Impairment of proteasome and anti-oxidative pathways in the induced pluripotent stem cell model for sporadic Parkinson's disease
Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is characterized by a slowness of movement (bradykinesia), difficulty in initiating movement (akinesia), rigidity and resting tremor [1]. The pathogenesis of PD is associated with the progressive degeneration of dopaminergic (DA) neurons and the presence of eosinophilic cytoplasmic inclusion bodies (Lewy bodies) with enrichment of α-synuclein in the ventral midbrain [2]. Interactions between environmental factors and genetic predisposition, most of which are as yet unknown, are thought to cause PD [3]. SNCA (α-synuclein), PARK2 (PARKIN), PINK1 (PTEN induced putative kinase 1), PARK7 (DJ-1), LRRK2 (leucine-rich repeat kinase 2), ATP13A2 (ATPase type 13A2), VPS35 (vacuolar protein sorting 35), EIF4G1 (eukaryotic translation initiation factor 4 gamma, 1), SYNJ1 (synaptojanin 1), DNAJC6 (DnaJ homolog, subfamily C, member 6), and DNAJC13 (DnaJ homolog, subfamily C, member 13) have been identified to be the causative genes for familiar and early-onset PD (EOPD) [4]. These genes have been implicated in the ubiquitin-proteasome and autophagolysosome protein degradation pathways, oxidative stress response, cell survival, apoptosis and mitochondrial function [5].
One of the critical challenges for PD research is the lack of live human neurons for mechanistic studies and the discovery of new drugs. The development of human induced pluripotent stem cells (iPSCs) has made it possible to generate patient-specific neurons to study their unique vulnerabilities in PD [6]. The iPSCs derived from PD patients (PD-iPSCs) can recapitulate disease phenotypes to serve as a platform for characterizing disease pathogenesis and discover new potential therapeutic strategies. In this study, we generated PD-iPSCs from a sporadic EOPD patient carrying a heterozygous deletion of exon 5 (Ex5del) in PARK2 gene [7]. The neurons derived from this PD patient's iPSCs demonstrated higher susceptibility to environmental triggers such as a proteasome inhibitor and oxidative stressor H2O2. These cells demonstrate a model to identify potential disease modifying strategies individually for PD patients.
Section snippets
Derivation of human skin fibroblasts
Fibroblasts were derived from a 26-year-old female EOPD patient carrying PARK2 Ex5del [7], and a 32-year-old female healthy volunteer after obtaining their informed consent under protocols approved by Chang Gung Memorial Hospital. Explants (1 cm3) of dermal biopsies were minced with a scalpel into pieces of less than 2 mm in diameter. Primary dermal fibroblasts were cultured in medium containing Glasgow Minimum Essential Medium (GMEM), 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 mg/ml
Establishment of iPSCs from a patient with sporadic EOPD
A 26-year-old female patient with early onset (at 20 years of age) typical l-DOPA (L-3,4-dihydroxyphenylalanine)-responsive PD heterozygous for Ex5del in PARK2 [7], and a 32-year-old female healthy volunteer, assented to skin biopsies for the derivation of iPSCs. Genetic screening confirmed the absence of mutations in ATP13A2, PARK7, GBA1 (β-glucosidase), HTRA2 (HtrA serine peptidase 2), LRRK2, PINK1, PLA2G6 (phospholipase A2, group VI), and UCHL1 in this patient ([10], [11], [12], [13] and
Discussion
In this study, we established a PD-iPSC model that recapitulated the critical phenotypes of PD from a sporadic EOPD patient. These PD-iPSC-derived neurons demonstrated an abnormal accumulation of α-synuclein and down-regulation of proteasome and anti-oxidative pathways. Overexpressing PARKIN in PD-iPSC-derived neurons reduced α-synuclein level and recovered proteasome activity. Treatment with the proteasome inhibitor MG132 or oxidative stressor H2O2 significantly increased cell death of the
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
We appreciate the technical assistance from Microscope Core Laboratory and Genomic Medicine Research Core Laboratory in Chang Gung Memorial Hospital, Linkou. This study was sponsored by grants from Chang Gung Memorial Hospital, Taipei, Taiwan (CMRPG 3E0571, 3A0691-93), the National Science Council, Executive Yuan (NSC 100-2314-B-182A-076–MY1-2) and the Ministry of Science and Technology, Taiwan (MOST 104-2314-B-182-060-MY2).
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