Journal: bioRxiv

Article Title: Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

doi: 10.1101/2024.03.24.586459

Figure Lengend Snippet: Mouse brain posterior cortex samples were homogenized and extracted for western blot analysis. Tau PS19 mouse group was normalized to the Non-Tg group for comparison. A-C, Tau PS19 mice show increased phosphorylation of the major mTORC1 subunit mTOR and several major mTORC1 downstream substrates (S6K1, S6, 4E-BP1). A, Western blot images of the total mTOR, S6K1, S6, 4E-BP1, and the associated phosphorylated mTOR (pS2448), S6K1 (pT421/S424), S6 (pS235/236), 4E-BP1 (pT37/46) are shown. B-E, Quantification analysis of A is shown for total and phosphorylated mTOR ( B ), S6K1 ( C ), S6 ( D ), and 4E-BP1 ( E ). F-I, Tau PS19 mice show increased phosphorylation of ULK1, p62, and decreased LC3-II/LC3-I ratio. F, Western blot images of total ULK1, p62, and the associated phosphorylated ULK1 (pS757), p62 (pS403), and LC3-I and LC3-II are shown. G-I, Quantification analysis of F is shown for total and phosphorylated ULK1 ( G ), p62 ( H ), and LC3-II/LC3-I ratio ( I ). n = 7-9 mice per genotype, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired t-test. Values are mean ± SEM.

Article Snippet: Primary antibodies used in this study include Tau-5 (NeoMarkers, #MS-247-PABX), HT-7 (Thermo ScientificTM, # MN1000), Tau pS202/T205 (AT8, Thermo ScientificTM, #MN1020), Tau pS214 (abcam, #ab170892), Tau-H150 (Santa Cruz Biotechnology, #sc-5587), Tau PS199/202 (AnaSpec, #54963-025), Tau pS396 (AnaSpec, #54977-025), ASS1 (Protein Tech, #16210-1-AP), ASL (Protein Tech, #16645-1-AP), GPRC6A (human, Gene Tex, #GTX108214), GPRC6A (mouse, LifeSpan BioSciences, #LS-B14489), AKT (Cell Signaling Tech, #4691X), AKT pT308 (Cell Signaling Tech, #2965S), AKT pS473 (Cell Signaling Tech, #4060X), ERK1/2 (Cell Signaling Tech, #9102S), ERK1/2 pT202/Y204 (Cell Signaling Tech, #9101), mTOR (Cell Signaling Tech, #2983S), mTOR pS2448 (Cell Signaling Tech, #5536S), S6K1 (Cell Signaling Tech, #2708), S6K1 pT421/S424 (Cell Signaling Tech, #9204S), S6 (Cell Signaling Tech, #2217S), S6 pS235/236 (Cell Signaling Tech, #2211S), S6 pS240/244 (Cell Signaling Tech, #2215S), 4E-BP1 (Cell Signaling Tech, #9644), 4E-BP1 pT37/46 (Cell Signaling Tech, #2855), 4E-BP1 pS65 (Cell Signaling Tech, #9451S), ULK1 (Cell Signaling Tech, #8054), ULK1 pS757 (Cell Signaling Tech, #14202), p62 (Protein Tech, #18420-1-AP), p62 pS403 (Cell Signaling Tech, #39786S), LC3 (Cell Signaling Tech, #L7543), Tau Dako (Agilent Technologies, #A002401-2), C-Myc (Cell Signaling Tech, #2272), GFP (abcam, #ab13970), tGFP (turbo GFP, Origene, #TA150075), GAPDH (Meridian Life Science, #H86504M), and β-actin (Sigma Aldrich, #A5441).

Techniques: Western Blot, Comparison, Phospho-proteomics

Journal: bioRxiv

Article Title: Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

doi: 10.1101/2024.03.24.586459

Figure Lengend Snippet: Arginine was supplemented in a concentration-dependent manner on the fourth-day culture of E18 wild-type mouse primary cortical neurons. The basal levels of arginine in the neurobasal medium (0.398 mM) served as the concentration for the control; two different concentrations of arginine (0.3 mM, 1 mM) were reconstituted into the medium. Data were normalized to the control for quantification analysis. After the treatment, cells were harvested and lysed as whole-cell lysate for western blot analysis. The arginine activated mTORC1 signaling, as measured by several downstream phosphorylated substrates. A, Representative western blot images of the total mTOR, S6, 4E-BP1, and the associated phosphorylated mTOR (pS2448), S6 (pS235/236), and 4E-BP1 (pT37/46), as well as total tau (Dako) are shown. B-E, Quantification analysis of western blots are shown for total and phosphorylated mTOR ( B ), S6 ( C ), 4E-BP1 ( D ), and total tau ( E ). n = 3 independent experiments, * p < 0.05, one-way ANOVA with Dunnett’s multiple-comparison test. Values are mean ± SEM.

