Rejuvenation of Mitochondrial Function and Remodeling of Mitochondria.
“Binding of SS-31 to the inner mitochondrial membrane induces curvatures and promote the assembly of respiratory supercomplexes to facilitate
electron transfer. Enhanced forward electron transfer increases mitochondrial respiration, P/O coupling, and ATP synthesis, while decreasing electron leak and production of reactive oxygen species (ROS).” (7)
- “SS-31 rapidly rejuvenates oxidative phosphorylation in aged skeletal muscle
- SS-31 remodels mitochondria cristae structure in aged mice
- SS-31 upregulates enzymes required for cardiolipin biosynthesis and remodeling
- SS-31 restores mitochondrial biogenesis
- SS-31 restores mitochondrial dynamics
- SS-31 repairs cellular structure and restore function during aging
- SS-31 prevents cell death and inflammation
- SS-31 boosts the body’s natural ability to heal itself”
“The immediate improvement in mitochondrial function suggests that it is not due to turnover of oxidatively damaged proteins or lipids. It is more likely explained by SS-31 binding to cardiolipin and promoting electron transfer by optimizing the heme environment of the cardiolipin/cytochrome c complex [81]. SS-31 induced redox regulation may also play a role as SS-31 caused a rapid increase in free glutathione in the muscle, consistent with the release of glutathione bound to proteins in the aged muscle.” (7)
“Resting ATP synthesis is lower in 30-month-old mice compared to 7-month-old mice, with no difference in oxygen consumption. This led to a 50% reduction in mitochondrial P/O coupling. A similar decrease in P/O coupling was found in elderly human subjects (67–85 years) compared to adults less than 50 years old. This mitochondrial uncoupling is associated with elevated ADP, and reduced ATP and phosphocreatine in aged mice and humans. It was quite surprising when Siegel et al. reported that a single treatment of SS-31 (3 mg/kg) in aged mice (27 months) rapidly reversed the decline in mitochondrial function. SS-31 is rapidly taken up by skeletal muscle, with maximal levels observed 30 min after subcutaneous (sc) administration. Maximal ATP production and P/ O coupling returned to levels seen in 5-month-old mice within 1 h after SS-31 treatment. SS-31 also restored maximal ATP production in aged mice but had no observable effect in young mice. The improvement in bioenergetics was associated with improved fatigue resistance. Treatment with SS-31 for 8 days significantly improved endurance capacity in the aged mice.” (7)
“SS-31 repairs mitochondrial cristae architecture in aged mice. Top panel: Representative transmission electron micrography images of murine cardiac mitochondria from (a) 6 months old, (b) 26 months old, and (c) 26 months old after 2 months treatment of SS-31. Bottom panel: Representative murine retinal pigment epithelium mitochondria from (d) 6 months old, (e) 26 months old, and (f) 26 months old after 2 months treatment of SS-31. SS-31 (1 mg/kg, sc) was administered daily starting at age of 24 months.” (7)
Reversing Loss of Diaphragm Strength After Mechanical Ventilation or Sepsis.
“SS31 prevents sepsis-induced diaphragm dysfunction, preserving force generation, endurance, and mitochondrial function.” (4)
“Diaphragmatic atrophy and contractile dysfunction can be seen after 12 h mechanical ventilation in rats. Mechanical ventilation significantly increased mitochondrial state 4 respiration, mitochondrial H2O2 release and oxidative damage to proteins and lipids that were completely blocked by SS-31 treatment during mechanical ventilation (Powers et al., 2011). As a result, diaphragmatic contractile dysfunction and fibre atrophy were also prevented by SS-31 treatment. Mitochondrial oxidative stress appears to be a required upstream signal for the activation of all key proteases, as SS-31 prevented activation of calpain and caspase-3, as well as 20S proteasome activity in the diaphragm.” (10)
“Efficacy of SS-31 in skeletal muscle aging: “Mitochondrial coupling efficiency and maximal ATP production in skeletal muscle of 30-monthold mice was significantly lower compared to 7-month-old mice (Marcinek et al., 2005). This was associated with higher resting ADP and decreased energy charge (ATP/ADP). Studies with isolated mitochondria revealed increased number of mitochondria but significantly reduced state 3 mitochondrial respiration and increased oxidative damage (Figueiredo et al., 2009). This age associated decline in skeletal muscle function and exercise intolerance results in significant health care costs, and there is no pharmacological treatment to rapidly reverse these mitochondrial deficits Siegel et al. (2013) recently reported that a single treatment with SS-31 restored in vivo mitochondrial energetics to ‘young’ levels in aged mice after only 1 h, while there was no effect in young mice. Age-related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation and cell energy state (phosphocreatine/ATP) were all rapidly reversed after SS-31 treatment. These effects of SS-31 in aged muscle were associated with a more reduced glutathione redox status and lower mitochondrial H2O2 emission. Skeletal muscle of aged mice were more fatigue-resistant after a single SS-31 treatment, and 8 days of SS-31 treatment led to increased whole-body endurance as measured by treadmill running. This rapid improvement in mitochondrial energetic cannot be due to repairing or replacing damaged mitochondria. Rather, it suggests that SS-31 can rapidly improve mitochondrial respiration by increasing the efficiency of the ETC, and this may be related to the ability of SS-31 to improve cardiolipin function and promote fluidity and the formation of supercomplexes on the IMM.” (10)
