Treatment with the mitochondrial‐targeted antioxidant peptide <scp>SS</scp>‐31 rescues neurovascular coupling responses and cerebrovascular endothelial function and improves cognition in aged mice

Normal functioning of the central nervous system (CNS) requires a continuous, tightly controlled supply of oxygen and nutrients as well as washout of harmful metabolites through uninterrupted cerebral blood flow (CBF). The energetic demands of neurons are very high, yet the brain has very little energetic reserves. During periods of intense neuronal activity, there is a requirement for adjusting oxygen and glucose delivery to local neuronal activity through rapid adaptive increases in CBF. This is ensured by a mechanism known as neurovascular coupling (NVC). The resultant functional hyperemia is a vital mechanism to maintain optimal microenvironment of cerebral tissue and thereby ensuring normal neuronal function. There is an increasing appreciation that (micro)vascular contributions to cognitive impairment and dementia in elderly patients are critical (Tarantini, Tran, Gordon, Ungvari &amp; Csiszar, 2016 ). Importantly, neurovascular coupling responses are impaired both in elderly patients (Fabiani et al., 2013 ; Stefanova et al., 2013 ; Topcuoglu, Aydin &amp; Saka, 2009 ; Zaletel, Strucl, Pretnar‐Oblak &amp; Zvan, 2005 ) and aged laboratory animals (Park, Anrather, Girouard, Zhou &amp; Iadecola, 2007 ; Toth et al., 2014 ), which likely contributes significantly to the progressive age‐related decline in higher cortical function, including cognition (Sorond, Hurwitz, Salat, Greve &amp; Fisher, 2013 ) and gait coordination (Sorond et al., 2011 ). Experimental studies support this concept, showing that pharmacologically induced neurovascular uncoupling in mice mimics important aspects of age‐related cognitive impairment (Tarantini et al., 2015 ). On the basis of these findings, we proposed that novel therapeutic interventions should be developed to rescue functional hyperemia in elderly patients to prevent/delay cognitive impairment (Toth et al., 2014 ). There is strong evidence that microvascular endothelial NO synthesis/release is stimulated during increased neuronal activity (via ATP‐mediated activation of endothelial purinergic receptors), which critically contribute to functional hyperemia under normal conditions (Toth, Tarantini, Davila et al., 2015 ). Previous studies demonstrate that aging exacerbates generation of reactive oxygen species (ROS) in the cerebromicrovascular endothelial cells, which contribute to age‐related neurovascular uncoupling in aged mice by promoting endothelial dysfunction (Toth et al., 2014 ). We hypothesize that pharmacological treatments, which attenuate endothelial oxidative stress and rescue NO mediation, will have the capacity to improve neurovascular coupling in aged individuals. The mitochondrial free radical theory of aging posits that mitochondria‐derived ROS (mtROS) production and related mitochondrial dysfunction are a critical driving force in the aging process. In support of this theory, it was demonstrated that attenuation of mitochondrial oxidative stress (by mitochondria‐targeted overexpression of catalase) increases mouse lifespan (Schriner &amp; Linford, 2006 ). Further, pharmacological treatments that attenuate mtROS production were shown to counteract some of the effects of aging in skeletal muscle (Siegel et al., 2013 ) and the heart (Dai, Chiao, Marcinek, Szeto &amp; Rabinovitch, 2014 ; Dai, Rabinovitch &amp; Ungvari, 2012 ; Dai et al., 2011 ). There is particularly strong evidence that mitochondrial oxidative stress is implicated in cardiovascular aging processes (Dai et al., 2012 ; Springo et al., 2015 ). Yet, although drugs that improve mitochondrial function have been shown to exert beneficial effects both on the vasomotor function of peripheral arteries (Gioscia‐Ryan et al., 2014 ; Pearson et al., 2008 ), their potential protective effects on the aged cerebral microvasculature has not been investigated. This study was designed to test the hypothesis that pharmacological attenuation of mtROS can restore cerebromicrovascular endothelial function and thus improve neurovascular coupling in aged mice. To achieve this goal, in aged mice mitochondrial oxidative stress was manipulated by treatment with the mitochondrial‐targeted Szeto‐Schiller (SS) peptide SS–31. SS–31 is a tetrapeptide with alternating aromatic‐cationic amino acid motif (H‐D‐Arg‐Dmt‐Lys‐Phe‐NH 2 ), which concentrates in the inner mitochondrial membrane more than 1,000‐fold compared with the cytosolic concentration (Bakeeva et al., 2008 ; Doughan &amp; Dikalov, 2007 ; Zhao et al., 2004 ) and is able to effectively reduce levels of O 2 − , H 2 O 2, hydroxyl radical and peroxynitrite both in vitro and in vivo (Graham et al., 2009 ; Szeto, 2008 ; Zhao et al., 2004 ). Mice were behaviorally evaluated on a battery of tests for characterization of cognitive function and motor coordination and functional tests for neurovascular coupling responses and cerebromicrovascular endothelial function. Markers of oxidative stress and expression of genes regulating neurovascular coupling responses in the cereb

