Impairment of proteasome and anti-oxidative pathways in the induced pluripotent stem cell model for sporadic Parkinson's disease

https://doi.org/10.1016/j.parkreldis.2016.01.001Get rights and content

Highlights

  • PD-iPSCs from a PD patient carrying a heterozygous deletion of exon 5 in Parkin were generated.

  • Abnormal α-synuclein accumulation in PD-iPSC-derived neurons.

  • Down-regulated proteasome and anti-oxidative pathways in PD-iPSC-derived neurons.

  • Pronounced cell death of PD-iPSC-derived neurons by proteasome inhibitor MG132 and oxidative stressor H2O2.

  • Proteasome enhancer benzamil and anti-oxidant genipin rescued above susceptibilities.

Abstract

Background

Parkinson's disease (PD) is associated with the progressive degeneration of dopaminergic neurons with abnormal accumulation of α-synuclein mainly in the ventral midbrain. However, the lack of live human neurons from PD patients and their heterogeneous pathogenic nature limit mechanistic studies and therefore the development of drugs to modify the disease progression of PD. The evolution of induced pluripotent stem cell (iPSC) technology makes it possible to generate patient-specific neurons to explore the pathogenesis in individual PD patients.

Methods

We generated PD-iPSCs from a sporadic early onset PD patient carrying a heterozygous deletion of exon 5 (Ex5del) in PARK2. The expression of α-synuclein and proteasome and anti-oxidative functions were examined in differentiated iPSC-derived neurons.

Results

The neurons derived from our PD-iPSCs demonstrated abnormal α-synuclein accumulation and down-regulation of the proteasome and anti-oxidative pathways. Environmental triggers such as proteasome inhibitor MG132 and H2O2 markedly induced cell death, while the proteasome enhancer benzamil and anti-oxidative compound genipin significantly rescued these increased susceptibilities.

Conclusions

These results demonstrate that unique genetic–environmental interactions are involved in neuronal death in PD patients. Our findings also provide a new model to identify potential disease-modifying strategies and an insight into personalized medicine for patients with PD.

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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