cytochrome-c-t and Parkinsonian-Disorders

Human ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved core subunit of mitochondrial respiratory chain complex III. We recently identified the disease-associated variants of UQCRC1 from patients with familial parkinsonism, but its function remains unclear. Here we investigate the endogenous function of UQCRC1 in the human neuronal cell line and the Drosophila nervous system. Flies with neuronal knockdown of uqcrc1 exhibit age-dependent parkinsonism-resembling defects, including dopaminergic neuron reduction and locomotor decline, and are ameliorated by UQCRC1 expression. Lethality of uqcrc1-KO is also rescued by neuronally expressing UQCRC1, but not the disease-causing variant, providing a platform to discern the pathogenicity of this mutation. Furthermore, UQCRC1 associates with the apoptosis trigger cytochrome c (cyt-c), and uqcrc1 deficiency increases cyt-c in the cytoplasmic fraction and activates the caspase cascade. Depleting cyt-c or expression of the anti-apoptotic p35 ameliorates uqcrc1-mediated neurodegeneration. Our findings identify a role for UQCRC1 in regulating cyt-c-induced apoptosis.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Cell Line, Tumor; Cytochromes c; Cytoplasm; Dopaminergic Neurons; Drosophila; Drosophila Proteins; Electron Transport Complex III; Gene Editing; Humans; Larva; Locomotion; Mitochondria; Parkinsonian Disorders; Protein Binding; Reactive Oxygen Species; RNA Interference

Parkinson's disease is a progressive neurodegenerative movement disorder with the cardinal symptoms of bradykinesia, resting tremor, rigidity, and postural instability, which lead to abnormal movements and lack of activity, which in turn cause muscular damage. Even though studies have been carried out to elucidate the causative factors that lead to muscular damage in Parkinson's disease, apoptotic events that occur in the skeletal muscle and a therapeutical approach to culminate the muscular damage have not been extensively studied. Thus, this study evaluates the impact of rotenone-induced SNPc lesions on skeletal muscle apoptosis and the efficacy of an ethyl acetate extract of Morinda citrifolia in safeguarding the myocytes. Biochemical assays along with apoptotic markers studied by immunoblot and reverse transcription-polymerase chain reaction in the current study revealed that the supplementation of Morinda citrifolia significantly reverted alterations in both biochemical and histological parameters in rotenone-infused PD rats. Treatment with Morinda citrifolia also reduced the expression of pro-apoptotic proteins Bax, caspase-3 and caspase-9 and blocked the release of cytochrome c from mitochondria induced by rotenone. In addition, it augmented the expression of Bcl2 both transcriptionally and translationally. Thus, this preliminary study paves a way to show that the antioxidant and anti-apoptotic activities of Morinda citrifolia can be exploited to alleviate skeletal muscle damage induced by Parkinsonism.

Topics: Animals; Antioxidants; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Creatine Kinase; Cytochromes c; Disease Models, Animal; L-Lactate Dehydrogenase; Male; Mitochondria; Morinda; Muscle, Skeletal; Oxidative Stress; Parkinsonian Disorders; Pars Compacta; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Rotenone; Up-Regulation

In response to apoptotic stimuli, mitochondria in mammalian cells release cytochrome c and other apoptogenic proteins, leading to the subsequent activation of caspases and apoptotic cell death. This process is promoted by the pro-apoptotic members of the Bcl-2 family of proteins, such as Bim and Bax, which, respectively, initiate and execute cytochrome c release from the mitochondria. Here we report the discovery of a small molecule that efficiently blocks Bim-induced apoptosis after Bax is activated on the mitochondria. The cellular target of this small molecule was identified to be the succinate dehydrogenase subunit B (SDHB) protein of complex II of the mitochondrial electron transfer chain (ETC). The molecule protects the integrity of the ETC and allows treated cells to continue to proliferate after apoptosis induction. Moreover, this molecule blocked dopaminergic neuron death and reversed Parkinson-like behavior in a rat model of Parkinson's disease.