Article Snippet: Primary antibodies used in this study include Tau-5 (NeoMarkers, #MS-247-PABX), HT-7 (Thermo ScientificTM, # MN1000), Tau pS202/T205 (AT8, Thermo ScientificTM, #MN1020), Tau pS214 (abcam, #ab170892), Tau-H150 (Santa Cruz Biotechnology, #sc-5587), Tau PS199/202 (AnaSpec, #54963-025), Tau pS396 (AnaSpec, #54977-025), ASS1 (Protein Tech, #16210-1-AP), ASL (Protein Tech, #16645-1-AP), GPRC6A (human, Gene Tex, #GTX108214), GPRC6A (mouse, LifeSpan BioSciences, #LS-B14489), AKT (Cell Signaling Tech, #4691X), AKT pT308 (Cell Signaling Tech, #2965S), AKT pS473 (Cell Signaling Tech, #4060X), ERK1/2 (Cell Signaling Tech, #9102S), ERK1/2 pT202/Y204 (Cell Signaling Tech, #9101), mTOR (Cell Signaling Tech, #2983S), mTOR pS2448 (Cell Signaling Tech, #5536S), S6K1 (Cell Signaling Tech, #2708), S6K1 pT421/S424 (Cell Signaling Tech, #9204S), S6 (Cell Signaling Tech, #2217S), S6 pS235/236 (Cell Signaling Tech, #2211S), S6 pS240/244 (Cell Signaling Tech, #2215S), 4E-BP1 (Cell Signaling Tech, #9644), 4E-BP1 pT37/46 (Cell Signaling Tech, #2855), 4E-BP1 pS65 (Cell Signaling Tech, #9451S), ULK1 (Cell Signaling Tech, #8054), ULK1 pS757 (Cell Signaling Tech, #14202), p62 (Protein Tech, #18420-1-AP), p62 pS403 (Cell Signaling Tech, #39786S), LC3 (Cell Signaling Tech, #L7543), Tau Dako (Agilent Technologies, #A002401-2), C-Myc (Cell Signaling Tech, #2272), GFP (abcam, #ab13970), tGFP (turbo GFP, Origene, #TA150075), GAPDH (Meridian Life Science, #H86504M), and β-actin (Sigma Aldrich, #A5441).

Techniques: Concentration Assay, Control, Western Blot, Comparison

Journal: bioRxiv

Article Title: Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

doi: 10.1101/2024.03.24.586459

Figure Lengend Snippet: Tetracycline inducible human HEK293T cells overexpressing tau (iHEK-Tau) were transfected with plasmids to overexpress GPRC6A-Myc or GFP control. The GPRC6A-Myc group was normalized to the GFP control for quantification analysis. A, Representative western blot images of overexpressed proteins (GPRC6A-Myc, GFP) and AKT/ERK signaling-associated proteins are presented. B-D, Quantification analysis of western blots are shown for GPRC6A-Myc ( B ), GFP ( B ), total and phospho-AKT (pS473, pT308, C ), and total and phospho-ERK (pT202/Y204, D ). E, Representative western blot images of mTORC1 signaling-associated proteins are presented. F-H, Quantification analysis of E is shown for total and phospho-mTOR (pS2448, F ), total and phospho-S6 (pS235/236, pS240/244, G ), and total and phospho-4E-BP1 (pT37/46, pS65, H ). I, Representative western blot images of autophagy-associated proteins and tau species are presented. J-L, Quantification analysis of I is shown for total and phospho-ULK1 (pS757, J ), LC3-II/LC3-I ratio ( K ), and total and phospho-tau (pS214, L ). n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, unpaired t-test. Values are mean ± SEM.