Efficacy of SS-31 in disuse muscle atrophy: “Disuse atrophy has been associated with loss of mitochondria, mitochondrial swelling, increased mitochondrial ROS production and impaired mitochondrial respiratory function (Muller et al., 2007; Powers et al., 2012). Mechanisms that have been proposed for increased mitochondrial ROS production in disuse atrophy include increased mitochondrial uptake of Ca2 , and increased fatty acid hydroperoxides (Bhattacharya et al., 2009; 2011). Free fatty acids can also decrease mitochondrial respiration and increase H2O2 production (Bhattacharya et al., 2011). The role of cardiolipin peroxidation in muscle atrophy has not been investigated, but swollen mitochondria lacking cristae membranes were observed in the rat soleus muscle after hindlimb suspension (Powers et al., 2012). Cardiolipin peroxidation could account for induction of the intrinsic apoptotic pathway. Recent studies have shown that mitochondrial oxidative stress is a requisite step towards the activation of muscle proteolysis. By promoting mitochondria respiration and reducing ROS generation, SS-31 has been shown to be effective in preventing disuse atrophy in animal models.” (10)
Muscle atrophy due to immobilization / “bed-ridden” cast: “When rats were subjected to casting for 7 days, significant atrophy was observed in both the soleus and plantaris muscle (Talbert et al., 2013). Casting caused significant reduction in state 3 mitochondrial respiration and increased mitochondrial H2O2 production. Immobilization was associated with activation of calpain and caspase-3 activity, as well as an increase in the proteasome system, with up-regulation of muscle-specific E3 ligases. Autophagy signalling was also increased, suggesting that all four proteolytic systems are involved in skeletal muscle atrophy. Daily SS-31 treatment prevented the decrease in state 3 respiration and increase in H2O2 production, and abolished the activation of all four proteolytic systems. SS-31 also prevented the downregulation of anabolic signalling molecules during immobilization, such as the Akt/mTOR pathway. Consequently, SS-31 treatment prevented immobilization-induced atrophy. Thus mitochondrial oxidative stress appears to play a requisite role in inhibiting protein synthesis and activating proteolytic systems to result in skeletal muscle atrophy. Previous research has shown that PGC-1α, a co-activator of the PPARγ, plays a major role in mitochondrial biogenesis and oxidative metabolism (Lin et al., 2005). PGC-1α expression in skeletal muscle is down-regulated in muscle atrophy from denervation and fasting, and overexpression of PGC-1α is sufficient to attenuate the muscle atrophy (Sandri et al., 2006). However, SS-31 did not prevent the down-regulation of PGC-1α during immobilization, suggesting that the protective effect of SS-31 is not mediated by PGC-1α-induced mitochondrial biogenesis.” (10)
“From a clinical perspective, the most important result from these experiments is the finding that treatment of animals with the mitochondrial-targeted antioxidant SS-31 prevents the rapid onset of MV-induced diaphragmatic atrophy and contractile dysfunction. Given that MV-induced diaphragmatic weakness may contribute to the failure-to-wean syndrome, this finding has significant clinical implications and suggests that mitochondrial-targeted antioxidants may have therapeutic potential in protecting the diaphragm from MV-induced weakness and reduce weaning problems in some patients. SS-31 is currently undergoing clinical development as a novel mitochondria-targeted therapy by Stealth Peptides Inc. The results of a recent Phase 1 clinical trial with intravenous infusion of SS-31 demonstrated safety and tolerability with predictable linear pharmacokinetics that included doses exceeding the expected patient dose by several fold. Clearly the use of SS-31 in prevention of MV-induced diaphragmatic weakness is an exciting possibility that warrants clinical investigation.” (11)
Biophysical Approaches Toward Understanding the Molecular Mechanism of Action of the Mitochondrial Therapeutic SS-31 (Elamipretide).
“Mitochondria orchestrate energy metabolism within eukaryotic cells. Mitochondrial disorders can arise from genetic defects, and are also associated with aging-related declines in cellular bioenergetic capacity and with complex diseases including cardiomyopathy, neurodegeneration, diabetes, and cancer. To date, there are no cures for mitochondrial diseases. Szeto-Schiller (SS) peptides are among the most promising mitochondrial therapeutics currently under investigation. They comprise a novel class of tetrapeptides characterized by aromatic-cationic motifs that selectively bind to mitochondria and display broad intrinsic therapeutic potential. Among SS peptides, the lead compound SS-31, under the proprietary name Elamipretide, is currently in advanced clinical trials. However, the molecular mechanism of action (MOA) of SS peptides remains virtually unknown. We are conducting a highly interdisciplinary study to elucidate how SS peptides interact with the cardiolipin-rich mitochondrial inner membrane and the effects that they have on membrane physical properties and structural assembly of oxidative phosphorylation (OXPHOS) complexes. With model membrane systems, we have used a host of approaches including fluorescence spectroscopy, calorimetry, solid state NMR, and small angle x-ray scattering to understand: (i) the driving forces, affinities, and thermodynamics of the peptide-bilayer interaction, and (ii) the effects that SS peptides have on lipid packing and order parameters. We have complemented these measurements with molecular dynamics approaches to analyze the conformations and lipid interactions of SS peptides in the bilayer-docked state. Finally, using disease models, we show that SS-31 restores the structural assembly of key OXPHOS complexes.” (6)
Heart Improvement via Mitochondria Optimization.
