Integrated Regulation of Dopaminergic and Epigenetic Effectors of Neuroprotection in Parkinson's Disease Models

Abstract

Whole exome sequencing of Parkinsons disease (PD) patient DNA identified single-nucleotide polymorphisms (SNPs) in the TNK2 gene. Although TNK2 encodes a non-receptor tyrosine kinase that has been shown to prevent the endocytosis of the dopamine reuptake transporter (DAT), a causal role for TNK2 in PD remains unresolved. We postulated that specific recessive mutations in patients resulted in aberrant or prolonged overactivity as a consequence of failed negative regulation by an E3 ubiquitin ligase, NEDD4. Interestingly, the sole Caenorhabditis elegans ortholog of TNK2, termed SID-3, is an established mediator of epigenetic gene silencing and systemic RNA interference facilitated by the SID-1 dsRNA transporter. While SID-3 had no prior association to dopamine neurotransmission in C. elegans, we hypothesized that TNK2/SID-3 represented a node of integrated dopaminergic and epigenetic signaling essential to neuronal homeostasis. Using genetic and chemical modifiers, including a TNK2 inhibitor (AIM-100) and NEDD4 activator (NAB2), in bioassays for dopamine uptake or RNAi in dopaminergic neurons of C. elegans, we determined that sid-3 mutants displayed neuroprotection from 6-hydroxydopamine (6-OHDA) exposure, as did wildtype animals treated with AIM-100 or NAB2. Additionally, NAB2 treatment of rat primary neurons correlated with a reduction of TNK2 levels and the attenuation of 6-OHDA neurotoxicity. Notably, CRISPR-modified nematodes engineered with genomic mutations in sid-3 analogous to PD patient-associated SNPs in TNK2 circumvented the resistance to RNAi characteristic of SID-3 dysfunction and furthermore exhibited enhanced susceptibility to neurodegeneration. This study describes a molecular etiology for PD whereby dysfunctional cellular dynamics, dopaminergic, and epigenetic signaling intersect to cause neurodegeneration. Significance StatementThe progressive loss of dopamine neurons is a pathological hallmark of Parkinsons disease (PD). Distinctions between resilience or susceptibility to neurodegeneration in PD are a combined consequence of genetic predisposition and environmental factors, the latter often manifesting as changes in gene expression that are coordinately controlled by small RNA molecules. This research reveals a functional convergence of proteins that modulate uptake of both dopamine and small RNAs, as a regulatory intersection for the integrated control of dopamine neuron health. Analysis of PD-patient mutations in the central protein associated with this functional interface further illustrated the clinical significance of this regulatory mechanism, as well as its potential for therapeutic intervention to prevent neurodegeneration through the fine-tuning of dopamine levels.