Article Snippet: Primary antibodies used in this study include Tau-5 (NeoMarkers, #MS-247-PABX), HT-7 (Thermo ScientificTM, # MN1000), Tau pS202/T205 (AT8, Thermo ScientificTM, #MN1020), Tau pS214 (abcam, #ab170892), Tau-H150 (Santa Cruz Biotechnology, #sc-5587), Tau PS199/202 (AnaSpec, #54963-025), Tau pS396 (AnaSpec, #54977-025), ASS1 (Protein Tech, #16210-1-AP), ASL (Protein Tech, #16645-1-AP), GPRC6A (human, Gene Tex, #GTX108214), GPRC6A (mouse, LifeSpan BioSciences, #LS-B14489), AKT (Cell Signaling Tech, #4691X), AKT pT308 (Cell Signaling Tech, #2965S), AKT pS473 (Cell Signaling Tech, #4060X), ERK1/2 (Cell Signaling Tech, #9102S), ERK1/2 pT202/Y204 (Cell Signaling Tech, #9101), mTOR (Cell Signaling Tech, #2983S), mTOR pS2448 (Cell Signaling Tech, #5536S), S6K1 (Cell Signaling Tech, #2708), S6K1 pT421/S424 (Cell Signaling Tech, #9204S), S6 (Cell Signaling Tech, #2217S), S6 pS235/236 (Cell Signaling Tech, #2211S), S6 pS240/244 (Cell Signaling Tech, #2215S), 4E-BP1 (Cell Signaling Tech, #9644), 4E-BP1 pT37/46 (Cell Signaling Tech, #2855), 4E-BP1 pS65 (Cell Signaling Tech, #9451S), ULK1 (Cell Signaling Tech, #8054), ULK1 pS757 (Cell Signaling Tech, #14202), p62 (Protein Tech, #18420-1-AP), p62 pS403 (Cell Signaling Tech, #39786S), LC3 (Cell Signaling Tech, #L7543), Tau Dako (Agilent Technologies, #A002401-2), C-Myc (Cell Signaling Tech, #2272), GFP (abcam, #ab13970), tGFP (turbo GFP, Origene, #TA150075), GAPDH (Meridian Life Science, #H86504M), and β-actin (Sigma Aldrich, #A5441).

Techniques: Transfection, Control, Western Blot

Journal: bioRxiv

Article Title: Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

doi: 10.1101/2024.03.24.586459

Figure Lengend Snippet: A-H , Human HeLa cells stably overexpressing tau were transfected with siRNA to down-regulate the expression of GPRC6A. The scrambled siRNA served as the control. The siRNA group against GPRC6A was normalized to the siRNA control group for quantification analysis. A, Representative western blot images of GPRC6A, mTORC1-autophagy signaling associated proteins, and tau are presented. B-H, Quantification analysis of western blots are shown for GPRC6A ( B ), total and phospho-mTOR (pS2448, C ), total and phospho-S6 (pS235/236, D ), total and phospho-4E-BP1 (pT37/46, E ), total and phospho-ULK1 (pS757, F ), LC3-II/LC3-I ratio ( G ), and total and phospho-tau (pS214, H ). n = 3 independent experiments. I-P, Tetracycline inducible human HEK293T cells overexpressing tau (iHEK-Tau) were transfected with either siRNA against GPRC6A or a scrambled target. The scrambled siRNA served as the control. The GPRC6A siRNA was normalized to the scramble siRNA control for quantification analysis. I, Representative western blot images of GPRC6A, mTORC1-autophagy signaling associated proteins, and tau are presented. J-P, Quantification analysis of I is shown for GPRC6A ( J ), total and phospho-mTOR (pS2448, K ), total and phospho-S6 (pS235/236, L ), total and phospho-4E-BP1 (pT37/46, M ), total and phospho-ULK1 (pS757, N ), LC3-II/LC3-I ratio ( O ), and total and phospho-tau (pS214, P ). n = 2 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired t-test. Values are mean ± SEM.

Article Snippet: Primary antibodies used in this study include Tau-5 (NeoMarkers, #MS-247-PABX), HT-7 (Thermo ScientificTM, # MN1000), Tau pS202/T205 (AT8, Thermo ScientificTM, #MN1020), Tau pS214 (abcam, #ab170892), Tau-H150 (Santa Cruz Biotechnology, #sc-5587), Tau PS199/202 (AnaSpec, #54963-025), Tau pS396 (AnaSpec, #54977-025), ASS1 (Protein Tech, #16210-1-AP), ASL (Protein Tech, #16645-1-AP), GPRC6A (human, Gene Tex, #GTX108214), GPRC6A (mouse, LifeSpan BioSciences, #LS-B14489), AKT (Cell Signaling Tech, #4691X), AKT pT308 (Cell Signaling Tech, #2965S), AKT pS473 (Cell Signaling Tech, #4060X), ERK1/2 (Cell Signaling Tech, #9102S), ERK1/2 pT202/Y204 (Cell Signaling Tech, #9101), mTOR (Cell Signaling Tech, #2983S), mTOR pS2448 (Cell Signaling Tech, #5536S), S6K1 (Cell Signaling Tech, #2708), S6K1 pT421/S424 (Cell Signaling Tech, #9204S), S6 (Cell Signaling Tech, #2217S), S6 pS235/236 (Cell Signaling Tech, #2211S), S6 pS240/244 (Cell Signaling Tech, #2215S), 4E-BP1 (Cell Signaling Tech, #9644), 4E-BP1 pT37/46 (Cell Signaling Tech, #2855), 4E-BP1 pS65 (Cell Signaling Tech, #9451S), ULK1 (Cell Signaling Tech, #8054), ULK1 pS757 (Cell Signaling Tech, #14202), p62 (Protein Tech, #18420-1-AP), p62 pS403 (Cell Signaling Tech, #39786S), LC3 (Cell Signaling Tech, #L7543), Tau Dako (Agilent Technologies, #A002401-2), C-Myc (Cell Signaling Tech, #2272), GFP (abcam, #ab13970), tGFP (turbo GFP, Origene, #TA150075), GAPDH (Meridian Life Science, #H86504M), and β-actin (Sigma Aldrich, #A5441).

