Regular articleTranscriptional alterations in skin fibroblasts from Parkinson's disease patients with parkin mutations
Introduction
Parkinson's disease (PD) is the most common neurodegenerative movement disorder affecting the 1%–2% of the population over the age of 65 years (Alves et al., 2008). The neuropathological hallmark of the disease is the loss of midbrain dopaminergic neurons (DAns) in the substantia nigra pars compacta (SNpc) and the presence of α-synuclein aggregates in Lewy bodies and Lewy neurites in the surviving DAns. Although the vast majority of cases are sporadic PD (sPD), patients about 10% present monogenic forms caused by pathogenic mutations in genes associated with PD (de Lau and Breteler, 2006, Simchovitz et al., 2016). Of these, loss-of-function mutations in the parkin gene (PRKN) are the most frequent cause of autosomal-recessive juvenile PD, accounting for up to 50% of familial PD and about 15% of the sPD patients with an age-at-onset before 45 years (Bonifati, 2014). These pathogenic mutations in PRKN have been described in all 12 exons of the gene and include point missense and null mutations, as well as insertions/deletions (Klein & Westenberger, 2012). The clinicopathological picture of PRKN-associated PD (PRKN-PD) typically includes early onset, slow progression, good and lasting response to levodopa, and DAn loss and gliosis in the SNpc without Lewy bodies (Bonifati et al., 2005, Klein and Westenberger, 2012).
The precise mechanisms by which PRKN loss-of-function mutations lead to neurodegeneration are not yet well understood. Wild-type PRKN encodes a multifunctional E3 ubiquitin ligase involved in key regulatory functions such as proteasomal-mediated protein turnover (Fiesel et al., 2015), mitochondrial function, and mitophagy (Clark et al., 2006b, Hasson et al., 2013, Park et al., 2006, Pickrell and Youle, 2015, Sarraf et al., 2013, Seirafi et al., 2015), thereby conferring neuroprotection and survival (Fiskin and Dikic, 2013, Zhang et al., 2015). Interestingly, recent studies have also suggested an association of PRKN with transcriptional deregulation of genes involved in the neurodegenerative process (da Costa et al., 2009, Duplan et al., 2013), suggesting that PRKN could be somehow involved in the regulation of the transcriptional activity of other genes (Tay et al., 2010, Unschuld et al., 2006). Using biological samples from PD patients, previous studies have reported transcriptomic alterations in postmortem tissues, peripheral blood mononuclear cells, and other tissues (Fernandez-Santiago et al., 2015, Grunblatt et al., 2004, Infante et al., 2015, Mutez et al., 2011). These studies have mainly focused on sPD or autosomal-dominant LRRK2-associated PD, but transcriptomic alterations potentially occurring in PRKN-PD are unknown.
In this context, here we have explored for the first time global gene expression changes in cultured skin fibroblasts from PRKN-PD patients. Skin fibroblasts from PD patients have been proposed as a cellular system to model disease (Auburger et al., 2012) able to reproduce several of the molecular alterations occurring in PD (Ambrosi et al., 2014, Hoepken et al., 2008, Romani-Aumedes et al., 2014, Yakhine-Diop et al., 2014). In addition, punch skin biopsies are minimally invasive and permit the possibility to establish primary cultures of self-propagating cells that preserve the genomic background of the patient without additional genetic manipulation. The goal of our study was to investigate whether gene expression changes occur in PD patients at peripheral non-neural tissues. More specifically, we have performed a whole-genome expression analysis by RNA sequencing (RNA-seq) in skin fibroblasts from PD patients carrying different loss-of-function mutations in PRKN and from genetically unrelated healthy controls. Our study identifies specific transcriptional changes, cellular processes, and molecular pathways, which are altered at the peripheral level in skin tissue from PRKN-PD patients.
Section snippets
Study approval
This study has been conducted conforming the principles of the Declaration of Helsinki and the Belmont Report. All participants gave written informed consent, and the study was approved by the Commission on Guarantees for Donation and Use of Human Tissues and Cells of the Instituto de Salud Carlos III and the local ethics committee at the Hospital Clínic de Barcelona. Personal data were anonymized, and subject samples were codified to preserve confidentiality.
Subjects and PRKN mutational screening
We selected 4 PD patients carrying
Gene expression analysis
To ascertain the potential impact of PRKN mutations on global gene expression, we performed an RNA-seq study in skin fibroblasts from PRKN-PD patients and healthy controls. We found detectable gene expression of 14,230 annotated transcripts. We then performed a principal component analysis of the top 500 most variable genes to inspect overall variability among samples (Fig. 1A). The first 2 principal components showed a strong gender effect, separating females and males. Differential expression
Discussion
We report for the first time the presence of a large number of gene expression alterations at the transcriptome level in skin fibroblasts from PD patients carrying mutations in the PRKN. We identified 343 DEGs associated with PRKN-PD of which 206 were upregulated DEGs, whereas 137 were downregulated. Biological enrichment analysis revealed that these DEGs were mainly involved in cell adhesion, cell growth, amino acid biosynthesis, and folate metabolism among others.
To date, other transcriptomic
Accession number
Gene expression data sets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession GSE90514.
Disclosure statement
The authors report no actual or potential conflict of interest including any financial, personal, or other relationships with other people or organizations within 3 years of beginning the work submitted that could inappropriately influence this work.
Acknowledgements
The authors thank the patients who participated in the study and their family members. We also want to thank the programs: Supports a Grups de Recerca de la Generalitat de Catalunya 2017–2019 (grant number SGR 893/2017) and CERCA Programme/Generalitat de Catalunya. This work was developed at the building Centre de Recerca Biomèdica Cellex, Barcelona.
Funding: This work was supported by funds from Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (ISCIII; grant number
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