A collection of excerpts extensively explaining many of SS-31’s effects on the heart can be found here: https://www.peptidesciences.com/blog/mir-133a-hypertrophy-ss31-pe2228
“…the results of this study show that the antioxidant peptide SS-31 markedly attenuated TAC-induced pulmonary arterial hypertension as well as lung parenchymal and right ventricular [heart] damage.” (2)
“Importantly, in addition to extending the findings of previous studies that SS-31 protected the heart from ischemia-reperfusion injury and reactive hypertrophic cardiomyopathy[16, 18-20], the previously unreported results of the present study further highlight the possibility that SS-31 may be a useful accessory agent in the clinical treatment of patients with PAH in the near future, especially for patients with left-sided heart failure-induced PAH” (2)
“The protein expression levels of NOX-1 and NOX-2, oxidative stress, ET-1R, γ-H2AX (Figure 7), cytosolic cytochrome c, HIF-1α, TRPC1, TRPC4, and TRPC6 (Figure 8) were significantly higher in TAC animals than those in SC animals and
in TAC animals with SS-31 treatment. The levels of these proteins were significantly higher in TAC animals that received
SS-31 treatment compared to SC animals, whereas mitochondrial cytochrome c showed a reverse pattern among the three groups of animals (Figure 8). The protein expression levels of inflammatory (ie, TNF-α, NF-κB, and MMP-9), apoptotic (ie, mitochondrial Bax, cleaved caspase 3, and PARP) and fibrotic (ie, Smad3 and TGF-β) biomarkers were significantly higher in TAC animals than in SC animals and were significantly lower in TAC animals after SS-31 treatment (Figure 9). On the other hand, protein expression levels of anti-fibrotic (Smad1/5, BMP-2), anti-inflammatory (Bcl-2) and endothelial function (eNOS) biomarkers showed an opposite pattern compared to that of the inflammatory biomarkers among the three groups (Figure 10). Furthermore, the protein expression levels of antioxidants (HO-1, NQO 1, GR, and GPx) were significantly increased in TAC animals and were further significantly increased in TAC animals that received SS-31 treatment compared with SC animals (Figure 10).” (2)
SS-31 Anti-fibrotic, Anti-inflammatory, Antioxidant and Endothelial Function Biomarkers’ Role in Heart Enlargement.
“Protein expression levels of anti-fibrotic, anti-inflammatory, antioxidant and endothelial function biomarkers in lung parenchyma at day 60
after the TAC procedure. (A) Protein expression of p-Smad1/5. (B) Protein expression of bone morphogenetic protein (BMP)-2. (C) Protein expression
of Bcl-2. (D) Protein expression of endothelial nitric oxide synthase (eNOS). (E) Protein expression of heme oxygenase (HO)-1. (F) Protein expression of
NAD(P)H quinone oxidoreductase (NQO) 1. (G) Protein expression of glutathione reductase (GR). (H) Protein expression of glutathione peroxidase (GPx).
All statistical analyses were performed by one-way ANOVA followed by Bonferroni multiple comparison post hoc test. SC=Sham control. TAC=Transverse
aortic constriction. Mean±SEM. n=8 for each group. **P<0.01 vs SC group. ##P<0.01 vs TAC group” (2)
“Figure 9. Protein expression levels of inflammatory, apoptotic and fibrotic markers in lung parenchyma at day 60 after the TAC procedure. (A) Protein expression of tumor necrosis factor (TNF)-α. (B) Protein expression of nuclear factor (NF)-κB. (C) Protein expression of matrix metalloproteinase (MMP)-9. (D) Protein expression of mitochondrial Bax (mit-Bax). (E) Protein expression of cleaved caspase (c-Casp) 3. (F) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP). (G) Protein expression of phosphorylated (p)-Smad3. (H) Protein expression of transforming growth factor (TGF)-β. All statistical analyses were performed by one-way ANOVA followed by Bonferroni multiple comparison post hoc test. SC=sham control. TAC=Transverse aortic constriction. Mean±SEM. n=8 for each group. **P<0.01 vs SC group. ##P<0.01 vs TAC group” (2)
“On day 60 after TAC, the changes in gene expression levels of NOX-1 and NOX-2 ( ie, two indicators of ROS generation) as well as TNF-α, MMP-9, and inducible nitric oxide synthase (iNOS) ( ie, three biomarkers of inflammation) were significantly higher in the TAC group than in the SC group (Figure 2). Cardiac hypertrophy is characterized by a switch from α- to β-myosin heavy chain (MHC) mRNA expression ( ie, reactivation of the fetal gene program). In the current study, mRNA expression of β-MHC in RV was significantly higher in the TAC group than in the SC group, whereas α-MHC in RV showed a reverse pattern between the two groups (Figure 2). Moreover, the protein expression levels of BNP ( ie, an indicator of pressure or volume overload) showed an identical pattern of ROS between the two groups (Figure 2). Again, these parameters were significantly reversed in TAC animals with SS-31 treatment.” (2)
“Masson’s trichrome staining showed that the RV fibrotic area was significantly increased in TAC group compared with that of the SC group (Figure 3). Consistently, Sirius red staining revealed that the RV collagen deposition area showed an identical pattern of fibrosis in these two groups (Figure 3). These two parameters were significantly reversed in TAC animals with SS-31 treatment.“
SS-31 Eye Drops Improve Visual Dysfunction in Diabetic Mouse.
”Daily administration of MTP-131 in eye drops reversed the severe visual dysfunction present later in the course of the disease.” (1)
“Reversal of visual decline was evident after 6 weeks of drug treatment (MTP-131 versus Veh: 0.211 c/d±0.003 versus 0.198±0.003 c/d, P<0.05); recovery to 80% normal was evident by 52 week.” (1)
Small Amounts of Oxidation is Required for Wound Healing.
