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Whole-exome sequencing of Parkinson’s disease (PD) patient DNA identified single-nucleotide polymorphisms (SNPs) in the tyrosine nonreceptor kinase-2 ( TNK2 ) gene. Although this kinase had a previously demonstrated activity in preventing the endocytosis of the dopamine reuptake transporter (DAT), a causal role for TNK2-associated dysfunction in PD remains unresolved. We postulated the dopaminergic neurodegeneration resulting from patient-associated variants in TNK2 were a consequence of aberrant or prolonged TNK2 overactivity, the latter being a failure in TNK2 degradation by an E3 ubiquitin ligase, neuronal precursor cell-expressed developmentally down-regulated-4 (NEDD4). Interestingly, systemic RNA interference protein-3 (SID-3) is the sole TNK2 ortholog in the nematode Caenorhabditis elegans , where it is an established effector of epigenetic gene silencing mediated through the dsRNA-transporter, SID-1. We hypothesized that TNK2/SID-3 represents a node of integrated dopaminergic and epigenetic signaling essential to neuronal homeostasis. Use of a TNK2 inhibitor (AIM-100) or a NEDD4 activator [N-aryl benzimidazole 2 (NAB2)] in bioassays for either dopamine- or dsRNA-uptake into worm dopaminergic neurons revealed that sid-3 mutants displayed robust neuroprotection from 6-hydroxydopamine (6-OHDA) exposures, as did AIM-100 or NAB2-treated wild-type animals. Furthermore, NEDD4 activation by NAB2 in rat primary neurons correlated to a reduction in TNK2 levels and the attenuation of 6-OHDA neurotoxicity. CRISPR-edited nematodes engineered to endogenously express SID-3 variants analogous to TNK2 PD-associated SNPs exhibited enhanced susceptibility to dopaminergic neurodegeneration and circumvented the RNAi resistance characteristic of SID-3 dysfunction. This research exemplifies a molecular etiology for PD whereby dopaminergic and epigenetic signaling are coordinately regulated to confer susceptibility or resilience to neurodegeneration.

Whole exome sequencing of Parkinson’s 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 Parkinson’s 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.

The fine-tuning of gene expression is critical for all cellular processes; aberrations in this activity can lead to pathology, and conversely, resilience. As their role in coordinating organismal responses to both internal and external factors have increasingly come into focus, small non-coding RNAs have emerged as an essential component to disease etiology. Using Systemic RNA interference Defective (SID) mutants of the nematode Caenorhabditis elegans, deficient in endogenous gene silencing, we examined the potential consequences of dysfunctional epigenomic regulation in the context of Parkinson’s disease (PD). Specifically, the loss of either the sid-1 or sid-3 genes, which encode a dsRNA transporter and an endocytic regulatory non-receptor tyrosine kinase, respectively, conferred neuroprotection to dopaminergic neurons in an established transgenic C. elegans strain wherein overexpression of human α-synuclein (α-syn) from a chromosomally integrated multicopy transgene causes neurodegeneration. We further show that knockout of a specific microRNA, mir-2, attenuates α-syn neurotoxicity; suggesting that the native targets of mir-2-dependent gene silencing represent putative neuroprotective modulators. In support of this, we demonstrated that RNAi knockdown of multiple mir-2 targets enhanced α-syn-induced dopaminergic neurodegeneration. Moreover, we demonstrate that mir-2 overexpression originating in the intestine can induce neurodegeneration of dopaminergic neurons, an effect that was reversed by pharmacological inhibition of SID-3 activity. Interestingly, sid-1 mutants retained mir-2-induced enhancement of neurodegeneration. Transcriptomic analysis of α-syn animals with and without a sid-1 mutation revealed 27 differentially expressed genes with human orthologs related to a variety of diseases, including PD. Among these was pgp-8, encoding a P-glycoprotein-related ABC transporter. Notably, sid-1; pgp-8 double mutants abolished the neurodegeneration resulting from intestinal mir-2 overexpression. This research positions known regulators of small RNA-dependent gene silencing within a framework that facilitates mechanistic evaluation of epigenetic responses to exogenous and endogenous factors influencing dopaminergic neurodegeneration, revealing a path toward new targets for therapeutic intervention of PD.Author SummaryThe progressive death of neurons that produce the neurotransmitter dopamine is a clinical hallmark of Parkinson’s disease (PD). An integrated response to environmental and genetic factors leads to expression changes in specific genes and non-protein-coding regulatory molecules called dsRNAs that influence the pathology underlying PD. Here we report, for the first time in an animal model of PD, that mutations in genes encoding proteins which function in the cellular import of dsRNA protect dopamine neurons from degeneration. By generating a profile of individual genes affected when dsRNA transport is incapacitated, we established a foundation for the systematic analysis of their distinct contributions to neuroprotection. One subclass of dsRNAs, termed microRNAs, function analogously to a conductor of an orchestra by controlling hundreds of genes, simultaneously, to coordinate cellular processes. We identified a single microRNA that suppresses the activity of numerous genes in dopamine neurons and contributes to neurodegeneration. Genomic knockout of this deleterious microRNA, or prevention of its transport into dopamine neurons, unmasked a set of previously unreported neuroprotective proteins. This study supports a hypothesis whereby mechanisms that “fine tune” dopamine availability intersect with regulators of dsRNA transport to cooperatively maintain an optimal balance between neuronal activity, survival, and neurodegeneration.