Regulation of TIR-1/SARM-1 by miR-71 Protects Dopaminergic Neurons in a C. elegans Model of LRRK2-Induced Parkinson's Disease

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by symptoms such as bradykinesia, resting tremor, and rigidity, primarily driven by the degradation of dopaminergic (DA) neurons in the substantia nigra. A significant contributor to familial autosomal dominant PD cases is mutations in the LRRK2 gene, making it a primary therapeutic target. This study explores the role of microRNAs (miRNAs) in regulating the proteomic stress responses associated with neurodegeneration in PD using C. elegans models. Our focus is on miR-71, a miRNA known to affect stress resistance and act as a pro-longevity factor in C. elegans. We investigated miR-71's function in C. elegans models of PD, where mutant LRRK2 expression correlates with dopaminergic neuronal death. Our findings reveal that miR-71 overexpression rescues motility defects and slows dopaminergic neurodegeneration in these models, suggesting its critical role in mitigating the proteotoxic effects of mutant LRRK2. Conversely, miR-71 knockout exacerbates neuronal death caused by mutant LRRK2. Additionally, our data indicate that miR-71's neuroprotective effect involves downregulating the toll receptor domain protein tir-1, implicating miR-71 repression of tir-1 as vital in the response to LRRK2-induced proteotoxicity. These insights into miR-71's role in C. elegans models of PD not only enhance our understanding of molecular mechanisms in neurodegeneration but also pave the way for potential research into human neurodegenerative diseases, leveraging the conservation of miRNAs and their targets across species.

Keywords: Parkinson; aging; elegans; miRNA; neurodegeneration.

Figure 1

miR-71 preserves dopaminergic neurons in LRRK2 worms. (A) Results from the dopaminergic neurodegeneration assay at different time points of adulthood (n = 10 worms). Error bars indicate SEM. One-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001 versus GFP control worms at each time point. † p < 0.05 and †† p < 0.01 versus G2019S worms at each time point. (B) Z-stack images from data shown in (B) were merged to identify DA neuronal cell bodies. Circles denote DA neurons and white arrows indicate abnormal or absent cephalic dopaminergic neurons. (C) Results from the basal slowing assay at different time points of adulthood (n = 20 worms). Error bars indicate SEM. One-way ANOVA: ** p < 0.01 and *** p < 0.005 versus GFP control worms at each time point. † p < 0.05 versus G2019S worms at each time point. (D) Kaplan–Meier survival analysis comparing LRRK2 expressing worms crossed with miR-71 mutants to GFP Control models. (E) Mean lifespan from data shown in (D). Bars represent mean ± SEM. One-way ANOVA: **** p < 0.0005 versus GFP Control. ^#### p < 0.0005 versus G2019S.

Figure 2

Loss of the toll-like receptor tir-1 is neuroprotective in LRRK2 expressing C. elegans. (A) The putative binding site between miR-71 and the toll-like receptor tir-1 3′ untranslated region (UTR). The minimum free energy of binding is −27.0 kcal/mol. (B) Results from qRT-PCR looking at relative expression of tir-1 at different time points of adulthood (n = 3 biological replicates). Error bars indicate SEM. One-way ANOVA: ns (not statistically significant), * p < 0.05, and *** p < 0.005. (C) Results from the dopaminergic neurodegeneration assay at different time points of adulthood (n = 10 worms). Error bars indicate SEM. One-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001 versus G2019S worms at each time point. † p < 0.05, †† p < 0.01, ††† p < 0.005 versus G2019S; miR-71 KO at each time point. # p < 0.05 and ## p < 0.01 versus GFP control at each time point. (D) Z-stack images from data shown in C were merged to identify DA neuronal cell bodies. Circles denote DA neurons and white arrows indicate abnormal or absent cephalic dopaminergic neurons. (E) Results from the basal slowing assay at different time points of adulthood (n = 20 worms). Error bars indicate SEM. One-way ANOVA: * p < 0.05 and ** p < 0.01 versus G2019S at each time point. † p < 0.05 and ††† p < 0.005 versus G2019S; miR-71 KO at each time point. # p < 0.05 versus GFP control at each time point.

Figure 3

miR-71 directly regulates the toll-like receptor tir-1 in LRRK2 expressing C. elegans. (A) Three miR-71 binding sites on the 3′ UTR of tir-1 were mutated to remove miR-71 regulation capability (tir-1 3′UTR(mut)). (B,C) Results from qRT-PCR looking at relative expression of tir-1 at different time points of adulthood (n = 3 biological replicates). Experiments for qRT-PCR were done in triplicate with about 50 worms per strain per experiment. Error bars indicate SEM. One-way ANOVA: * p < 0.05 and ** p < 0.01. (D) Z-stack images from data shown in B were merged into a singular image to identify DA neuronal cell bodies. Circles denote DA neurons and white arrows indicate abnormal or absent cephalic dopaminergic neurons. (E) Results from the basal slowing assay at different time points of adulthood (n = 20 worms). Error bars indicate SEM. One-way ANOVA: * p < 0.05, ** p < 0.01, *** p < 0.001 versus GFP control worms at each time point. † p < 0.05 and †† p < 0.01 versus G2019S at each time point. # p < 0.05 versus G2019S;m71 OE at each time point.