Fig 2: USP24 regulates autophagy and neurite length in human iPSC-derived dopaminergic neurons. (a) Western blot illustrating the changes in LC3-II, NBR1, and phospho-ATG14 (P-ATG14) in human iPSC-derived dopaminergic neurons following USP24 knockdown. Dopaminergic precursor cells were differentiated into dopaminergic neurons for 2 wk, transduced with indicated lentiviral shRNAs and evaluated after an additional week in culture. (b) Quantification of USP24:tubulin levels from figure (a). (c) Quantification of LC3-II:tubulin levels from figure (a) (d) Quantification of NBR1:tubulin levels from figure (a) (2 independent experiments; 4–6 replicates) (e and f) Levels of LC3-II following USP24 knockdown are further increased with 100 µM chloroquine (chq) treatment (overnight). (e) Western blot illustrating change in LC3-II levels in human iPSC-derived dopaminergic neurons following USP24 knockdown. (f) Quantification of LC3-II:loading control from figure (e) (4–6 replicates). (g) Representative images of human iPSC-derived neurons at 6 wk after lentiviral transduction, showing increased neurite density in cultures with USP24 knockdown. Cells were transduced with indicated shRNAs; after additional 6 wk cultures were fixed and stained with antibodies against TUBB3/tubulin B3/TUJ1; all neurons) and TH (tyrosine hydroxylase; dopaminergic neurons). Images were acquired at 20X; bar: 25 µm. (h) Quantification of length of neuronal processes over time after lentiviral transduction with indicated shRNAs. All data are normalized to nt shRNA at 1-week time point. Statistical analysis at 1-week time point is presented in Figure S4 and at 6-weeks in (i-j). (i) Quantification of neurite length:neuron from figure (h) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. (j) Quantification of neurite length:neuron for the dopaminergic neurons (TH positive) at 6-week time point. Data are normalized to nt shRNA at 6-week time point. All data are presented as ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001 n = 3–10 (median cell number 634/group).

Fig 3: USP24 regulates protein stability of ULK1. (a) Western blot demonstrating increased levels of ULK1 and phospho-ATG13 (P-ATG13) in cells with USP24 knockdown. (b) Quantification of ULK1:ACTB from figure (a). (c) Quantification of P-ATG13:ACTB from figure (a). (d) Real time q-PCR quantification of ULK1:GAPDH mRNA expression following USP24 knockdown (e) Protein stability assay illustrating that USP24 knockdown decreases the rate of ULK1 degradation. Cells were treated with cycloheximide (50µg/µL) to inhibit protein synthesis. (f) Quantification of ULK1:ACTB from figure (e) at 0 and 3-hour time points. (g) IP demonstrating increased ubiquitination of ULK1 after USP24 knockdown. (h) Quantification of the ubiquitin (HA):ULK1 in figure (g). (i) Western blot demonstrating decreased levels of LC3-II in H4 cells with USP24 knockdown treated with ULK1 inhibitor MRT67307 (10 µM, 4 h) and BafA (100 nM, 3 h), as compared to BafA treatment alone. (j) Quantification of LC3-II:ACTB in BafA and BafA+MRT67307 conditions from figure (i). (k) Western blot demonstrating decreased levels of LC3-II in Hela cells with USP24 knockdown treated with MRT67307 (10 µM, 4 h) and BafA (100 nM, 3 h) as compared to BafA treatment alone. (l) Quantification of LC3-II:ACTB in BafA and BafA+MRT67307 conditions from figure (k). (m) Representative fluorescent images demonstrating attenuated accumulation of autophagosomes in H4 GFP-LC3 cells with USP24 knockdown after MRT68921 treatment. All images were acquired at 20X; bar: 25 µm. (n) Quantification of autophagosome intensity from figure (m). All data are presented as ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–14 (median cell number 43/group).

Fig 4: USP24 is differentially expressed in PD patient substantia nigra. (a) q-PCR quantification of USP24:GAPDH mRNA expression in PD patient SN versus unaffected control substantia nigra. All data points correspond to individuals (n = 4); group means are indicated with horizontal lines; error bars are SE; red circle marks a major statistical outlier (defined as a value above Q1-Q3 outer fence) with significantly increased expression of USP24 mRNA (z-score = 4.6). (b) Western blot illustrating USP24 protein levels in 4 PD patient and 4 unaffected age matched control human substantia nigra. (c) Quantification of USP24:ACTB levels from figure (b). Red circles mark major statistical outliers with significantly increased USP24 protein expression (z-scores = 4.5 and 11.0).

Fig 5: USP24 regulates the class III PtdIns3K activity. (a) Representative images of phosopho-RPS6 (P-RPS6) staining in H4 cells following USP24 knockdown, MTOR knockdown, or treatment with 100 nM rapamycin, demonstrating that USP24 does not regulate MTORC1 activity. (b) Quantification of P-RPS6 intensity from (a) (2 independent experiments, 6–12 replicates). (c) Representative images of H4 cells stably expressing the FYVE-dsRed reporter following USP24 knockdown, demonstrating increased accumulation of the class III PtdIns3K product, PtdIns3P. All images were acquired at 20X; bar: 25 µm. (d) Quantification of FYVE-dsRed accumulation in cells with USP24 knockdown from a representative experiment (4–8 replicates). (e) Western blot demonstrating that LC3-II accumulation in USP24 knockdown cells is attenuated following inhibition of the class III PtdIns3K with spautin (20 µM). (f) Quantification of LC3-II:ACTB from figure (e). (g) Representative fluorescence images demonstrating attenuated accumulation of autophagosomes in H4 GFP-LC3 cells with USP24 knockdown after spautin treatment. All images were acquired at 20X; bar: 25 µm. (h) Quantification of autophagosome intensity from figure (g). All data are presented as ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3–8 (median cell number 421/group).