Topics: Animals; Antiparkinson Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Behavior, Animal; Cell Proliferation; Cytochromes c; Disease Models, Animal; Dopaminergic Neurons; Dose-Response Relationship, Drug; Electron Transport; HeLa Cells; Humans; Mitochondria; Motor Activity; Neuroprotective Agents; Oxidopamine; Parkinsonian Disorders; Protein Binding; Pyridones; Rats; RNA Interference; Signal Transduction; Succinate Dehydrogenase; Sulfones; Time Factors; Transfection

The pathological features of Parkinson's disease (PD) include an abnormal accumulation of α-synuclein in the surviving dopaminergic neurons. Though PD is multifactorial, several epidemiological reports show an increased incidence of PD with co-exposure to pesticides such as Maneb and paraquat (MP). In pesticide-related PD, mitochondrial dysfunction and α-synuclein oligomers have been strongly implicated, but the link between the two has not yet been understood. Similarly, the biological effects of α-synuclein or its radical chemistry in PD is largely unknown. Mitochondrial dysfunction during PD pathogenesis leads to release of cytochrome c in the cytosol. Once in the cytosol, cytochrome c has one of two fates: It either binds to apaf1 and initiates apoptosis or can act as a peroxidase. We hypothesized that as a peroxidase, cytochrome c leaked out from mitochondria can form radicals on α-synuclein and initiate its oligomerization.. Samples from controls, and MP co-exposed wild-type and α-synuclein knockout mice were studied using immuno-spin trapping, confocal microscopy, immunohistochemistry, and microarray experiments.. Experiments with MP co-exposed mice showed cytochrome c release in cytosol and its co-localization with α-synuclein. Subsequently, we used immuno-spin trapping method to detect the formation of α-synuclein radical in samples from an in vitro reaction mixture consisting of cytochrome c, α-synuclein, and hydrogen peroxide. These experiments indicated that cytochrome c plays a role in α-synuclein radical formation and oligomerization. Experiments with MP co-exposed α-synuclein knockout mice, in which cytochrome c-α synuclein co-localization and interaction cannot occur, mice showed diminished protein radical formation and neuronal death, compared to wild-type MP co-exposed mice. Microarray data from MP co-exposed wild-type and α-synuclein knockout mice further showed that the absence of α-synuclein per se or its co-localization with cytochrome c confers protection from MP co-exposure, as several important pathways were unaffected in α-synuclein knockout mice.. Altogether, these results show that peroxidase activity of cytochrome c contributes to α-synuclein radical formation and oligomerization, and that α-synuclein, through its co-localization with cytochrome c or on its own, affects several biological pathways which contribute to increased neuronal death in an MP-induced model of PD.

Topics: alpha-Synuclein; Animals; Cell Death; Cytochromes c; Free Radicals; Immunohistochemistry; Male; Maneb; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Neurons; Oligonucleotide Array Sequence Analysis; Oxidative Stress; Paraquat; Parkinsonian Disorders; Pesticides

Ropinirole is a pharmacologically active agent used in the treatment of Parkinson's disease (PD) that directly acts on dopamine receptors. Recent studies reported that ropinirole has neuroprotective potential. However, there have been no detailed studies on apoptosis. Here we demonstrated the protective effects of ropinirole against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity in a mouse model of PD. Unlike previous studies, we focused on apoptotic pathways. To confirm the protective effect of ropinirole, we conducted behavioral tests and tyrosine hydroxylase (TH)-immunohistochemistry. In the pole test and the rotarod test, commonly used behavioral tests in the mouse model of PD, ropinirole treatment (0.5, 1, or 2 mg/kg) maintained movement ability against MPTP-induced changes in motor coordination and postural balance, and bradykinesia. Our histological analyses illustrated that ropinirole significantly reduced MPTP-induced dopaminergic neuron damage in both the substantia nigra pars compacta (SNpc) and the striatum. In addition, we performed Western blot or kit analysis to measure apoptosis-related protein levels in the SNpc. Ropinirole increased the Bcl-2/Bax ratio, transcription factor A, and nuclear respiratory factor 1 and inhibited cytosolic cytochrome c release and caspase-3 activity, indicating that ropinirole inhibited the apoptotic cascade. These results suggest that ropinirole has neuroprotective effects in a mouse model of PD by inhibiting apoptosis.

