Loss of MAGEL2 in Prader-Willi syndrome leads to decreased secretory granule and neuropeptide production

Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 (MAGEL2). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2pΔ/m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient–derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.


Quantitative Proteomic / Mass Spectrometry
For adrenal gland, pituitary, liver, brain stem and WAT: the TMT labeled samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher). Samples were injected directly onto a 25 cm, 100 µm ID column packed with BEH 1.7 µm C18 resin (Waters). Samples were separated at a flow rate of 400 nL/min on EASY-nLC (ThermoFisher). Buffer A and B were 0.1% (v/v) formic acid in water and acetonitrile, respectively. A gradient of 1-25% B over 180 min, an increase to 40% B over 40 min, an increase to 90% over 10 min and held at 90% B for 10 min was used for a 240 min total run time. The column was re-equilibrated with 20 μL of buffer A prior to the injection of the sample. Peptides were eluted directly from the tip of the column and nanospray directly into the mass spectrometer by application of 2.8 kV voltage at the back of the column.
The Lumos was operated in a data-dependent mode. Full MS1 scans were collected in the Orbitrap at 120k resolution. The cycle time was set to 3 s, and within this 3 s the most abundant ions per scan were selected for CID MS/MS in the ion trap. MS3 analysis with multi-notch isolation (SPS3) was utilized for detection of TMT reporter ions at 60k resolution. Monoisotopic precursor selection was enabled and dynamic exclusion was used with an exclusion duration of 10 sec.
For hypothalamus: the TMT labeled samples were analyzed on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher). Samples were injected directly onto a 25 cm, 100 µm ID column packed with BEH 1.7 µm C18 resin (Waters). Samples were separated at a flow rate of 300 nL/min on EASY-nLC (ThermoFisher). Buffer A and B were 0.1% formic acid in water and acetonitrile, respectively. A gradient of 1-25% B over 200 min, an increase to 50% B over 120 min, an increase to 90% over 30 min and held at 90% B for 10 min was used for a 360 min total run time. The column was re-equilibrated with 20 μL of buffer A prior to the injection of the sample. Peptides were eluted directly from the tip of the column and nanospray directly into the mass spectrometer by application of 2.8 kV voltage at the back of the column. The Fusion was operated in a data-dependent mode. Full MS1 scans were collected in the Orbitrap at 120k resolution. The cycle time was set to 3 s, and within this 3 s the most abundant ions per scan were selected for CID MS/MS in the ion trap. MS3 analysis with multi-notch isolation (SPS3) was utilized for detection of TMT reporter ions at 60k resolution. Monoisotopic precursor selection was enabled and dynamic exclusion was used with an exclusion duration of 10 sec.
For peptide identification, tandem mass spectra were searched against a database including the UniProt mouse database one entry per gene (21982 entries released 12/1/2019) with common contaminants and reversed sequences using ProLuCID (69). The search was set with 50 and 600 ppm for precursor and fragments mass tolerance, respectively. The precursor mass range was set from 600 to 6000 with half or fully tryptic status. The N-term static modification was considered as (+229.1629) for TMT labeling and the amino acid residue specific static modifications were (+57.02146) on cysteine for carbamidomethylation and (+229.1629) on lysine for TMT labeling.

Figure S4. Endosomal protein recycling is not impaired in undifferentiated DPSC and DPSC-derived adipocytes. A)
There is no difference in proportion of cells with impaired M6PR trafficking between control and PWS deletion DPSC and DPSC-derived adipocytes. Each data point represents one individual, plotted as mean±SD; >75 cells/data point, and analyzed by t-test. B) Quantification of western deletion analysis showed no difference in the expression of alpha-5, alpha-V and beta-1 between control and PWS deletion DPSC. Each target protein is first normalized to GAPDH, and then PWS deletion DPSC is normalized to averaged control. Each data point represents one individual, plotted as mean±S.D. and analyzed by unpaired, two-tailed student t-test. C-D) There is no difference in the fluorescence intensity of F-actin (C) or ArpC5 (D) on VPS35-marked endosomes between control and PWS deletion DPSC, and DPSC-derived adipocytes. Each data point represents one individual, plotted as mean±SD; >75 cells/data point, and analyzed by unpaired, two-tailed student t-test. 20wk (B) old Magel2 pΔ/m+ and Magel2 +/+ mouse hypothalami. Each data point represents one animal, plotted as mean±S.D. and analyzed by unpaired, two-tailed student t-test, ****p<0.0001. (n>3 per genotype) C) Transcript levels of PCSK1, PCSK2, CHGB and CPE are unaltered between control and PWS iN at 3d, 7d, 10d and 14d post-induction. Each data point represents one induction experiment (n=2), plotted as mean±S.D. and analyzed by ANOVA. D) Representative images of immunofluorescence staining with CHGB and LAMP1 in control and PWS iN. E) Transcript levels of PCSK1, PCSK2, CHGB and CPE are unaltered between control #3 and PWS #3 iN at 14d post-induction. Each data point represents one induction experiment (n=3), plotted as mean±S.D. and analyzed by t-test. Figure S6. SG proteins colocalize with WASH and retromer components within the soma of human iN. A) Western blot analysis of Pcsk1, Pcsk2, Chgb and Magel2 in 2wk old Washc1 cKO mouse hypothalami. Gapdh served as loading control. Each data point represents one animal, n=4 per genotype, plotted as mean±S.D. and analyzed by unpaired, two-tailed student t-test. B) Representative images of immunofluorescence staining with CHGB and FAM21 at both soma and neurite in control iN. Scale bars=10 µm. C) Representative images of immunofluorescence staining with CHGB and VPS35 at both soma and neurite in control iN. Scale bars= 10 µm. D) Representative images of immunofluorescence staining with CHGA and WASH at both soma and neurite in control iN. Scale bars represent 10 µm. E) Representative images of immunofluorescence staining with CHGA and VPS35 at both soma and neurite in control iN. Scale bars=10 µm. Figure S7. Neuropeptide transcript levels are not altered in Magel2 pΔ/m+ mice. Transcript levels of various neuropeptides between 2wk (A) and 20wk (B) old Magel2 pΔ/m+ and Magel2 +/+ mouse hypothalami. Each data point represents one animal, (n>3 per genotype), plotted as mean±S.D. and analyzed by unpaired, two-tailed student t-test. C) Transcript levels of various neuropeptides between Ctrl#3 and PWS#3 iN at 14d post-induction. Each data point represents one induction experiment (n=3), plotted as mean±S.D. and analyzed by t-test. Tables   Table S1. Protein expression differences between Magel2 +/+ (WT) and Magel2 p-/m+ (KO) (n=5 each) as determined by TMT proteomics for the indicated tissues. Table S2. Gene ontology (GO) analysis of TMT proteomics data for the indicated tissues. Angelman, deletion F 6.8 unknown Table S4. Information on PWS patients and control subjects included in Fig. 8J.