“Growing evidence suggests that reactive oxygen species (ROS) are crucial regulators of several phases of healing processes. ROS are centrally involved in all wound healing processes as low concentrations of ROS generation are required for the fight against invading microorganisms and cell survival signaling. Excessive production of ROS or impaired ROS detoxification causes oxidative damage, which is the main cause of non healing chronic wounds.” (3)
“Oxygen is required to disinfect wounds and provide adequate fuel for healing. In addition, oxygen (O2)-dependent redox signaling is crucial for wound repair. Physiologically, hydrogen peroxide (H2O2) and superoxide serve as intracellular messengers stimulating key phases of wound healing including cell recruitment, production of cytokines and angiogenesis. Of note, H2O2, a reactive species produced by dismutation of superoxide, acts as the principal secondary messenger in wound healing and is present at low concentrations (100–250 µM) in normal wounds.” (3)
“Generation of ROS during the hemostatic phase of wound healing is related to NADPH oxidases (NOX) located in vascular cells, which are activated by tissue factor expression secreted by platelets. During inflammation, ROS production by NOX enzymes plays a central role in microorganism killing by neutrophil and macrophage oxidative burst. Whereas the NOX2 isoform is responsible for the large amounts of ROS produced during the respiratory burst, NOX4 isoform has been implicated in phagocyte recruitment.” (3)
SS-31 Reduces Mitochondrial Oxidation while still Promoting Wound Healing.
“Tissue repair is blunted in aging due to mitochondrial dysfunction and senescence of resident stem cells. Recent research showed that repletion of NAD+ with nicotinamide riboside rejuvenated mitochondrial function in muscle stem cells in aged mice and improved musclefunction in muscular dystrophy. NAD+ repletion improved mitochondrial function in aged mice and prevented skeletal muscle stem cell senescence. These recent studies suggest that promoting mitochondrial bioenergetics can improve the function of resident stem cells and reduce their senescence in old age. There is extensive evidence that SS-31 can promote healing of damaged tissues. Treatment with SS-31 in acute renal ischemia significantly accelerated the rate of tubular cell regeneration as shown by proliferation markers. SS-31 has been reported to promote normal healing of injured tissues and ameliorate fibrotic changes in kidneys and hearts. SS-31 was even able to heal terminally differentiated podocytes and restore their complex foot processes that form the filtration barrier in the kidney [95]. SS-31 was shown to reduce senescent markers in glomerular parietal epithelial cells. Parietal epithelial cells are known to serve as podocyte progenitors, making it possible that SS-31 can help prevent senescence of other resident stem cells. (7)
SS-31 may also promote tissue healing by increasing local microvascular blood flow in the injured tissue. Tissue repair and regeneration is dependent on local microvascular blood flow. Microvascular rarefaction, or microvascular dropout, has been described in many tissues in response to hypertension, diabetes, ischemia and aging. Microvascular rarefaction leads to chronic tissue hypoxia that could be detrimental to stem cell function. A major contributor to microvascular rarefaction is endothelial injury, and renal capillary endothelial mitochondria have been shown to undergo swelling and loss of cristae membranes in response to ischemia. Treatment with SS-31 protected mitochondrial structure in capillary endothelial cells and prevented endothelial cell swelling, cell detachment, and cell death. The protection of endothelial mitochondria by SS-31 significantly reduced capillary dropout in the kidney. SS-31 has also been shown to prevent cardiac and renal arteriolar dropout and tissue hypoxia in renal hypertension and metabolic syndrome. SS-31 may also protect against age-associated changes in microvascular blood flow that contribute to the decline in healing power with age. The ability of SS-31 to protect the microvasculature has also been extended to the neurovasculature. During periods of intense neuronal activity, it is necessary to adjust cerebral blood flow to meet energy needs. This neurovascular coupling is impaired in elderly patients and animals. Treatment with SS-31 (10 mg/kg) for 10 days restored neurovascular coupling in 24-month-old mice. This improvement of the aged neurovasculature was associated with significantly improved spatial working memory, motor learning skill, and gait coordination.” (7)
“A wealth of preclinical studies supports the ability of these compounds to ameliorate diverse chronic conditions. Clinical trials with SS-31 (elamipretide) have shown promising results in age-related conditions including heart failure, muscle weakness, chronic kidney disease and age-related macular degeneration” (7)
“Mitochondrial-targeted peptides such as elamipretide have the potential to mitigate mitochondrial dysfunction and aberrant inflammatory response through activation of nucleotide-binding oligomerization domain (NOD)-like family receptors, such as the pyrin domain containing 3 (NLRP3) inflammasome, nuclear factor-kappa B (NF-κB) signaling pathway inhibition, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2).” (3)
“Deregulation of the inflammatory phase of wound healing and persistence of the pro-inflammatory macrophages in the diabetic wounds has been related to sustained NLRP3 inflammasome activity.” (3)
“Mitochondria dysfunction elicits inflammasome activation. Injured mitochondria release molecular pattern that are recognized by cell membrane receptors and cytosolic toll-like receptor (TLR) 9. NLRP3 inflammasomes are activated by a myriad of stimuli that include danger-associated molecular patterns (DAMPs). Once activated, NLRP3 forms a multimeric protein complex with associated speck- like protein containing a caspase activation and recruitment domain (CARD; ASC) and caspase-1 (CASP1) termed the inflammasome. Caspase-1 is activated in the inflammasome complex, which cleaves pro-IL-1β (pro-interleukin-1β) and pro-IL-18 into their bioactive mature forms. Mitochondrial DNA (mtDNA), N-formyl proteins, ATP and mitochondrial reactive oxygen species (mtROS), have all been shown to promote NLRP3 inflammasome activation either directly or via specific receptor such as formyl peptide receptor 1 (FPR1) and P2X purinoceptor 7 (P2RX7). TLR9 preferentially binds DNA motifs present in mitochondria and triggers signaling cascades that lead to a pro-inflammatory cytokine response.” (3)
SS-31 Targets the Mitochondria Dysfunction Seen in Amyotrophic Lateral Sclerosis (ALS) Disease.
“Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder, which results in a rapidly progressive paralysis due to degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord. About 90% of cases occur sporadically and 10% are familial. Among the familial cases, 10–20% can be attributed to point mutations in the gene coding for the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1) (Rosen et al. 1993). This discovery led to the generation of mouse models overexpressing common human SOD1 mutations. These mice develop a phenotype which closely mimics human ALS (Gurney et al. 1994). The precise molecular mechanisms leading to motor neuron death have not been fully elucidated; however, oxidative damage may play a role. In the G93A mouse model, as well as in human familial and sporadic ALS, markers of oxidative damage of proteins, lipids and DNA [protein carbonyls, nuclear DNA 8-hydroxy-2¢-deoxyguanosine (OH8dG) levels, 3-nitrotyrosine and 3-nitro-4-hydroxyphenylacetic acid, malondialdehyde, heme oxygenase-1, 4-hydroxy-2-nonenal] are elevated in the brain and spinal cord (Beal et al. 1997; Ferrante et al. 1997a,b; Simpson et al. 2004; Aguirre et al. 2005; Casoni et al. 2005; Perluigi et al. 2005). Antioxidants, such as metal porphyrins, and agents which improve bioenergetics, such as creatine, are neuroprotective in ALS mouse models (Klivenyi et al. 1999, 2004; Wu et al. 2003; Crow et al. 2005; Petri et al. 2006).” (8)
“There is evidence for a role of mitochondrial dysfunction in both sporadic and familial ALS (Beal 2005). The expression of mutant SOD1 in neuronal cell lines leads to mitochondrial dysfunction, including a loss of mitochondrial membrane potential and elevated cytosolic calcium (Carri et al. 1997; Menzies et al. 2002). Structural and functional alterations of mitochondria preceding disease onset have been found in ALS mouse models (Wong et al. 1995; Kong and Xu 1998; Mattiazzi et al. 2002; Higgins et al. 2003; Damiano et al. 2006). Mutant SOD1 is recruited to spinal cord mitochondria and is present in the outer mitochondrial membrane, intermembrane space and the mitochondrial matrix, where it can form aggregates and therefore potentially contribute to mitochondrial dysfunction (Higgins et al. 2003; Liu et al. 2004; Vijayvergiya et al. 2005). Entrapment of the anti-apoptotic and cytoprotective protein Bcl-2 in these aggregates has been shown in vivo and in vitro, and may provide a mechanism for increased apoptotic cell death in ALS (Pasinelli et al. 2004).” (8)
“In the present experiments, we tested a novel cellpermeable peptide antioxidant (SS-31), which is targeted to the inner mitochondrial membrane. It is part of a recently developed group of small peptides, including the prototype SS-02 (Dmt-D-Arg-Phe-Lys-NH2, where Dmt ¼ 2¢,6¢-dimethyltyrosine), SS-20 (Phe-D-Arg-Phe-Lys-NH2) and SS-31 (D-Arg-Dmt-Lys-Phe-NH2), all of which consist of alternating aromatic and basic amino acid residues with dimethyltyrosine providing ROS scavenging properties” (8)
“SS-31 decreases mitochondrial ROS production and inhibits MPT and swelling. It also prevents cytochrome c release induced by calcium and inhibits the 3-nitropropionic acid (3-NP)-induced activation of MPT in isolated mitochondria. In cell cultures, it protects against 3-NP toxicity and tert-butyl-hydroperoxide (tBHP) treatment, which increases intracellular ROS and induces markers of apoptotic cell death, such as phosphatidylserine translocation, nuclear condensation and increased caspase activity (Zhao et al. 2005). It is effective against myocardial ischemia–reperfusion injury in both ex vivo and in vivo models (Zhao et al. 2004; Song et al. 2005). Structural and functional abnormalities of mitochondria resulting in the generation of ROS, aggregate formation and the release of pro-apoptotic factors are implicated in ALS
pathogenesis. SS-31 is localized to the inner mitochondrial membrane, where it may be particularly effective in protecting against mitochondrial oxidative damage. In the present study, we therefore examined the efficacy of SS-31 in both in vitro and in vivo models of ALS associated with G93A SOD1 mutations.” (8)
“Treatment with 5 mg/kg SS-31 intraperitoneally, starting at 30 days of age, led to a significant delay in disease onset in G93A SOD1 mice, as defined by the appearance of tremor and hind limb clasping, as well as deterioration in the rotarod performance. The average age of onset was 88 ± 7 days in the control group and 95 ± 6 days in the SS-31-treated group [p < 0.05; log-rank (Mantel–Cox)]. Survival was significantly increased by SS-31 treatment from 130 ± 12 to 142 ± 12 days (i.e. 9%)” (8)
“Treatment was well tolerated and no side-effects were observed. There was a gender effect on survival, which has been observed in previous studies with this model, as well, with males having a shorter lifespan than females (average of 5 days). This gender difference was seen in both groups and was not modified by the treatment. Motor performance was significantly improved in SS-31-treated mice between days 100 and 130 (p < 0.005; repeated measures ANOVA followed by Fisher’s protected leastsignificant differences [PLSD] post hoc test) (Fig. 3). There was a trend towards a decrease in weight loss in the SS-31-treated group which was not statistically significant (data not shown).” (8)
Reduction of motor neuron loss:
“Attenuation of motor neuron loss by SS-31 in the ventral horn of the lumbar spinal cord of G93A mice. Photomicrographs show cresyl violet-stained sections through the ventral horn of the lumbar spinal cord from non-transgenic control (a) and G93A mice treated with vehicle (PBS) (b) or SS-31 (c) at 110 days of age. Stereological analysis revealed significantly decreased numbers of surviving neurons in G93A mice treated with vehicle relative to non-transgenic controls (***p < 0.001). This cell loss was significantly ameliorated by treatment with SS-31 (**p < 0.01). Values are means ± SEM. Differences between means were analyzed using ANOVA followed by the Newman–Keuls post hoc test.” (8)