To further ascertain the endothelial protective effects of SS‐31, endothelium‐dependent vasodilator responses were tested in isolated, cannulated branches of the MCA. Pressurized MCAs developed spontaneous myogenic tone (~30%), the magnitude of which did not differ between the experimental groups. In young vessels, administration of acetylcholine (Figure 2 c) and ATP (Figure 2 d) resulted in significant dilation, whereas these responses were significantly attenuated in vessels derived from aging mice (Figure 2 c,d). Treatment of aged mice with SS‐31 significantly improved both acetylcholine‐ and ATP‐induced vasodilation, restoring responses to the level observed in vessels of young mice. To assess the role of endothelium‐derived NO, L‐NAME was applied. L‐NAME significantly inhibited acetylcholine‐ and ATP‐induced dilator responses, eliminating the differences between the three groups. These finding suggests that SS‐31 significantly improves endothelial function by restoring endothelial NO mediation in aged cerebral vessels. Vasodilator responses elicited by administration of the NO donor sodium nitroprusside (SNP) did not differ significantly among the experimental groups (Figure 2 e), suggesting that the dilator capacity of smooth muscle cells in the aged cerebral vasculature is preserved and is unaffected by SS‐31.

Treatment with SS‐31 did not alter the expression of Nos1 and Nos3 in the aged mouse brain (Figure 2 h). The expression of arginase 1 was increased in aged mice and tended to be downregulated by SS‐31 treatment. NADPH oxidases are also important sources of ROS in the cerebral vasculature, whose increased expression and activity may contribute to impaired neurovascular coupling (Park et al., 2007 ; Toth et al., 2014 ). We found that in the cortex of aged mice mRNA expression of the NADPH‐oxidase subunits Nox1 and Nox2 were upregulated compared to young mice (Figure 2 i), extending recent findings (Park et al., 2007 ; Toth et al., 2014 ). Expression of Nox2 in SS‐31‐treated aged CMVECs was statistically not different from that in young CMVECs (Figure 2 h). Treatment with SS‐31 did not alter the expression of Nox1, Sod1 and Sod2 in the aged mouse brain (Figure 2 h).

To investigate the effects of age and SS‐31 treatment on the motor performance of mice, we measured grip strength and inverted grid hang time (data not shown), which evaluates muscle strength and endurance. We found that neither aging nor SS‐31 treatment affected the outcome of these tests. Motor skill learning was assessed using a modified rotarod test. During a 4‐day‐long experimental period, young mice displayed a steeper motor skill learning curve compared to aged control mice. After the first day, aged control animals consistently fell off the accelerating rotarod earlier compared to young controls, showing impaired acquisition of motor skill learning. Treatment with SS‐31 restored motor skill learning behavior to the young control levels (Figure 4 a). 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 &lt; .05 vs. young control; # p &lt; .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 &lt; .001 young vs. aged; p &lt; .0001 young vs. aged treated). c) Scheme showing proposed role for increased mitochondrial oxidative stress in cerebromicrovascular endothelial impairment and neurovascular dysfunction in aging

