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. Author manuscript; available in PMC: 2021 Mar 4.
Published in final edited form as: Neuron. 2019 Dec 30;105(5):813–821.e6. doi: 10.1016/j.neuron.2019.12.003

Figure 4. ALFY depletion increases endogenous mHtt aggregation in human neurons directly reprogrammed from HD patient fibroblasts.

Figure 4.

(A) Schematic depicting the conversion of human fibroblasts isolated into cells analogous to medium spiny neurons (MSNs) using brain-enriched microRNAs, miR-9/9*−124, combined with striatum-enriched factors. shRNA against Alfy (shALFY) or a control sequence (shCTRL) was administered after replating.

(B) Representative image from converted fibroblasts at post-induction day (PID) 19, immunostained for TUBB3 (green) and the nuclear stain DAPI (blue).

(C-F) The percentage of MSNs positive for mHtt inclusions. HD-MSNs treated with (C) shCTRL or (D) shALFY immunostained with MW8 antibody at PID 19. (E) Quantification of MSNs positive for mHtt inclusion bodies relative to unaffected patient fibroblasts. Data shown as mean±sem, with individual data points. ANOVA revealed a significant difference of shALFY(F(5,12)= 19.88, P<0.0001). Post hoc Tukey’s test comparison outcomes indicated. N=average of 400 cells/genotype/shRNA. (F) Quantification of percentage of MSNs positive for mHtt inclusion bodies across different patient fibroblast lines. Pairwise t-tests are performed within each fibroblast line. n=219–450 cells/cell line/shRNA. ***, p<0.001; **, p<0.01; *, p<0.05; n.s.=not significant. See also Figure S4.