LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1

Garikoitz Azkona,Nerea Vazquez,Georg Auburger,Rakel López De Maturana,Patricia Del Rio,Blas Morales,Rosario Sánchez-Pernaute,Amaia Zubiarrain,José Matias Arbelo,Daniel Jimenez-Blasco,Amaya Sousa,Juan P Bolaños

doi:10.1007/s12035-016-0303-7

Abstract

Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson’s disease. Fibroblasts from Parkinson’s disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson’s disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson’s disease.

Highlights

Rare monogenic forms of Parkinson’s disease (PD) have been correlated to specific gene mutations [1], providing the opportunity to identify novel pathogenic pathways or molecular mechanisms that may contribute to more frequent forms of the disease [2] and to examine interactions between PARK genes
With the aim to define a PINK1-related phenotype for in vitro studies, we investigated expression pattern of genes involved in mitochondrial dynamics and PARK genes in fibroblasts and induced pluripotent stem cell-derived dopamine neurons from a PINK1-PD Spanish kindred [14]
PDP3; boxed) after electroporation with PINK1.GFP. c RNA levels analyzed by quantitative RT-PCR in fibroblasts after PINK1 over-expression

Summary

Introduction

Rare monogenic forms of Parkinson’s disease (PD) have been correlated to specific gene mutations [1], providing the opportunity to identify novel pathogenic pathways or molecular mechanisms that may contribute to more frequent forms of the disease [2] and to examine interactions between PARK genes. PD-related mutations in PINK1 (PTEN-induced kinase 1, PARK6) compromise kinase function or protein stability [3]; the phenotype is thought to result from a loss of function. PINK1 regulates mitochondrial fusion and fission dynamics [9], the net effect of PINK1 mutations is currently a matter of controversy because both increased fusion and increased fission have been reported in different species [10]

Methods

Results

Conclusion