Topics: Animals; Antiparkinson Agents; Apoptosis; bcl-2-Associated X Protein; Behavior, Animal; Caspase 3; Cytochromes c; DNA-Binding Proteins; Dopamine Agonists; Dopaminergic Neurons; High Mobility Group Proteins; Indoles; Male; Mice; Mice, Inbred C57BL; MPTP Poisoning; Neuroprotective Agents; Nuclear Respiratory Factor 1; Parkinsonian Disorders; Proto-Oncogene Proteins c-bcl-2; Substantia Nigra

An association between excessive zinc (Zn) accumulation in brain and incidences of Parkinson's disease (PD) has been shown in several epidemiological and experimental investigations. The involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and glutathione (GSH) in the pathogenesis of PD has also been proposed in a few studies. Despite the implicated role of oxidative stress in PD, the entire mechanism of Zn-induced dopaminergic neurodegeneration has not yet been clearly understood. The present study aimed to investigate the involvement of NADPH oxidase and GSH in Zn-induced dopaminergic neurodegeneration and also to assess its similarity with paraquat (PQ)-induced rat model of PD. Male Wistar rats were treated either with Zn (20 mg/kg; i.p.) or PQ (5 mg/kg; i.p.) in the presence and absence of NADPH oxidase inhibitor, apocynin (10 mg/kg; i.p.) and a GSH precursor, N-acetyl cysteine (NAC; 200 mg/kg; i.p.) either alone or in combination along with the respective controls. Apocynin and/or NAC pre-treatment significantly alleviated Zn- and PQ-induced changes in neurobehavioral deficits, number of dopaminergic neurons and contents of the striatal dopamine and its metabolites. Apocynin and/or NAC also mitigated Zn- and PQ-induced alterations in oxidative stress, NADPH oxidase activation and cytochrome c release, caspases-9 and -3 activation and CD11b expression. The results obtained thus suggest that Zn induces oxidative stress via the activation of NADPH oxidase and depletion of GSH, which in turn activate the apoptotic machinery leading to dopaminergic neurodegeneration similar to PQ.

Topics: Acetophenones; Acetylcysteine; Animals; Apoptosis; Caspases; CD11b Antigen; Corpus Striatum; Cytochromes c; Dopamine; Dopaminergic Neurons; Enzyme Inhibitors; Glutathione; Male; Membrane Glycoproteins; Mitochondria; Motor Activity; NADPH Oxidase 2; NADPH Oxidases; Oxidative Stress; Paraquat; Parkinsonian Disorders; Phosphoproteins; Rats; Rats, Wistar; Serotonin; Substantia Nigra; Tyrosine 3-Monooxygenase

Multiple system atrophy (MSA) is an adult-onset sporadic neurodegenerative disorder of unknown etiology featuring parkinsonism, ataxia, and autonomic failure in any combination. Because disease progression in MSA is rapid and no drug treatment consistently benefits MSA patients in the long term, neuroprotective or regenerative strategies may be invaluable in the management of MSA patients. In this study, we investigated whether human mesenchymal stem cells (hMSCs) had a protective effect on MSA using an animal model of double-toxin-induced MSA parkinsonism (MSA-P). MSA-P was established with coinjections of MPTP and 3-NP; hMSCs were injected into the tail vein 1 day after the last toxin injection. Three groups of mice were compared (i.e., control, MPTP + 3-NP, and MPTP + 3-NP with hMSC treatment) through histopathological, behavioral, and Western blot analyses. In the substantia nigra (SN) and the striatum, 2.0% and 3.8% of total injected hMSCs were observed, respectively. Compared with double-toxin-treated mice, hMSC treatment in double-toxin-treated mice significantly increased survival of TH- and NeuN-immunoreactive cells in the SN and the striatum, with coincident improvement in motor behavior. Additionally, hMSC treatment significantly decreased double-toxin-induced microglial and astroglial activation in the SN and striatum. Western blot analysis showed that hMSC administration in double-toxin-treated mice increased the expression of p-Akt and Bcl-2 and decreased Bax and cytochrome c expression. This study demonstrates that hMSC treatment protected against loss of neurons in the SN and the striatum induced by double toxin exposure, which may be mediated by modulation of inflammatory and cell survival and death signaling-pathway as the hMSCs migrated from the peripheral circulation into the SN and striatum.

