Journal: Cell death & disease
Article Title: Opposing roles for GSK3β and ERK1-dependent phosphorylation of huntingtin during neuronal dysfunction and cell death in Huntington's disease.
doi: 10.1038/s41419-025-07524-0
Figure Lengend Snippet: Fig. 1 The proteomic network on HTT containing light membranes is dramatically altered in HD iPSC-derived neurons. A Representative images from healthy/normal (WT, Q17) or diseased (HD, Q109) human iPSCs stained with the pluripotent marker OCT-4, the neuronal precursor (NPC) marker Nestin and the mature neuronal markers MAP2 and βIII-Tubulin. Hoechst stains nuclei. Scale = 25 μm. Differentiated neurons show Synaptophysin (SYP) positive staining. Scale = 10 μm. B Electrophysiological analysis of WT and HD human neurons differentiated from iPSCs show action potentials, which are abolished in the presence of TTX or TEA. C Schematic diagram of human iNeuron lysate fractionation into perinuclear supernatant (PNS), light membrane (LM), soluble (SF), and heavy membrane (P1) fractions by ultra- centrifugation and sucrose gradient separation. D Workflow for quality control and quantification of unique peptides identified from LC-MS of HTT-IPs from WT or HD human iNeurons. E Hierarchical cluster heat map showing the avg. relative abundance (spectral count; SpC) of 800 proteins (≥3 unique peptides/trial across ≥2 biological replicates) quantified across the WT and HD HTT-IPs with a normalized fold change (FC) threshold of ±2X and a significance threshold of p < 0.05 determined by a Welch’s t test across three independent biological replicates. Increased in HD HTT-IP = red, decreased in HD HTT-IP = blue. In addition, proteins were identified in only WT HTT-IP (lost = green) or in only HD HTT-IP (gained = orange). F Volcano plot with the y axis depicting significance (−log10[p value]) and the x axis depicting fold change of individual peptides between HD and WT HTT-IPs (log2[FC]). Three independent biological replicates were performed for each genotype. A negative, no-antibody IP was performed to account for non-specific peptide association with magnetic beads. G Representative western blot of HTT-IP from WT or HD LMs, probed against HTT, KIF5A, KIF5B, KIF5C, DNCT, MAP1B, MAP2, RAB2, RAB5, RAB7, VPS35, or SUMO2. Except for KIF5A, all show presence in WT and HD HTT-IP. No bands are seen in the negative no antibody control (−Crtl). n = 3. Statistical analysis was conducted using the two-sample two-sided Student’s t test comparing signal/noise intensity between bands in WT and HD conditions normalized to WT. Data represented as mean ± SEM. ns = p > 0.05, *p < 0.05, **p < 0.005.
Article Snippet: Blots were blocked using TBST with 5% BSA for 60 mins at 25 °C and incubated with primary antibodies (SYT1 (Thermofisher 1:1000), Rab4 (Abcam 1:1000), Rab5 (Abcam 1:1000), Rab2 (SCBT 1:500), Rab7 (SCBT 1:500), VPS35 (SCBT 1:500), SUMO2/3 (Cytoskeleton 1:500), KIF5A (Goldstein 1:250), KIF5B (Goldstein 1:250), KIF5C (Goldstein 1:250), DIC (Abcam 1:1000), DNCT (Abcam 1:1000), Actin (ThermoFisher 1:1000), Tubulin (Abcam 1:2000), HTT rabbit polyclonal (Abcam 1:1000), HTT mouse monoclonal (EMD Millipore 1:1000), Golgi (Millipore Sigma 1:1000), Cytochrome C (Santa Cruz 1:1000), TOM20 (CellSignaling Technology 1:500), MAP1B (SCBT 1:1000), MAP2 (BD Pharmigen 1:1000), Total AKT1 (CellSignaling Technology 1:1000), pAKT1 (Ser473, CellSignaling Technology 1:1000), Total GSK3α/β (CellSignaling Technology 1:1000), pGSK3α/β (pY279/pY216; Abcam 1:1000), or ERK (pan-ERK; BD Transduction Laboratories 1:1000) for 16 h at 4 °C.
Techniques: Derivative Assay, Staining, Marker, Fractionation, Membrane, Centrifugation, Control, Liquid Chromatography with Mass Spectroscopy, Magnetic Beads, Western Blot
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![Fig. 1 The proteomic network on HTT containing light membranes is dramatically altered in HD iPSC-derived neurons. A Representative images from healthy/normal (WT, Q17) or diseased (HD, Q109) human iPSCs stained with the pluripotent marker OCT-4, the neuronal precursor (NPC) marker Nestin and the mature neuronal markers MAP2 and βIII-Tubulin. Hoechst stains nuclei. Scale = 25 μm. Differentiated neurons show Synaptophysin (SYP) positive staining. Scale = 10 μm. B Electrophysiological analysis of WT and HD human neurons differentiated from iPSCs show action potentials, which are abolished in the presence of TTX or TEA. C Schematic diagram of human iNeuron lysate fractionation into perinuclear supernatant (PNS), light membrane (LM), soluble (SF), and heavy membrane (P1) fractions by ultra- centrifugation and sucrose gradient separation. D Workflow for quality control and quantification of unique peptides identified from LC-MS of HTT-IPs from WT or HD human iNeurons. E Hierarchical cluster heat map showing the avg. relative abundance (spectral count; SpC) of 800 proteins (≥3 unique peptides/trial across ≥2 biological replicates) quantified across the WT and HD HTT-IPs with a normalized fold change (FC) threshold of ±2X and a significance threshold of p < 0.05 determined by a Welch’s t test across three independent biological replicates. Increased in HD HTT-IP = red, decreased in HD HTT-IP = blue. In addition, proteins were identified in only WT HTT-IP (lost = green) or in only HD HTT-IP (gained = orange). F Volcano plot with the y axis depicting significance (−log10[p value]) and the x axis depicting fold change of individual peptides between HD and WT HTT-IPs (log2[FC]). Three independent biological replicates were performed for each genotype. A negative, no-antibody IP was performed to account for non-specific peptide association with magnetic beads. G Representative western blot of HTT-IP from WT or HD LMs, probed against HTT, KIF5A, KIF5B, KIF5C, DNCT, <t>MAP1B,</t> MAP2, RAB2, RAB5, RAB7, VPS35, or SUMO2. Except for KIF5A, all show presence in WT and HD HTT-IP. No bands are seen in the negative no antibody control (−Crtl). n = 3. Statistical analysis was conducted using the two-sample two-sided Student’s t test comparing signal/noise intensity between bands in WT and HD conditions normalized to WT. Data represented as mean ± SEM. ns = p > 0.05, *p < 0.05, **p < 0.005.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_3294/pm40263294/pm40263294__page3_image1.jpg)
