Parkinson's disease genetic mutations increase cell susceptibility to stress: Mutant α-synuclein enhances H2O2- and Sin-1-induced cell death
Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder with high prevalence in the aging, and affects 2% of the population over the age of 60 years [24]. PD is characterized clinically by tremor, muscle rigidity, disturbances of balance, and bradykinesia. The two pathological hallmarks of PD are loss of dopaminergic neurons and the presence of cytoplasmic eosinophilic inclusions named Lewy bodies [24]. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors [8], [24], [35], [87]. There are at least five well-defined genetic forms of PD including α-synuclein- and Leucine rich repeat kinase 2 (LRRK2)- parkin-, DJ-1- and PINK-1-linked PD [11], [12], [42], [61], [65], [86], [89].
α-Synuclein, the first identified PD-associated protein, is a major protein component in Lewy bodies and Lewy neurites in sporadic PD [24]. The function of α-synuclein is not well understood. Data from knock-out mice suggests that α-synuclein may play a role in neuronal plasticity [15] and may regulate synaptic maturation and maintenance [1], [13], [17]. Three mutations (A53T, A30P, and E46K) in the α-synuclein gene cause rare familial forms of PD [45], [65], [86]. Genetic duplication or triplication at the α-synuclein locus leading over-expression of α-synuclein protein also cause genetic PD [72]. Variation in levels of α-synuclein inherited by promoter variants may also contribute to the risk of developing PD [21], [63]. α-Synuclein over-expression results in the degeneration of dopaminergic neurons and motor deficits in Drosophila melanogaster and transgenic mice [6], [14], [22], [30], [47]. These findings indicate that α-synuclein may play a role in both familial and sporadic PD pathogenesis.
Proteasome inhibition, oxidative stress and nitrative damages are increased in aging and PD [8], [27], [53], [76], [78]. Studies from PD patients and animal models indicate involvement of free radicals, oxidative, nitrative stress and proteasome inhibition in the pathogenesis of PD [2], [3], [19], [71]. We previously demonstrated that inducible expression of A30P, but not wild type α-synuclein in media containing 1% HS and 0.5% FBS is only toxic in combination with subtoxic doses of a proteasome inhibitior, lactacystin [81]. Recently we determined that in media without serum but containing N2 supplement and nerve growth factor (NGF, induction and differentiation media), expression of A53T α-synuclein alone caused endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and led to cell death. By contrast, induction of wild type or A30P α-synuclein expression in similarly cultured cells with the same media was not toxic [74]. In this study, we investigated the combination of genetic alteration of α-synuclein and cellular stress using α-synuclein (wild type, A30P and A53T) inducible PC12 cell models. We demonstrated that using regular growth media (10% HS and 5% FBS), induction of either wild type or mutant (A30P or A53T) α-synuclein expression was not toxic, but induced biochemical changes indication of cell vulnerability, including decrease of proteasome activity, and increase of intracellular reactive oxygen species (ROS) and 3-nitrotyrosine levels. We further found that inducible expression of mutant α-synuclein (A30P or A53T) enhances lactacystin-, H2O2- and Sin-1-induced cell death, compared with non-induced conditions. These results suggest that genetic alterations in α-synuclein may lead to increased neuronal vulnerability to oxidative and nitrative damage in PD pathogenesis.
Section snippets
Cell culture, inducible cell lines, cell death and viability assays
Media and N2 supplements for cell culture were from Invitrogen (Carlsbad, CA).
PC12 cell lines expressing inducible wild type, A30P, or A53T α-synuclein were described previously [74], [81], and grown in DMEM containing 10% horse serum and 5% FBS in a 5% CO2 atmosphere. Trypan blue exclusion was used to measure cell death by counting the number of dead (blue) and live cells in the cultures after induction of expression. Lactate dehydrogenase (LDH) cytotoxicity assay was performed according to
Induction of α-synuclein expression in PC12 cells
To study the role of α-synuclein in PD pathogenesis, we employed PC12 cell lines in which α-synuclein (wild type, A30P or A53T) was inducible using the Tet-off gene regulatory system as described [74], [81]. In the absence of doxycycline (Dox), the tetracycline-controlled transactivator (tTA) binds to the tetracycline-responsive element (TRE) of the α-synuclein construct, inducing expression of the protein. Conversely, in the presence of Dox, tTA does not bind to the promoter, suppressing
Discussion
Expression of α-synuclein has been achieved in a number of cellular systems, with results ranging from a lack of any effect after over-expression [43], to adverse effects of varying severity [38], [39], [40], [46], [48], [57], [60], [64], [68], [77], [80], [81], [84], [85]. We have shown that the expression of A30P mutant α-synuclein heightens the cytotoxic response of cells challenged with lactacystin in media containing 1% HS and 0.5% FBS [81]. Recently we have found that cell toxicity was
Acknowledgement
This research was funded by NINDS NS38377, Udall PD Research Center, National Institutes of Health.
References (89)
- et al.
Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system
Neuron
(2000) Oxidatively modified proteins in aging and disease
Free Radic Biol Med
(2002)- et al.
Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation
Mol Cell
(2005) - et al.
Ubiquitin-proteasome system and Parkinson's diseases
Exp Neurol
(2005) - et al.
The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease
Trends Neurosci
(1998) - et al.
To live or die by the sword: the regulation of apoptosis by the proteasome
Dev Cell
(2004) - et al.
Immunodetection of 3-nitrotyrosine in the liver of zymosan-treated rats with a new monoclonal antibody: comparison to analysis by HPLC
Free Radic Biol Med
(2001) - et al.
Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction
J Biol Chem
(1999) - et al.
Effect of wild-type or mutant Parkin on oxidative damage, nitric oxide, antioxidant defenses, and the proteasome
J Biol Chem
(2002) - et al.
Human alpha-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine
Neurosci Lett
(2002)
Enhanced vulnerability to oxidative stress by alpha-synuclein mutations and C-terminal truncation
Neuroscience
Dopamine induces proteasome inhibition in neural PC12 cell line
Free Radic Biol Med
Disease-related prion protein forms aggresomes in neuronal cells leading to caspase activation and apoptosis
J Biol Chem
The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity
J Biol Chem
Synuclein, dopamine and oxidative stress: co-conspirators in Parkinson's disease?
Brain Res Mol Brain Res
Proteasomal function is impaired in substantia nigra in Parkinson's disease
Neurosci Lett
Metalloenzyme-like activity of Alzheimer's disease beta-amyloid. Cu-dependent catalytic conversion of dopamine, cholesterol, and biological reducing agents to neurotoxic H(2)O(2)
J Biol Chem
Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease
Neuron
The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B
Cell
Parkin protects against the toxicity associated with mutant alpha-synuclein: proteasome dysfunction selectively affects catecholaminergic neurons
Neuron
The ubiquitin-proteasome pathway in Parkinson's disease and other neurodegenerative diseases
Trends Cell Biol
The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53
Cell
Neurobiology of Huntington's disease
Neurobiol Dis
Dityrosine cross-linking promotes formation of stable alpha-synuclein polymers. Implication of nitrative and oxidative stress in the pathogenesis of neurodegenerative synucleinopathies
J Biol Chem
Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease
Free Radic Biol Med
alpha-Synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro
Free Radic Biol Med
Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection
J Biol Chem
Differential cytotoxicity of dopamine and H2O2 in a human neuroblastoma divided cell line transfected with alpha-synuclein and its familial Parkinson's disease-linked mutants
Neurosci Lett
Oxidative stress and genetics in the pathogenesis of Parkinson's disease
Neurobiol Dis
DNA damage by nitrite and peroxynitrite: protection by dietary phenols
Meth Enzymol
Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology
Neuron
A generalised increase in protein carbonyls in the brain in Parkinson's but not incidental Lewy body disease
J Neurochem
Oxidative DNA damage in the parkinsonian brain: an apparent selective increase in 8-hydroxyguanine levels in substantia nigra
J Neurochem
Mitochondrial dysfunction and oxidative stress in aging and neurodegenerative disease
J Neural Transm Suppl
Amyotrophic lateral sclerosis associated with mutations in the CuZn superoxide dismutase gene
Curr Neurol Neurosci Rep
Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease
Science
Impairment of the ubiquitin-proteasome system by protein aggregation
Science
Autosomal recessive early onset parkinsonism is linked to three loci: PARK2, PARK6, and PARK7
Neurol Sci
Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism
Science
Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein
J Neurosci
alpha-Synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease
Nat Neurosci
Programmed axon death, synaptic dysfunction and the ubiquitin proteasome system
Curr Drug Targets CNS Neurol Disord
Resistance of alpha-synuclein null mice to the parkinsonian neurotoxin MPTP
Proc Natl Acad Sci USA
Decreased ferritin levels in brain in Parkinson's disease
J Neurochem
Cited by (53)
Characterization of β-cyclodextrin/myrtenol complex and its protective effect against nociceptive behavior and cognitive impairment in a chronic musculoskeletal pain model
2020, Carbohydrate PolymersCitation Excerpt :Furthermore, catalase is an enzyme that metabolizes H2O2, releasing water and oxygen, making H2O2 one of the main indicators of brain homeostasis. Disturbance in H2O2 content in cerebral structures has been shown to be related to brain diseases (Jiang et al., 2007). Thus, the maintenance of oxidative homeostasis induced by β-cyclodextrin/myrtenol could potentially be underlying the reduced behavioral impairment observed in the treated animals.
Autophagy as a Cellular Stress Response Mechanism in the Nervous System
2020, Journal of Molecular BiologyA sticky situation: Aberrant protein–protein interactions in Parkinson's disease
2020, Seminars in Cell and Developmental BiologyApoptosis signal regulating kinase 1 deletion mitigates α-synuclein pre-formed fibril propagation in mice
2020, Neurobiology of Aging