The key finding of his study is that the mitochondria‐targeted antioxidative peptide SS‐31 exerts significant beneficial effects on neurovascular coupling responses and cognitive function in a mouse model of aging that recapitulates cerebromicrovascular dysfunction and deficits of higher brain function manifested in elderly patients. Extending previous findings in elderly patients and aged laboratory animals, we demonstrate that aging leads to significant neurovascular dysregulation in mice, characterized by diminished CBF changes in response to increased neuronal activity (Figure 1 ). Here, we demonstrate for the first time that aging‐induced decline in neurovascular coupling is restored by treatment with SS‐31 (Figure 1 ). There is strong experimental evidence that endothelial NO production contributes importantly to neurovascular coupling (Toth et al., 2014 ). This concept is supported by our observation that both genetic depletion of eNOS (Toth, Tarantini, Davila et al., 2015 ) and pharmacological inhibition of NO synthesis (Figure 2 a,b) dramatically reduce neurovascular coupling in young mice. The findings that in aged mice inhibition of endothelial NO synthesis does not attenuate functional hyperemia (Toth et al., 2014 ) (Figure 2 a) suggest that cerebromicrovascular endothelial dysfunction significantly contributes to age‐related neurovascular uncoupling (Figure 2 b)(Park et al., 2007 ). Importantly, treatment with SS‐31 rescues NO mediation of neurovascular coupling in aged animals (Figure 2 a) supporting the concept that potent endothelial protective effects of SS‐31 play a key role in its anti‐aging action. Additional evidence in support of this concept comes from the observations that SS‐31 treatment rescued acetylcholine‐ and ATP‐induced, endothelial NO‐mediated vasodilation in aged mice (Figure 2 c,d). It should be noted that endothelium‐derived NO also modulates cellular metabolism and mitochondrial function, is an important inhibitor of platelet aggregation, smooth muscle cell proliferation and leukocyte adhesion, and exerts potent anti‐inflammatory, anti‐apoptotic, and pro‐angiogenic effects. Therefore, SS‐31 mediated improvement in microvascular NO production likely has clinical significance beyond rescue of NVC response, potentially exerting multifaceted protective effects on neuronal, astrocytic and microglial functions. Previous studies demonstrate that the mechanism(s) by which aging impairs cerebromicrovascular endothelial function involves an increased breakdown of NO by elevated levels of ROS (Toth et al., 2014 ). On the basis of the findings that SS‐31 treatment effectively attenuates oxidative stress in aged CMVECs (Figure 2 f–g), we propose that in aged mice SS‐31 treatment restores NVC responses and endothelium‐dependent cerebromicrovascular dilation by preventing oxidative stress‐mediated scavenging of NO in the microvascular endothelial cells. Recovery of endothelial function in aged peripheral arteries has also been reported using the structurally different mitochondria‐targeted antioxidant MitoQ (Gioscia‐Ryan et al., 2014 ). The mechanisms by which SS‐31 attenuates mtROS production in aging are likely multifaceted. Age‐related loss of efficiency in mitochondrial electron transport likely increases electron leak and mtROS production. SS‐31 is known to concentrate in the inner mitochondrial membrane more than 1,000‐fold compared with the cytosolic concentration (Bakeeva et al., 2008 ; Doughan &amp; Dikalov, 2007 ; Zhao et al., 2004 ). While SS‐31 may scavenge ROS directly at the site of their production, recent studies show that SS‐31 binds to cardiolipin via electrostatic and hydrophobic interactions and thereby protects the structure of mitochondrial cristae, increasing the efficiency of mitochondrial electron transport (Siegel et al., 2013 ) and attenuating mtROS production (Szeto, 2014 ). Increased efficiency of ATP generation and reduction in ROS are thereby coupled. Based on the findings that SS‐31 improves mitochondrial respiration in cerebromicrovascular endothelial cells (Figure 2 h), while reducing ROS generation (Figure 2 f,g) we posit that such a mechanism is operational in the cerebral microcirculation of SS‐31‐treated aged mice. There are studies suggesting that upregulation of the tissue renin–angiotensin II system (Wang et al., 2007 ) contributes to increased vascular oxidative stress in aging (Modrick, Didion, Sigmund &amp; Faraci, 2009 ). Importantly, SS‐31 was also shown to effectively attenuate angiotensin II‐induced mtROS production (Dai et al., 2011 ). The microvascular endothelium in the brain, which maintains the blood–brain barrier and exhibits controlled transcellular transport systems, has high energy demands. Future studies should determine how SS‐31‐induced changes in cellular energetics impact barrier and transport function in aged CMVECs. In addition, there is growing evidence that mitochondria‐derived ROS regulate the expression and activity of

After the treatment period, behavioral tasks were performed to characterize the effect of SS‐31 treatment on learning and memory, sensory‐motor function, gait and locomotion ( n = 20 in each group).

Motor coordination was assessed using the rotarod as described (Tarantini et al., 2015 ). Analysis of day‐to‐day changes in performance on the accelerating rotarod test was used to evaluate the motor skill learning. The grid hanging test was also used to evaluate balance and motor function. A grip strength test was used to measure the maximal muscle strength of forelimbs of the mice.

After behavioral testing, mice in each group were anesthetized with isoflurane (4% induction and 1% maintenance), endotracheally intubated, and ventilated. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow above the left barrel cortex using laser speckle contrast imaging in response to contralateral whisker stimulation (Toth et al., 2014 ; Toth, Tarantini, Ashpole et al., 2015 ; Toth, Tarantini, Davila et al., 2015 ). In a separate cohort of mice, the role of NO mediation in NVC responses was tested. In brief, in anesthetized, intubated and ventilated mice equipped with an open cranial window, changes in CBF were assessed above the left barrel cortex using a laser Doppler probe in response to electric stimulation of the contralateral whisker pad, as described (Tarantini et al., 2015 ; Toth et al., 2014 ; Toth, Tarantini, Ashpole et al., 2015 ). Changes in CBF were averaged and expressed as percent (%) increase from the baseline value. To assess the role of NO mediation, CBF responses to whisker stimulation were repeated in the presence of the nitric oxide synthase inhibitor N ω ‐Nitro‐L‐arginine methyl ester (L‐NAME; 3 × 10 −4 M, 20 min).

A quantitative real‐time RT–PCR technique was used to analyze mRNA expression in cortical samples using validated TaqMan probes (Applied Biosystems) and a Strategen MX3000 platform, as previously reported (Toth, Tarantini, Ashpole et al., 2015 ).

To substantiate the endothelium‐protective effect of SS‐31, we performed real‐time measurements of the oxygen consumption rate (OCR; a marker of oxidative phosphorylation) in young and aged CMVECs after treatment with SS‐31 (10 −5 M, for 48 hr) using a Seahorse XF96 extracellular flux analyzer.

ST, AY, EF, AC, and ZU designed research; MNVA, ST, AY, GAF, PH, and TG performed experiments; ST, MNVA, AY, GAF, PH, EF, AP, AC, and ZU analyzed data; ST, AC, and ZU wrote the paper. AY, WES, PR, and EF revised the paper.