LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction

Abstract

Parkinson's disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

Keywords: LRRK2; Mitochondrial DNA damage; Parkinson's disease; Stem cells.

Fig. 1

LRRK2 neural cells exhibit greater levels of mtDNA damage than neural cells from healthy subjects. (A) Neural cells were differentiated from iPSCs derived from LRRK2 mutation carriers with the G2019S (black bars) and R1441C (gray bars) mutations and from healthy subjects (white bars). Mitochondrial DNA lesions were increased in neural cells from individual iPSC clones carrying LRRK2 mutations (clones L1–3 and L5–6, Supplemental Table 1) relative to neural cells from healthy subjects’ iPSCs (clones C1–3, Supplemental Table 1). (B) In parallel, neural cells from individuals carrying LRRK2 mutations (black and grey bars) contained a similar number of mtDNA copies as neural cells from healthy subjects. QPCR-based assay was performed in triplicate with three experimental replicates. Data are presented as mean ± SEM.

Fig. 2

Neuroprogenitor cells carrying the LRRK2 G2019S mutation show more mtDNA damage than neuroprogenitor cells from iPSCs of a healthy sibling without the LRRK2 G2019S mutation. (A) Neuroprogenitor cells were differentiated from iPSCs carrying the LRRK2 G2019S mutation (black bar) and from a healthy sibling (white bar). mtDNA lesions were increased in neuroprogenitor cells carrying the LRRK2 G2019S mutation (clone L4a, Supplemental Table 1) relative to mtDNA from healthy sibling (clone C3, Supplemental Table 1). QPCR-based assay was performed in triplicate with three experimental replicates. Data are presented as mean ± SEM, *p = 0.0002.

Fig. 3

(A–D) Representative images of immunocytochemistry show that differentiated neural cells derived from both mutant LRRK2^G2019S/WT and gene-corrected LRRK2^WT/WT iPSCs show equivalent expression of general neuronal markers (synapsin and alpha-synuclein) as well as robust expression of dopaminergic markers (TH & VMAT2). While GABA-containing neurons were also seen, astroglia (GFAP) were not (data not shown). Also note that our previous work provided compelling electrophysiological evidence that our protocol produces a true neuronal phenotype (Cooper et al., 2012).

Fig. 4

Genomic repair of the LRRK2 G2019S mutation reduced mtDNA damage in neuroprogenitor cells and neural cells. NPCs (A) and neural cells (B) differentiated from iPSCs that retained LRRK2 G2019S mutation after ZFN transfection (clone L4e^Unmod, black bar, Supplemental Table 1) exhibited greater levels of mtDNA damage than cells differentiated from ZFN-corrected iPSCs (clone L4b^WT/WT, white bar, Supplemental Table 1, *p < 0.002). QPCR-based assay was performed in triplicate with three experimental replicates. Data are presented as mean ± SEM.