Fig 5. Intestinal overexpression of mir-2 enhances DA neurodegeneration and is attenuated by AIM-100 when sid-1 is wildtype.
(A, D) DA neurons were scored for neurodegeneration at day 4 post-hatching in an α-syn model background with mir-2 OE in the intestine (Pges-1::mir-2). In (A), mir-2(gk259) is examined (mutant and wildtype) while in (D), both sid-1(pk3321) and mir-2(gk259) mutants and wildtype are examined. Values represent mean + S.D. (n = 30 worms per genotype/group per replicate, 3 independent replicates of 3 independent stable lines). One-Way ANOVA with Dunnett’s post hoc analysis was used to compare worm classes where mir-2 is OE in the intestine (Pges-1::mir-2) to the α-syn (with or without sid-1 mutation) worm classes; * P < 0.05, ** P < 0.01, *** P <0.001, **** P < 0.0001. (B, C, E, F) DA neurons were scored for degeneration on day 4 post-hatching. Values represent mean + S.D. (n = 30 worms per genotype/group per replicate, 3 independent replicates of 1 independent stable line). Worms were either exposed to the 0.1% ethanol solvent control or AIM-100 (100 μM), and the α-syn control model or the α-syn model with mir-2 OE in the intestine (Pges-1::mir-2) alone (B), with mir-2(gk259) (C), sid-1(pk3321) (E), or both sid-1(pk3321) and mir-2(gk259) mutants (F) were examined. Two-way ANOVA with Tukey’s post hoc analysis was used to compare all conditions to each other; ns P ≥ 0.05, * P < 0.05, ** P < 0.01, *** P <0.001. (G) DA neurons were scored for degeneration on days 5 and 7 post-hatching. Values represent mean + S.D. (N = 3; n = 30 worms, per genotype). Two-Way ANOVA with Šídák’s post hoc analysis was used to compare α-syn expressed in neurons to α-syn + sid-1(pk3321) + mir-2(gk259) at each time point; **** P < 0.0001. (H) Expression levels of α-syn in α-syn only worms and α-syn worms administered AIM-100. Expression is normalized to the α-syn only group. Total RNA was isolated at day 4 post-hatching. Unpaired, two-tailed Student’s t-test was used to compare α-syn + solvent to α-syn + AIM-100; * P < 0.05. (I) An illustrative triptych (created with Biorender.com) representing an interpretation of the results obtained for SID-3 regulation of mir-2 import, modeling the differential impact of α-syn-induced DA neurodegeneration. The triptych displays conditions in which α-syn is expressed in DA neurons and mir-2 is overexpressed (OE) in the worm intestine. In the left panel, sid-1 and sid-3 are wildtype, and SID-3 is depicted as inhibiting the endocytosis of SID-1, thus maintaining wildtype SID-1 levels for transport of mir-2 and suppress the expression of its target genes, leaving DA neurons vulnerable to α-syn-induced neurodegeneration. The middle pane depicts these same animals treated with the potent and selective SID-3 inhibitor, AIM-100. When SID-3 is inhibited by AIM-100, evidence suggests that endocytosis of SID-1 is increased, resulting in a decrease of mir-2 import into cells, and conversely increasing the abundance of upregulated transcripts that contribute to the DA neuroprotection observed. The right pane reflects the same scenario as does the middle, except under conditions where sid-3 is wildtype, sid-1 is mutant, and SID-3 is inhibited by AIM-100. If SID-3 activity is inhibited, increased internalization SID-1 from cell surfaces would be predicted, along with a corresponding decrease of mir-2 import would be expected, aside from the fact that any SID-1 on cell surfaces is non-functional due to mutation. Strikingly, although mir-2 import into cells via SID-1 is eliminated in this latter example, an enhancement of α-syn-induced neurodegeneration is still observed experimentally. This result suggests that an alternate mechanism of dsRNA uptake may exist and is revealed in the absence of functional SID-1.