Topics: Animals; bcl-2-Associated X Protein; Corpus Striatum; Cytochromes c; Disease Models, Animal; Humans; Male; Mesenchymal Stem Cell Transplantation; Mice; Mice, Inbred C57BL; Motor Activity; MPTP Poisoning; Multiple System Atrophy; Neurotoxins; Nitro Compounds; Parkinsonian Disorders; Propionates; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Substantia Nigra

Mutations in the ATP13A2 gene are associated with Kufor-Rakeb syndrome (KRS) and are found also in patients with various other types of parkinsonism. ATP13A2 encodes a predicted lysosomal P5-type ATPase that plays important roles in regulating cation homeostasis. Disturbance of cation homeostasis in brains is indicated in Parkinson disease pathogenesis. In this study, we explored the biological function of ATP13A2 as well as the pathogenic mechanism of KRS pathogenic ATP13A2 mutants. The results revealed that wild-type ATP13A2, but not the KRS pathogenic ATP13A2 mutants, protected cells from Mn(2+)-induced cell death in mammalian cell lines and primary rat neuronal cultures. In addition, wild-type ATP13A2 reduced intracellular manganese concentrations and prevented cytochrome c release from mitochondria compared with the pathogenic mutants. Furthermore, endogenous ATP13A2 was up-regulated upon Mn(2+) treatment. Our results suggest that ATP13A2 plays important roles in protecting cells against manganese cytotoxicity via regulating intracellular manganese homeostasis. The study provides a potential mechanism of KRS and parkinsonism pathogenesis.

Topics: Animals; Brain; Cell Death; Cytochromes c; Cytosol; HEK293 Cells; Homeostasis; Humans; Manganese; Mitochondria; Mutation; Nerve Tissue Proteins; Neurons; Parkinsonian Disorders; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley

Proteasomal dysfunction and apoptosis are major hallmarks in the pathophysiology of Parkinson's disease (PD). PARK6 which is caused by mutations in the mitochondrial protein kinase PINK1 is a rare autosomal-recessively inherited disorder mimicking the clinical picture of PD. To investigate the cytoprotective physiological function of PINK1, we used primary fibroblasts from three patients homozygous for G309D-PINK1 as well as SHEP neuroblastoma cells stably overexpressing GFP-tagged wild type (wt) PINK1. Here we demonstrate that overexpression of wt PINK1 inhibits activation of Bax and release of cytochrome c, thereby diminishing caspase 9 processing and effector caspase activity after induction of proteasomal stress with the proteasome inhibitor (PI) MG132 in SHEP cells. Conversely, effector caspase activation induced by PIs, but not by the unrelated apoptotic stimulus staurosporine was potently enhanced in primary fibroblasts from homozygous PARK6 patients in comparison to those of heterozygous carriers or unaffected siblings. SHEP cells overexpressing wt PINK1 showed an elevated expression of the cytoprotective gene parkin, whereas PARK6 fibroblasts displayed significantly decreased expression of parkin in comparison to wild type control cells. Interestingly, overexpressed GFP-PINK1 was exclusively localized in the mitochondria of SHEP cells, but was redistributed to the cytoplasm under conditions of proteasomal stress. Our data indicate that PINK1 plays an important and specific physiological role in protecting cells from proteasomal stress, and suggest that PINK1 might exert its cytoprotective effects upstream of mitochondria engagement.

Topics: Analysis of Variance; Apoptosis; Blotting, Western; Cell Fractionation; Cysteine Proteinase Inhibitors; Cytochromes c; Cytoplasm; Fibroblasts; Flow Cytometry; Humans; Leupeptins; Microscopy, Confocal; Mitochondria; Parkinsonian Disorders; Proteasome Endopeptidase Complex; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Skin