Techniques: Stable Transfection, Transfection, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Nutrient Sensing Receptor GPRC6A Regulates mTORC1 Signaling and Tau Biology

doi: 10.1101/2024.03.24.586459

Figure Lengend Snippet: A-D , Mouse N2a cells were transfected with two different shRNA against endogenous mouse Gprc6a and a scrambled sequence as the control. The two shRNA groups against Gprc6a (shGprc6a-1, shGprc6a-2) were normalized to the shRNA control group (shScramble) for quantification analysis. A, Representative western blot images of GPRC6A, total and phosphorylated mTOR, and S6 are shown. B-D, Quantification analysis of western blots for ( A ) is shown for GPRC6A ( B ), total and phospho-mTOR (pS2448, C ), and total and phospho-S6 (pS235/236, pS240/244, D ). n = 4 independent experiments. one-way ANOVA with Dunnett’s multiple-comparison test. E, Shows a schematic timeline diagram of the two rAAV constructs encoded with either shScramble or shGprc6a-2 injected into the brains of 6-month-old Non-Tg and Tau PS19. Four months post-incubation, brain tissue was harvested from 10-month-old mice and subjected to sequential sarkosyl-based protein extraction for western blot analysis. F, Western blot images of turbo green fluorescent protein (tGFP), GPRC6A, and total and phospho-tau are represented. G-H, Quantification analysis of F is shown for tGFP ( G ), GPRC6A ( H ) from the S1 soluble fraction and P1 membrane fraction of Non-Tg and Tau PS19 mice (n = 5-6 mice per group from Non-Tg and Tau PS19 mice). Two-way ANOVA followed by pairwise comparisons. I-K, Quantification analysis of F is shown for total tau (HT-7), phospho-tau at S202/T205 (AT8), and phospho-tau at S214 from protein fractions of soluble tau (S1, I ), sarkosyl soluble tau (S2, J ), and sarkosyl insoluble tau (P3, K ) in Tau PS19 mice. n = 6 mice per group from Tau PS19 mice. Unpaired t-test. * p < 0.05, ** p < 0.01, Values are mean ± SEM.

Article Snippet: Primary antibodies used in this study include Tau-5 (NeoMarkers, #MS-247-PABX), HT-7 (Thermo ScientificTM, # MN1000), Tau pS202/T205 (AT8, Thermo ScientificTM, #MN1020), Tau pS214 (abcam, #ab170892), Tau-H150 (Santa Cruz Biotechnology, #sc-5587), Tau PS199/202 (AnaSpec, #54963-025), Tau pS396 (AnaSpec, #54977-025), ASS1 (Protein Tech, #16210-1-AP), ASL (Protein Tech, #16645-1-AP), GPRC6A (human, Gene Tex, #GTX108214), GPRC6A (mouse, LifeSpan BioSciences, #LS-B14489), AKT (Cell Signaling Tech, #4691X), AKT pT308 (Cell Signaling Tech, #2965S), AKT pS473 (Cell Signaling Tech, #4060X), ERK1/2 (Cell Signaling Tech, #9102S), ERK1/2 pT202/Y204 (Cell Signaling Tech, #9101), mTOR (Cell Signaling Tech, #2983S), mTOR pS2448 (Cell Signaling Tech, #5536S), S6K1 (Cell Signaling Tech, #2708), S6K1 pT421/S424 (Cell Signaling Tech, #9204S), S6 (Cell Signaling Tech, #2217S), S6 pS235/236 (Cell Signaling Tech, #2211S), S6 pS240/244 (Cell Signaling Tech, #2215S), 4E-BP1 (Cell Signaling Tech, #9644), 4E-BP1 pT37/46 (Cell Signaling Tech, #2855), 4E-BP1 pS65 (Cell Signaling Tech, #9451S), ULK1 (Cell Signaling Tech, #8054), ULK1 pS757 (Cell Signaling Tech, #14202), p62 (Protein Tech, #18420-1-AP), p62 pS403 (Cell Signaling Tech, #39786S), LC3 (Cell Signaling Tech, #L7543), Tau Dako (Agilent Technologies, #A002401-2), C-Myc (Cell Signaling Tech, #2272), GFP (abcam, #ab13970), tGFP (turbo GFP, Origene, #TA150075), GAPDH (Meridian Life Science, #H86504M), and β-actin (Sigma Aldrich, #A5441).

Techniques: Transfection, shRNA, Sequencing, Control, Western Blot, Comparison, Construct, Injection, Incubation, Protein Extraction, Membrane