“Cell death following incubation with H2O2 was significantly decreased by treating the cells with SS-31 (at 1, 10 or 100 lM concentrations) after exposure to H2O2. No dose dependence of the protective effect of SS-31 was seen in our study; the maximum effect was observed at the lowest concentration (1 lM) of SS-31, suggesting that SS-31 is effective in protecting both wild-type and G93A mutant SOD1 cells at submicromolar concentrations.” (8)
“We also presented the first in vivo study with this compound in an animal model of chronic neurodegeneration. SS-31 has previously demonstrated efficacy at doses of 1 and 3 mg/kg in animal models of cerebral ischemia–reperfusion (Cho et al. 2005) and renal fibrosis resulting from ureteral obstruction (Felsen et al. 2005). Administration of SS-31 in G93A ALS mice before the onset of symptoms led to a significant increase in survival and improvement of motor performance. The gain in motor function can be explained by a significantly decreased cell loss in the lumbar spinal cord in the SS-31-treated group. This amelioration of the ALS-like phenotype in the G93A mice in our study was associated with a decrease in the levels of markers of lipid peroxidation and protein nitration (4-hydroxynonenal and 3-nitrotyrosine). 4-Hydroxynonenal and malondialdehyde are the main aldehyde reaction products of the peroxidation of polyunsaturated fatty acids (Esterbauer et al. 1991). 4-Hydroxynonenal accumulates in neurons and can induce apoptosis, inhibit glucose transport and disrupt the cytoskeleton…” (8)
“Although SS-31 may also scavenge ROS in non-mitochondrial sites, such as lysosomes or peroxisomes, it was shown to concentrate approximately 5000-fold in mitochondria (Zhao et al. 2004), and non-mitochondrial concentrations of SS-31 are expected to be too low to exert much scavenging action. Further mechanisms of action of SS-31 which have been shown in vitro, such as a decrease in mitochondrial swelling and cytochrome c release, may contribute to its neuroprotective effects in G93A mice. Our results support the assumption that dysfunctional mitochondria are important contributors in the generation of oxidative stress in ALS associated with SOD1 mutations. Direct targeting of ROS production at the inner mitochondrial membrane, and therefore the prevention of further mitochondrial damage, is an interesting new approach to treat neuronal damage induced by oxidative stress.” (8)
SS-31 Reduced the Area and Sizes of Western Diet-induced Atherosclerotic Plaques.
“SS-31 administration reduced the area and sizes of western diet-induced atherosclerotic plaques and changed the composition of the plaques in ApoE-/- mice. Oxidative stress was suppressed, as evidenced by the reduced DHE stain, down-regulated 8-OHDG expression, and increased SOD activity after chronic SS-31 administration. Moreover, systemic inflammation was ameliorated as seen by decreasing serum ICAM-1, MCP-1, and IL-6 levels. Most importantly, SS-31 administration inhibited cholesterol influx by down-regulating expression of CD36 and LOX-1 to prevent lipid accumulation to further suppress the foam cell formation and atherosclerotic progression… Administration of SS-31 prevents against atherosclerotic formation in ApoE-/- mice suggesting that SS-31 might be considered to be a potential drug to prevent atherosclerotic progression. (9)
“Oxidative stress probably due to the combination of highly reactive oxygen species (ROS) generation and impaired antioxidant defense is deeply involved in the pathogenesis of atherosclerosis [5]. ROS can modulate atherosclerosis progression partially by promoting DNA damage and accelerating cellular senescence. Meanwhile, mitochondrial DNA damage and dysfunction can augment ROS production, therefore, forming a positive feedback loop [6]. ROS can up-regulate the expression of oxidized low-density lipoprotein (ox-LDL) receptors on cell surface [7] including cluster of differentiation 36 (CD36) and lectin-like ox-LDL receptor-1 (LOX-1), which play important roles to take in ox-LDL [8]. Up-regulating CD36 expression promotes atherosclerosis in ApoE-/- mice [9]. LOX-1 knockout mice exhibit reduced intima thickness, inflammation and atherosclerosis.” (9)
“The peptide Szeto-Schiller (SS)-31 (D-Arg-dimethylTyr-Lys-Phe-NH2), belonging to a family of aromatic-cationic peptides, selectively targets to mitochondrial inner membrane reacting with cardiolipin [19], preventing ROS generation, improving ATP production, and decreasing oxidative stress [20]. These anti-oxidative effects have been shown to reduce ischemia-reperfusion injury [20], protect against neurodegeneration [21], and ameliorate insulin resistance caused by high-fat diet in several animal models [22]. Recently, we reported its inhibition of foam cell formation in RAW264.7 cells [23], which are a common cellular model of atherosclerosis. Here, we further investigate the in vivo effect of SS-31 on preventing the development of atherosclerosis in a mouse model.” (9)
“Fig 1. Chronic administration of SS-31 is associated with reduced atherosclerotic lesion area in ApoE-/- mice. ApoE-/- mice fed the Western diet were treated with either 1 mg/kg SS-31 (M1 group) or 3 mg/ kg (M3 group) SS-31, or saline (P group) by subcutaneous injection daily for twelve weeks. Representative images of Oil Red O staining of aorta and aortic root are shown in (A) and (C). Quantification of positive area are shown in (B) for (A) and (D) for (C). Data represent the mean ± SEM. * p < 0.05. n = 5 for each group. The black scale bars represent 100 μm.” (9)
“Fig 2. SS-31 administration modulates the composition of atherosclerotic plaques in ApoE-/- mice.