Pramipexole, an agonist for dopamine (DA) D2/D3-receptors, has been used to treat both early and advanced Parkinson's disease (PD). In this study, we examined the effect of pramipexole on DA neurons in a PD model of C57BL/6 mice, which were treated with rotenone (30 mg/kg, p.o.) daily for 28 days. Pramipexole (1 mg/kg, i.p.) was injected daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone caused a loss of DA neurons in the substantia nigra pars compacta (SNpc), motor deficits and the up-regulation of alpha-synuclein immunoreactivity in some surviving DA neurons. Pramipexole inhibited rotenone-induced DA neuronal death and motor deficits, and reduced immunoreactivity for alpha-synuclein. In addition, pramipexole inhibited the in vitro oligomerization of human wild-type alpha-synuclein by H(2)O(2)plus cytochrome c. To examine the neuroprotective effect of pramipexole against oxidative stress, we used a DJ-1-knockdown SH-SY5Y cell line and electron spin resonance (ESR) spectrometry. Simultaneous treatment with H(2)O(2) and pramipexole resulted in the significant protection of DJ-1-knockdown cells against cell death in a concentration-dependent manner. A high concentration of pramipexole directly scavenged hydroxyl radical (*OH) generated from H(2)O(2) and Fe(2+). Furthermore, pramipexole increased Bcl-2 immunoreactivity in DA neurons in the SNpc. These results suggest that pramipexole may protect DA neurons against exposure to rotenone by chronic oral administration, and this effect is mediated by multiple functions including scavenging of *OH and induction of Bcl-2 protein.

Topics: alpha-Synuclein; Animals; Antiparkinson Agents; Apoptosis; Benzothiazoles; Cell Line, Tumor; Corpus Striatum; Cytochromes c; Dopamine; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Hydroxyl Radical; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neural Pathways; Neurotoxins; Oxidative Stress; Parkinsonian Disorders; Pramipexole; Proto-Oncogene Proteins c-bcl-2; Rotenone; Substantia Nigra; Uncoupling Agents

Mutations in the PTEN induced putative kinase 1 (PINK1) gene cause an autosomal recessive form of Parkinson disease (PD). So far, no substrates of PINK1 have been reported, and the mechanism by which PINK1 mutations lead to neurodegeneration is unknown. Here we report the identification of TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone also known as heat shock protein 75 (Hsp75), as a cellular substrate for PINK1 kinase. PINK1 binds and colocalizes with TRAP1 in the mitochondria and phosphorylates TRAP1 both in vitro and in vivo. We show that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Moreover, we find that the ability of PINK1 to promote TRAP1 phosphorylation and cell survival is impaired by PD-linked PINK1 G309D, L347P, and W437X mutations. Our findings suggest a novel pathway by which PINK1 phosphorylates downstream effector TRAP1 to prevent oxidative-stress-induced apoptosis and implicate the dysregulation of this mitochondrial pathway in PD pathogenesis.

Topics: Animals; Apoptosis; Base Sequence; Cytochromes c; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; Mitochondria; Oxidative Stress; Parkinsonian Disorders; PC12 Cells; Phosphorylation; Point Mutation; Protein Binding; Protein Kinases; Rats; Recombinant Proteins; RNA, Small Interfering; Substrate Specificity; Transfection

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). We investigated molecular mechanisms underlying PINK1 neuroprotective function and PARK6 mutation-induced loss of PINK1 function. Overexpression of wild-type PINK1 blocked mitochondrial release of apoptogenic cytochrome c, caspase-3 activation and apoptotic cell death induced by proteasome inhibitor MG132. N-terminal truncated PINK1 (NDelta35), which lacks mitochondrial localization sequence, did not block MG132-induced cytochrome c release and cytotoxicity. Despite mitochondrial expression, PARK6 mutant (E240K), (H271Q), (G309D), (L347P), (E417G) and C-terminal truncated (CDelta145) PINK1 failed to inhibit MG132-induced cytochrome c release and caspase-3 activation. Overexpression of wild-type PINK1 blocked cytochrome c release and cell death caused by atractyloside, which opens mitochondrial permeability transition pore (mPTP). PARK6 PINK1 mutants failed to inhibit atractyloside-induced cytochrome c release. These results suggest that PINK1 exerts anti-apoptotic effect by inhibiting the opening of mPTP and that PARK6 mutant PINK1 loses its ability to prevent mPTP opening and cytochrome c release.

Topics: Apoptosis; Brain; Caspase 3; Cell Line; Cytochromes c; Cytoprotection; Energy Metabolism; Enzyme Inhibitors; Humans; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mutation; Neurons; Parkinsonian Disorders; Protein Kinases

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Caspases; Cytochromes c; Disease Models, Animal; Dopamine; Drug Resistance; Genetic Predisposition to Disease; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; MPTP Poisoning; Nerve Degeneration; Neurons; Parkinsonian Disorders; Proteins; Signal Transduction; Substantia Nigra; Up-Regulation