Fig 2: Quantitative proteomics reveals loss of SG proteins in hypothalamus of Magel2p?/m+ mice.(A) Volcano plots showing proteins that are significantly changed in 8-week-old Magel2p?/m+ mouse hypothalamuses as detected by TMT. The horizontal lines denote P value thresholds (P = 0.05; analyzed by unpaired, 2-tailed t test), and vertical lines denote log2 fold change thresholds (>0.5 and <–0.5), n = 5 per genotype. (B) Gene ontology categories that are enriched based on proteins with significantly decreased abundance in Magel2p?/m+ mouse hypothalamuses. (C) Peptide abundances of various SG proteins are significantly reduced in 8-week-old Magel2p?/m+ mouse hypothalamuses. The Magel2p?/m+ animal is normalized to averaged Magel2+/+ mice; n = 5 per genotype. Each data point represents 1 animal, plotted as mean ± SD and indicated P values as analyzed by unpaired, 2-tailed t test with Bonferroni’s correction. (D) Western blot analysis confirmed reduced expression of Pcsk1, Pcsk2, and Chgb in 8-week-old Magel2p-/m+ mouse hypothalamuses. Asterisks mark a nonspecific band. Gapdh served as loading control. (E) Quantification of Western blot analysis showed reduced expression of Pcsk1, Pcsk2, and Chgb in Magel2p?/m+ mouse hypothalamuses at 2, 4, 8, and 20 weeks; n > 4 per genotype. Each target protein is first normalized to Gapdh, and then the Magel2p-/m+ animal is normalized to averaged Magel2+/+ mice. Each data point represents 1 animal, plotted as mean ± SD and analyzed by 2-tailed t test with Bonferroni’s correction. (F–H) Volcano plots showing proteins that are significantly changed in Magel2p–/m+ mouse (F) pituitary (8 weeks old), (G) liver (5.4 months old), and (H) white adipose tissue (5.4 months old), as detected by TMT. The horizontal lines denote P value thresholds (P = 0.05; analyzed by unpaired, 2-tailed t test), and vertical lines denote log2 fold change thresholds (>0.5 and <–0.5). n = 5 per genotype. *P < 0.05 and **P < 0.01.

Fig 3: SG protein levels are reduced in pancreatic islets, but not hypothalamic tissue, from Snord116p–/m+ mice.(A and B) Western blot analysis of Pcsk1, Pcsk2, and Chgb in 8-week-old Snord116p–/m+ mouse hypothalamic tissue. Gapdh served as a normalization loading control. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test. (n > 4 per genotype.) (C and D) Western blot analysis of Pcsk1, Pcsk2, and Chgb in 8-week-old Snord116p–/m+ mice islets. Gapdh served as normalization loading control. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test, *P > 0.05. (n > 4 per genotype.) (E) Snord116 transcript levels are comparable between 8-week-old Magel2p?/m+ and Magel2+/+ mouse hypothalamuses. Each data point represents 1 animal (n = 3 per genotype), plotted as mean ± SD and analyzed by unpaired, 2-tailed t test.

Fig 4: DPSC-derived neurons from PWS patients have impaired endosome-mediated protein trafficking and decreased SG protein abundance.(A) Representative images of immunofluorescence staining of M6PR in DPSC-derived neurons. Proper M6PR trafficking shows clustered juxtanuclear localization. (B) Proportion of cells with impaired M6PR trafficking is significantly increased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (C) Representative images of immunofluorescence staining of integrin a5 in DPSC-derived neurons. Yellow arrows indicate increased intracellular (endosomal) pools. (D) Quantification of Western blot analysis showed reduced expression of integrin a5, aV, and ß1 in PWS deletion DPSC-derived neurons. Each target protein is first normalized to GAPDH, then normalized to averaged control. Each data point represents 1 individual (n = 6), plotted as mean ± SD and analyzed by unpaired, 2-tailed t test. (E) Representative images of immunofluorescence staining of phalloidin on VPS35-marked endosomes in DPSC-derived neurons. (F) Fluorescence intensity of F-actin on VPS35-marked endosomes is significantly decreased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (G) Fluorescence intensity of ArpC5 on VPS35-marked endosomes is significantly decreased in PWS and SYS DPSC-derived neurons. Each data point represents 1 individual, plotted as mean ± SD; more than 75 cells/data point, analyzed by 1-way ANOVA. (H) Western blot analysis of M6PR, a5, aV, and ß5 between control and PWS iN. GAPDH served as loading control. (I) Quantification of Western blot analysis showed reduced expression of PCSK1, PCSK2, and CHGB in PWS deletion and SYS DPSC-derived neurons. Each target protein is first normalized to GAPDH, then normalized to averaged control. Each data point represents 1 individual, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test. *P < 0.05 and **P < 0.01. Scale bars: 10 µm.

Fig 5: WASH-mediated protein trafficking prevents lysosomal degradation of SG proteins.(A) Transcript levels of Pcsk1, Pcsk2, Chgb, and Cpe in 8-week-old Magel2p?/m+ and Magel2+/+ mouse hypothalamuses, n > 3 per genotype. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test, ****P < 0.0001. (B) Transcript levels of PCSK1, PCSK2, CHGB, and CPE between control and PWS iN at 14 days postinduction. Each data point represents 1 induction experiment (n = 3), plotted as mean ± SD and analyzed by 1-way ANOVA. (C) Western blot analysis shows rescued expression of PCSK1, PCSK2, CHGB, and CPE in PWS iN at 14 days postinduction following inhibition of lysosomes by Baf (1 µM, 7 hours) but not inhibition of proteasomes by MG132 (10 µM, 7 hours). DMSO served as vehicle control. GAPDH served as loading control. (D) Quantification of Western blot analysis confirmed reduced expression of Pcsk1 and Pcsk2 in 2-week-old Washc1 conditional knockout (cKO) mouse hypothalamuses, n = 4 per genotype. Gapdh served as loading control. Each data point represents 1 animal, plotted as mean ± SD and analyzed by unpaired, 2-tailed t test, *P < 0.05. (E) Representative images of immunofluorescence staining with CHGB and WASH at both soma and neurite in control iN. Scale bars indicate 10 µm.