Representative images of immunohistochemical staining are shown in (A) for CD68 (as a marker of macrophages), α-SMA (as a marker of smooth muscle), and Masson staining for collagenous fibers in aorta root. Quantification of stained area as a percentage of lesion area is given in (B), (C), and (D), respectively. Data represent the mean ± SEM. * p < 0.05. n = 5 for each group. The black scale bars represent 50 μm.” (9)
“The results showed a significant reduction of the 8-OHDG-positive area of aortic root in mice treated with SS-31 (11.74 ± 1.96% vs. 15.24 ± 2.60%, p = 0.043 for M1 vs. P; 10.75 ± 2.85% vs. 15.24 ± 2.6%, p = 0.031 for M3 vs. P, Fig 3E) compared to that in mice treated with saline. Collectively, our results indicate that SS-31 provides protective effects against atherosclerotic development most likely through reducing oxidative stress to improve energetics of aorta.” (9)
“Fig 3. Chronic administration of SS-31 improves ATP production, reduces ROS accumulation and increases the competence against oxidative stress and damage in aorta. Representative images of DHE stain are shown in (A). ATP production (B) in aorta was measured immediately after the samples were prepared in 5 mice from each group. Western blotting analysis of SOD2 expression in aorta is shown in (C) from 4 independent experiments. Enzymatic activity of SOD (n = 5 for each group) is shown in (D). Representative images of immunohistochemical staining for 8-OHDG and quantification of stained area as a percentage of lesion area are presented in E (n = 5 for each group). Data represent the mean ± SEM. * p < 0.05, ***p < 0.001. The white scale bars represent 20 μm. The black scale bars represent 50 μm.” (9)
SS-31 Modulates the Lipid Uptake of Macrophages In The Aorta of ApoE-/- Mice.
“Foam cell formation is a critical event of early atherosclerotic plaque. Uncontrolled uptake of oxidized low-density lipoprotein (ox-LDL), excessive cholesterol esterification and impaired cholesterol release contribute to accumulation of cholesterol ester (CE) stored as lipid droplets and subsequently trigger the formation of foam cells. Therefore, we used immunohistochemical staining to detect the expression of CD36 and LOX-1, two principal receptors responsible for ox-LDL influx, and ABCA1, one of the important transporter mediating cholesterol efflux. As shown in Fig 5, CD36 (M1 vs. P, p = 0.038 and M3 vs. P, p = 0.024 in Fig 5B) and LOX-1“ (9)
SS-31 Ameliorates Systemic Inflammation in ApoE-/- mice.
Chronic inflammation is one of the pathogenic features of atherosclerosis [13]. ICAM-1 and MCP-1 are the major chemokines involved in accelerating the adhesion of monocytes/macrophages onto endothelium and subsequent transmigration into intima [16, 26]. In consistence with the reduction of macrophages in plaques, ICAM-1 and MCP-1 were significantly decreased in serum of mice treated with SS-31 ( p = 0.012 and p = 0.030 for M1 and M3 vs. P, respectively (comparison order same in the following figures), in Fig 4A; p = 0.026 and p = 0.046; Fig 4B). Macrophages within the vessel wall could release pro-inflammatory cytokines including IL-6, IL-1β, and tumor necrosis factor (TNF)-α, which will further mediate distant inflammatory effects, such as activating hepatic genes encoding acute phase reactant fibrinogen, C-reactive protein (CRP) and serum amyloid A[27]. The levels of CRP and IL-6 were determined and found to be dropped in serum of mice treated with SS-31 significantly for IL-6 ( p = 0.044; p = 0.027; Fig 4C), not for CRP (p = 0.133; p = 0.369; Fig 4D). Taken together, the results suggest that daily injection of SS-31 could overall ameliorate systemic inflammation in ApoE-/- mice.” (9)
SS-31 Reduced Beta Amyloid Accumulation and Cognitive Decline Caused by Senescence Acceleration — Both Are Implicated in Alzheimer’s and Dementia.
“SS-31 also rescued learning and memory deficits in senescence-accelerated (SAMP8) mice. In addition, SS-31 repaired mitochondria structure, reduced expression of fission-related proteins, and lowered amyloid-β levels in the neurons of hippocampal CA1 of SAMP8 mice. These results suggest that restoration of mitochondrial bioenergetics can improve protein quality control…”
“Mitochondria-Targeted Antioxidant SS31 Prevents Amyloid Beta-Induced Mitochondrial Abnormalities and Synaptic Degeneration in Alzheimer’s Disease.” (13)
SS-31 Reversed Cognitive Decline by Restoring Mitochondria Function in Hippocampus, the Short-Term Memory Center of the Brain.
