A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A

Lydia Reznichenko,Tali Fishman-Jacob,Silvia A. Mandel,Moussa B.H. Youdim

doi:10.1074/jbc.m109.034223

Abstract

The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.

Highlights

Tel.: 972-4-8295289; Fax: 972-4-8513145; E-mail: mandel@ tx.technion.ac.il. 2 The abbreviations used are: PD, Parkinson disease; ALDH1A1, aldehyde dehydrogenase family 1, subfamily A1; Hsc-70, 70 kDa heat-shock cognate protein; DA, dopamine; DA transporter (DAT), dopamine transporter; Lewy bodies (LBs), Lewy body; MPPϩ, 1-methyl-4-phenylpyridinium ion; shRNA, short hairpin RNA; LV, lentivirus; SNpc, substantia nigra pars compacta; VMAT2, vesicular monoamine transporter-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; dysregulation of genes involved in biological processes linked to protein aggregation and catabolism, mitochondrial dysfunction, oxidative stress, cell cycle, vesicle trafficking, synaptic transmission, dopamine (DA) metabolism, and cell adhesion/ cytoskeleton maintenance [1,2,3,4,5]
Small Interference RNA-mediated Down-regulation of SKP1A Gene and Protein in SN4741 Cells—Two different sequences of shRNA-encoding LVs targeting the transcript of murine SKP1A were assayed to endogenously generate small interference RNA-mediated silencing of SKP1A
SKP1A Silencing Induces Cell Morphology and Cell Cycle Alterations—SKP1A silenced cells display distinctive phenotypic characteristics when compared with the control

Summary

Introduction

Dysregulation of genes involved in biological processes linked to protein aggregation and catabolism, mitochondrial dysfunction, oxidative stress, cell cycle, vesicle trafficking, synaptic transmission, dopamine (DA) metabolism, and cell adhesion/ cytoskeleton maintenance [1,2,3,4,5]. Disease-linked point mutations in parkin, which retain their catalytic E3 ligase activity, have a propensity to mislocalize within the cell and aggregate in inclusion bodies [17] This implies that disruption of the cytoplasmic co-localization with essential protein partners may constitute the molecular basis of the loss of parkin function. Besides SKP1A, our pioneer large scale transcriptomics analysis of gene expression profile of sporadic PD SNpc [1] has identified a number of differentially expressed genes participating in the neurotoxic cascade of DA neurons death, in particular those related to the handling of proteins, dopaminergic transmission, and metabolism One such gene is HSPA8 (heat shock 70-kDa protein 8), coding for Hsc-70 (70 kDa heat-shock cognate protein), a member of the Hsp family whose expression was severely reduced in PD SN. These highly reactive, neurotoxic aldehydes can accumulate in case of decreased levels of ALDH and ADH, as occurs in SNpc of PD, and can promote neuronal death [22, 23]

Objectives

Results

Conclusion