Elsevier

Neurobiology of Aging

Volume 28, Issue 11, November 2007, Pages 1709-1717
Neurobiology of Aging

Parkinson's disease genetic mutations increase cell susceptibility to stress: Mutant α-synuclein enhances H2O2- and Sin-1-induced cell death

https://doi.org/10.1016/j.neurobiolaging.2006.07.017Get rights and content

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. α-Synuclein is a major component of Lewy bodies in sporadic PD, and genetic alterations in α-synuclein cause autosomal-dominant hereditary PD. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Here we investigated the effect of α-synuclein expression on cell susceptibility to proteasome inhibition, oxidative and nitrative stresses by using a PC 12-Tet-off regulatory system. We found that inducible expression of A30P or A53T mutant α-synuclein decreased the proteasome activity, increased intracellular ROS levels, and enhanced lactacystin- and H2O2-induced cell death. Furthermore, 3-nitrotyrosine levels increased in cells expressing α-synuclein, and further increased after Sin-1 (a NO donor) treatment compared with untreated or treated non-induced cells. Expression of α-synuclein (mutant more than wild type) significantly enhances Sin-1 toxicity. These results indicate that genetic mutations in α-synuclein may increase neuronal vulnerability to cellular stress in aging and PD pathogenesis.

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.

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