Regulation of MCCC1 expression by a Parkinson's disease-associated intronic variant: implications for pathogenesis

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuron loss and α-synuclein aggregation. While some familial cases result from single-gene mutations, most are sporadic, involving complex genetic and environmental interactions. Among PD risk loci identified through genome-wide association studies, MCCC1 encodes a mitochondrial enzyme essential for leucine catabolism; however, the causal variant remains unclear. Here, we investigated whether the intronic variant rs12637471 regulates MCCC1 mRNA expression and influences PD risk. Postmortem brain analysis revealed significantly elevated MCCC1 mRNA levels in G-allele carriers, consistent with peripheral tissue eQTL data from GTEx. Using CRISPR/Cas9-edited induced pluripotent stem cells, we generated isogenic lines differing only at rs12637471 and observed increased MCCC1 expression in G-allele dopaminergic neurons. Given MCCC1's mitochondrial role, its dysregulation may impact mitochondrial homeostasis, autophagy, or inflammation, potentially contributing to PD pathogenesis.

Fig. 1

Risk allele G–associated upregulation of MCCC1 mRNA in human postmortem brain and iPSC-derived isogenic neurons. A qRT-PCR analysis of MCCC1 mRNA expression in postmortem frontal cortex, stratified by rs12637471 genotype. Center lines show the medians and box limits indicate the 25th and 75th percentiles. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05 vs. the protective-allele A homozygote group (GG: n = 4, AG: n = 12, AA: n = 15)

Fig. 2

Generation of isogenic iPSC lines differing only at rs12637471 using CRISPR/Cas9-mediated genome editing. A Sanger sequencing chromatograms of genome-edited iPSC lines targeting rs12637471. “AG” represents the wild-type (201B7) genotype, while “AA” and “GG” indicate single-nucleotide knock-in clones. The rs12637471 position is highlighted in yellow. B Pie charts depicting the knock-in outcomes (AA or GG) from CRISPR/Cas9-mediated editing of wild-type AG iPS cells at rs12637471, as determined by TaqMan genotyping and Sanger sequencing. C Schematic representation of isogenic cell-line generation from single human iPS cells using CRISPR/Cas9-based genome editing, followed by neuronal differentiation and MCCC1 mRNA expression analysis

Fig. 3

Analysis of in vitro–differentiated human iPSC-derived neurons. A Overview of the neural differentiation protocol from human iPS cells. B Immunostaining of differentiated neurons on day 42, showing TH/FOXA2/DAPI (top) and TH/TUJ1/DAPI (bottom). Insets highlight double-positive cells. Scale bar, 50 μm. C Quantification of TH + /FOXA2+ and TH + /TUJ1+ cells as a percentage of total cells on day 42. Data are presented as mean ± SE (AA: n = 3, AG: n = 3, GG: n = 3). D qRT-PCR analysis of MCCC1 mRNA expression in iPSC-derived isogenic neural cultures on day 42, stratified by rs12637471 genotype. Center lines show the medians and box limits indicate the 25th and 75th percentiles. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05; **P < 0.01 vs. the protective-allele A homozygote group (GG: n = 4, AG: n = 4, AA: n = 4)