Mitochondria targeted peptides protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity

Abstract

A large body of evidence suggests that mitochondrial dysfunction and oxidative damage play a role in the pathogenesis of Parkinson's disease (PD). A number of antioxidants have been effective in animal models of PD. We have developed a family of mitochondria-targeted peptides that can protect against mitochondrial swelling and apoptosis (SS peptides). In this study, we examined the ability of two peptides, SS-31 and SS-20, to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice. SS-31 produced dose-dependent complete protection against loss of dopamine and its metabolites in striatum, as well as loss of tyrosine hydroxylase immunoreactive neurons in substantia nigra pars compacta. SS-20, which does not possess intrinsic ability in scavenging reactive oxygen species, also demonstrated significant neuroprotective effects on dopaminergic neurons of MPTP-treated mice. Both SS-31 and SS-20 were very potent (nM) in preventing MPP+ (1-methyl-4-phenylpyridinium)-induced cell death in cultured dopamine cells (SN4741). Studies with isolated mitochondria showed that both SS-31 and SS-20 prevented MPP+-induced inhibition of oxygen consumption and ATP production, and mitochondrial swelling. These findings provide strong evidence that these neuroprotective peptides, which target both mitochondrial dysfunction and oxidative damage, are a promising approach for the treatment of PD.

FIG. 1.

The neuroprotective effect of SS-31 and SS-20 on mice treated with MPTP. (A) In a moderate MPTP modality (10 mg/kg, three doses in 2 h interval), dopamine depletion in striatum by MPTP toxin was 44%, and SS-31 showed a significant dose-respondent protection effect on MPTP-induced dopamine depletion. DOPAC and HVA levels showed dose-respondent protection effects of SS-31. (B) 4 mg/kg SS-20 in a moderate MPTP modality (10 mg/kg, three doses in every 2 h) significantly protected dopamine, DOPAC, and HVA from MPTP-induced depletion. #p < 0.01 compared with normal control. *p < 0.05 compared with MPTP alone, n = 10.

FIG. 2.

SS-31 and SS-20 protect dopaminergic neurons in substantia nigra against MPTP damage. (A) Representative slides showed TH-positive neuronal loss in SNpc caused by MPTP toxicity and protected by either SS-31 or SS-20. (B) TH-positive neuronal counts in SNpc showed SS-31 (0.5, 1, and 5 mg/kg) dose-dependently protected neurons from MPTP (10 mg/kg, three doses in 2 h interval) caused cell loss and SS-20 (4 mg/kg) also attained significant protection. (C) Nissl stained neuron counts in SNpc verified the enzyme stained TH-positive neuronal loss. #p < 0.05 compared with normal control. *p < 0.05 compared with MPTP alone, n = 10.

FIG. 3.

SS-31 and SS-20 reduced MPP⁺-induced cell death in SN4741 dopamine cells. SN4741 cells were treated with MPP⁺ (100 μM) for 48 h in the absence or presence of SS peptides. Cell viability was quantified by the Almar Blue assay. (A) Concurrent treatment with SS-31 or SS-20 dose-dependently attenuated the reduction in cell viability induced by MPP⁺ *p < 0.05; **p < 0.01 compared to treatment with MPP⁺ alone. (B) SS-31 and S-S20 reduced apoptotic cell death induced by 100 μM MPP⁺. Apoptotic cells were evaluated by microscopy using the Hoechst 33324 dye and cells with fragmented or condensed nuclei were scored as apoptotic. **p < 0.01 compared to treatment with MPP⁺ alone.

FIG. 4.

SS-31 and SS-20 reduced MPP⁺-induced inhibition of mitochondrial oxygen consumption and ATP production. Isolated mouse liver mitochondria were incubated in respiratory buffer and oxygen consumption was measured by a Clark-type oxygen electrode. State 3 respiration was initiated by the addition of ADP. Co-incubation with 50 μM SS-31 or 10 μM and 50 μM SS-20 significantly attenuated the inhibition of oxygen consumption induced by 100 μM MPP⁺. ATP production was determined by using luciferase bioluminescent assay. Co-incubation with both doses of 10 μM and 50 μM of either SS-31 or SS-20 significantly attenuated the inhibition of ATP production induced by 100 μM MPP⁺. *p < 0.05; **p < 0.01 compared to treatment with MPP⁺ alone. Incubation with SS-31 or SS-20 alone did not alter oxygen consumption and ATP production (data not shown).

FIG. 5.

SS-31 and SS-20 reduced mitochondrial swelling induced by 300 μM MPP⁺ in the presence of 50 μM Ca²⁺ and Pi. Mitochondrial swelling was measured by decrease in absorbance at 540 nm. Addition of MPP⁺ alone dose-dependently increased mitochondrial swelling (A). The light gray line indicates no MPP⁺ added. The swelling induced by 300 μM MPP⁺ was prevented by addition of 50 μM SS-31 (B) or 50 μM SS-20 (C).