“We showed that cognitive deficits induced by exposure of the aging mice to isoflurane were accompanied by mitochondrial dysfunction in hippocampus due to loss of the enzymatic activity of complex I. This loss resulted in the increase of reactive oxygen species production, decrease of ATP production and mitochondrial membrane potential, and opening of mitochondrial permeability transition pore. Further, we provided evidence that the BDNF signaling pathway was involved in this process to regulate synaptic plasticity related proteins, for instance, downregulation of synapsin 1, PSD-95 and p-CREB, and upregulation of NR2A, NR2B, CaMKII and CaMKII. Of note, the isoflurane-induced cognitive deficits were rescued by SS-31 through reversal of mitochondrial dysfunction, which facilitated the regulation of BDNF signaling including the expression reversal of aforementioned important synaptic-signaling proteins in aging mice. Our data demonstrate that reversing mitochondrial dysfunction by SS-31 enhances BDNF signaling pathway and synaptic plasticity, and provides protective effects on cognitive function, thereby support the notion that SS-31 may have therapeutic benefits for elderly humans undertaking anesthesia.” (5)
- “SS-31 attenuated isoflurane-Induced cognitive deficits in
aging mice - SS-31 reversed isoflurane-induced mitochondrial dysfunction
by improving ETC enzymatic activities in aging mice - SS-31 protected hippocampus against isoflurane-induced
mitochondrial dysfunction by maintaining ROS, ATP, and MMP
levels and mPTP opening in aging mice - Reversal of isoflurane-induced mitochondrial dysfunction
enhanced hippocampal BDNF pathway and synaptic plasticity in
aging mice - Intact mitochondria maintained by SS-31 consolidated
hippocampal NMDA-CaMKII- CREB signaling in aging mice after
anesthesia”
“Regulatory role of the protected mitochondria by SS-31 pretreatment on BDNF pathway and synaptic plasticity in isoflurane-treated aging mice. BDNF/TrkB pathway (A) and two kinds of synaptic-structural proteins (B) were detected. Values are presented as mean ± SEM (n = 6). *p < 0.05, versus the control group; #p < 0.05 versus the isoflurane group.” (5)
“Protective effects of SS-31 on mitochondria concerning ROS, ATP, and MMP levels and mPTP opening in isoflurane-anesthetized aging mice. The ROS levels (A), ATP production (B), MMP (C), and Opening of mPTP (D) were measured. Values are presented as mean ± SEM (n = 6). *p < 0.05, versus the control group; #p < 0.05 versus the isoflurane group.” (5)
“Current researches have firmly established the role of CaMKII as a major mediator of the postsynaptic mechanisms of hippocampal LTP. However, we found the increases of CaMKII and CaMKII expression and impairment of the hippocampus dependent memory after exposure to isoflurane. These results might provide evidence that CaMKII would also mediate a signaling required for long-term depression (LTD). Similar results were presented by Coultrap and colleagues that CaMKII indeed induces both LTP and LTD, two opposing forms of synaptic plasticity. Meanwhile, p-CREB level was found to be decreased in the hippocampus after exposure to isoflurane as reported previously, which is in consistence with the hypothesis that phosphorylation of CREB is necessary for the maintenance of LTP. Thus, SS-31 pretreatment consolidated the hippocampal NMDA-CaMKII-CREB signaling by selectively reversing the protein expression or phosphorylation, i.e. protection of mitochondria specifically rescues the structure and function of synapses against cognitive deficits” (5)
“BDNF/TrkB has also been shown to regulate the phosphorylation, trafficking, and expression of N-methyl-D-aspartate (NMDA) receptor subunits. The actions of BDNF on NMDA receptor in the hippocampus have direct implications in concerning its ability to facilitate Ca2+ influx. Thus, in turn, CaMKII/CREB intracellular signaling pathway could be activated, as observed in cellular models of learning and memory, such as long-term potentiation (LTP). Our data showed that NR2A, NR2B, CaMKII, and CaMKII were upregulated and p-CREB was downregulated, in accompany with the downregulated BNDF/TrkB signaling after the aging mice were exposed to isoflurane. Moreover, SS-31 pretreatment timely regulated and rectified NMDA-CaMKII-CREB signaling with an exception of NR2A. The different response between NR2A and NR2B to SS-31 administration may reflect the age-related expression and suggest a role of NR2B as a direct recruiter of CaMKII to the synapse.” (5)
“Integrative mitochondria consolidated NMDA-CaMKII-CREB signaling in the anesthetized aging mice. Two NMDA receptor subunits (A) and two CaMKII isoforms and cAMP response element binding protein (B) were determined. Values are presented as mean ± SEM (n = 6). *p < 0.05, versus the control group; #p < 0.05 versus the isoflurane group.” (5)
Sourced Studies:
(3) Cano Sanchez, Mariola, et al. “Targeting Oxidative Stress and Mitochondrial Dysfunction in the Treatment of Impaired Wound Healing: A Systematic Review.” Antioxidants, vol. 7, no. 8, 24 July 2018, p. 98, 10.3390/antiox7080098.
(9) Zhang, Meng, et al. “Chronic Administration of Mitochondrion-Targeted Peptide SS-31 Prevents Atherosclerotic Development in ApoE Knockout Mice Fed Western Diet.” PLoS ONE, vol. 12, no. 9, 29 Sept. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5621700/, 10.1371/journal.pone.0185688.
(13) Europe PMC. “Europe PMC.” Europepmc.Org, 2019, europepmc.org/article/pmc/pmc3513393.