mice, 14-day treatment duration, and inability to separate cell types within microvessels. Future studies should include both sexes, examine time course, and assess functional outcomes.