Objective: To identify novel genes and pathways acting as modifiers of pathogenic Parkin variant (PV) penetrance in affected compared to unaffected carriers of heterozygous Parkin PVs in a CRISPR/Cas9- and stem cell-based model of purified, patient-derived dopaminergic midbrain neurons.

Background: PVs of Parkin, an E3 ubiquitin ligase, cause autosomal recessive Parkinson’s disease (PD). Remarkably, heterozygous Parkin PVs may increase the risk of developing PD. Inheritance could hence also be interpreted as dominant, albeit with highly reduced penetrance. Thus, identifying modifiers of reduced heterozygous Parkin PV penetrance may uncover genes and pathways acting either as a second hit or in a protective manner.

Method: We utilized induced pluripotent stem cells (iPSC) from affected (n=3) and unaffected (n=2) heterozygous Parkin PV carriers edited via CRISPR/Cas9 to express mCherry in tandem with tyrosine hydroxylase (TH). Upon differentiation into dopaminergic neurons, neuronal cultures were sorted using fluorescence-activated cell sorting to obtain enriched IPSC-derived dopaminergic midbrain neurons (mDANs). Total RNA from enriched TH-positive (TH+) and TH-negative (TH-) neurons from two independent differentiation rounds was sequenced in bulk and analyzed for differentially expressed genes (DEGs).

Results: The TH+ neurons comprised 43.13% ± 25.37% of the input cell population. By contrasting RNA sequencing results of TH+ to TH- neurons, we identified 1790 DEGs and enrichment of neuron-specific gene sets within the TH+ population. Gene set enrichment analyses of sorted TH+ neurons primarily implicate dysregulation of pathways involved in neuronal development and microtubule function in affected carriers. Top DEGs for the two sets of differentiated mDANs include HSPA1A and TYR, respectively. Furthermore, combining both datasets highlights SLC25a43, encoding a mitochondrial carrier, as a consistent DEG and potential modifier of Parkin PV penetrance.

Conclusion: Here we show that limited and variable yield of mDANs after differentiation could be successfully overcome, using TH-mCherry reporter lines followed by FACS of resulting dopaminergic neuronal cultures. The identified DEGs and enriched gene sets may act as modifiers of heterozygous Parkin PV penetrance. HSPA1A, TYR, and SLC25a43, implicated in PD-relevant pathways of protein stabilization, dopamine toxicity, and mitochondrial function, are of particular interest.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/novel-modifiers-of-heterozygous-pathogenic-parkin-variant-penetrance-in-parkinsons-disease/