Treatment with the mitochondrial-targeted antioxidant peptide SS-31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice

Abstract

Moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling has an essential role in maintenance of healthy cognitive function. In advanced age, increased oxidative stress and cerebromicrovascular endothelial dysfunction impair neurovascular coupling, likely contributing to age-related decline of higher cortical functions. There is increasing evidence showing that mitochondrial oxidative stress plays a critical role in a range of age-related cellular impairments, but its role in neurovascular uncoupling remains unexplored. This study was designed to test the hypothesis that attenuation of mitochondrial oxidative stress may exert beneficial effects on neurovascular coupling responses in aging. To test this hypothesis, 24-month-old C57BL/6 mice were treated with a cell-permeable, mitochondria-targeted antioxidant peptide (SS-31; 10 mg kg^-1 day^-1 , i.p.) or vehicle for 2 weeks. Neurovascular coupling was assessed by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that neurovascular coupling responses were significantly impaired in aged mice. Treatment with SS-31 significantly improved neurovascular coupling responses by increasing NO-mediated cerebromicrovascular dilation, which was associated with significantly improved spatial working memory, motor skill learning, and gait coordination. These findings are paralleled by the protective effects of SS-31 on mitochondrial production of reactive oxygen species and mitochondrial respiration in cultured cerebromicrovascular endothelial cells derived from aged animals. Thus, mitochondrial oxidative stress contributes to age-related cerebromicrovascular dysfunction, exacerbating cognitive decline. We propose that mitochondria-targeted antioxidants may be considered for pharmacological microvascular protection for the prevention/treatment of age-related vascular cognitive impairment (VCI).

Keywords: aging; cerebral circulation; endothelial dysfunction; oxidative stress; vascular cognitive impairment.

Figure 1

Treatment with SS‐31 rescues neurovascular coupling in aged mice. Panel a–b: Representative pseudocolor laser speckle flowmetry maps of baseline CBF (Panel a) and CBF changes in the whisker barrel field relative to baseline during contralateral whisker stimulation (Panel b, right oval, 30 s, 5 Hz) in young (3 months old), aged (24 months old), and SS‐31‐treated aged mice. Color bar represents CBF as percent change from baseline. Panel c shows the time course of CBF changes after the start of contralateral whisker stimulation (horizontal bars). Summary data are shown in panel d. Data are mean ± SEM. (n = 6–8 in each group), *p < .05 vs. young; ^# p < .05 vs. aged. (one‐way ANOVA with post hoc Tukey's tests)

Figure 2

Treatment with SS‐31 improves microvascular endothelial function and rescues NO mediation of neurovascular coupling response in aged mice. Panel a: Representative traces of cerebral blood flow (CBF; measured with a laser Doppler probe above the whisker barrel cortex) during contralateral whisker stimulation (30 s, 5 Hz) in the absence and presence of the NO synthase inhibitor L‐NAME in young (3 months old), aged (24 months old), and SS‐31‐treated aged mice. Summary data are shown in panel b. *p < .05 vs. young; ^# p < .05 vs. aged. (one‐way ANOVA with post hoc Tukey's tests) Panel c–d: Dilation of cannulated branches of the middle cerebral artery, isolated from young, aged, and SS‐31‐treated aged mice, in response to the endothelium‐dependent vasodilator acetylcholine (c) and ATP (d) in the absence and presence of the NO synthase inhibitor L‐NAME (3 × 10⁻⁴ M). Panel e: Vascular dilations to the endothelium‐independent aged.; n.s.: not significant. Panel f: Time course for changes in mtROS production in aged CMVECs induced by treatment with SS‐31 (10⁻⁵ M). Data are mean ± SEM. (n = 8 in each group). *p < .05 vs. young control; ^# p < .05 vs. aged control. Panel g: Attenuation of mtROS production in aged CMVECs was associated with significant improvement in cellular oxygen consumption rate (OCR; a marker of oxidative phosphorylation; see Methods). Data are mean ± SEM. (n = 8 in each group). *p < .05 vs. young control; ^# p < .05 vs. aged control. Panel h: qPCR data showing cortical mRNA expression of nitric oxide synthases Nos3 and Nos1, arginases (Arg1, Arg2), NADPH oxidases (Nox1, Nox2), and superoxide dismutases (Sod1, Sod2) in young, aged, and SS‐31‐treated aged mice. Data are mean ± SEM. (n = 6–7 in each group). *p < .05 vs. young control; ^# p < .05 vs. aged control

Figure 3

In SS‐31‐treated aged mice, rescue of neurovascular coupling responses associates with improved radial‐arm water maze (RAWM) performance. Young (3 months old), aged (24 months old), and SS‐31‐treated aged mice were tested in the RAWM. Panel a: Heatmap representing the percentage of time spent in different locations in the maze for a randomly selected animal from each group during experimental day 3. Note that the untreated aged mouse required a greater amount of time and a longer path length in order to find the hidden escape platform. Older mice also re‐enter a previously visited arm multiple time, accruing working memory errors. Panel b: Older animals have higher combined error rates throughout days 2 and 3 of the learning phase and retrieval day 10 (p < .0001). Combined error rate is calculated by adding 1 error for each incorrect arm entry as well as for every 15 s spent not exploring the arms. Panel c: Older animals make significantly more working memory errors (repetitive incorrect arm entries) as compared to young mice. In contrast, aged mice treated with SS‐31 perform this task significantly better than untreated aged mice (p = .04). Panel d: The ratio of successful escapes, averaged across trial blocks, is shown for each group. Note day‐to‐day improvement in the performance of young mice, which was significantly delayed in aged mice. Aged mice treated with SS‐31 were more successful at finding the hidden escape platform in comparison with untreated age‐matched controls (p < .0001). Panel e: Average path length required to reach the hidden platform in the RAWM for trial blocks 1–6 and for probe trial on day 10. Young mice find the hidden platform while swimming significantly less than aged animals (p < .0001). In aged mice treated with SS‐31, the average path length required to reach the hidden platform did not differ from that in young mice. Panel f: Escape latencies to find the hidden platform in the RAWM for aged control mice are significantly higher when compared to young and aged treated mice (p < .0001 for both comparisons). Panel g: There was no significant difference in swimming speed among all experimental groups. Panel h: SS‐31 treatment reduced the time spent engaged in nonexploratory behavior in control‐treated old mice as compared to young animals. (p < .0001). n = 20 in each group. All data are shown as mean ± SEM. Statistical significance was calculated using one‐way ANOVA with Tukey's post hoc test to determine differences among groups

Figure 4

SS‐31 treatment in aged mice improves motor skill learning but not gait coordination. a) Motor skill learning on the accelerating rotarod. Daily changes in mean latencies to fall for young (3 months old), aged (24 months old) and SS‐31‐treated aged mice are shown. Data are mean ± SEM. (n = 20 in each group). *p < .05 vs. young control; ^# p < .05 vs. aged control. b) 3D triplot of first three principal components (PC) identified by PCA on the correlation matrix of spatial and temporal indices of gait. Each point represents an individual mouse, and centroids are shown for each experimental group. Note that mice in the same age groups clustered together. Distance between centroids is shown inset. Whole differences between young and aged mouse gait were evident, rescue of NVC responses by SS‐31 treatment did not reverse age‐related changes in mouse gait (MANOVA; p = .47 aged vs. aged treated; p < .001 young vs. aged; p < .0001 young vs. aged treated). c) Scheme showing proposed role for increased mitochondrial oxidative stress in cerebromicrovascular endothelial impairment and neurovascular dysfunction in aging