Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Representative images of dose dependent increase of BrdU incorporation in hi NSC upon TGR5 activation (Scale bar 25 µm). (B) Quantitation of BrdU incorporation in hi NSC as shown in (A). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (***p = 0.0001, Veh vs TC-G 1005 1µM), (****p < 0.0001 Veh vs TC-G 1005 10µM) n =3 independent experiment. (C) Representative western blot of PCNA showing its dose dependent increase in expression upon TGR5 activation. (D) Quantitation of PCNA shown in (C). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (*p = 0.0172, Veh vs TC-G 1005 1µM), (**p = 0.0047 Veh vs TC-G 1005 10µM) n =10 - 11 /group. (E) Representative western blot of PCNA showing no effect on proliferation upon TGR5 knockdown. (F) Quantitation of immunoblot shown in (E). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Sidak’s multiple comparisons test, (*p = 0.0372, Scr-ShRNA vs Scr-ShRNA + TC-G 1005 1µM, p = ns TGR5-ShRNA vs TGR5-ShRNA + TC-G 1005 1µM) n = 5 /group.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: BrdU Incorporation Assay, Activation Assay, Quantitation Assay, Western Blot, Expressing, Knockdown, shRNA

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Illustration of Quiescence establishment and TC-G 1005 treatment paradigm. (B) Representative western blots of FOXO3a, PCNA and total and phosphor EGFR in BMP4 & TC-G 1005 treated hiNSC showing dose dependent decrease in levels of FOXO3a & increase in levels of PCNA and Phospho EGFR depicting activation of hiNSC. (C) Quantitation of FOXO3a Expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (*p = 0.0308; Veh vs BMP4, p = ns BMP4 vs BMP4 + TC-G 1005 100 nM, *p = 0.0225; BMP4 vs BMP4 + TC-G 1005 1 µM, *p = 0.0394; BMP4 vs BMP4 + TC-G 1005 10 µM) n = 6 - 9 /group. (D) Quantitation of PCNA Expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p=0.0067; Veh vs BMP4, p = ns; BMP4 vs BMP4 + TC-G 1005 100 nM, p = ns; BMP4 vs BMP4 + TC-G 1005 1 µM, *p = 0.0148; BMP4 vs BMP4 + TC-G 1005 10 µM), n = 8 – 10 /group. (E) Quantitation of phospho EGFR and Total EGFR Expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (****p<0.0001; Veh vs BMP4, p = ns BMP4 vs BMP4 + TC-G 1005 100 nM, p = ns; BMP4 vs BMP4 + TC-G 1005 1 µM, *p = 0.0454; BMP4 vs BMP4 + TC-G 1005 10 µM), n = 7 - 10 /group. (F) Immunostaining of Ascl 1 in BMP4 & TC-G 1005 treated hiNSC showing its reappearance upon treatment (scale bar 8 µm). (G) Quantitation of expression of Ascl1 upon treatment as shown in (F). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0028; Veh vs BMP4, **p = 0.0068 BMP4 vs BMP4 + TC-G 1005 1 µM) n = 3 independent experiment.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Western Blot, Activation Assay, Quantitation Assay, Expressing, Immunostaining

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Representative western blots of p62, Beclin 1 & LC3 in BMP4 & TC-G 1005 treated hiNSC showing dose dependent decrease in levels of p62 & LC3-II, increase in beclin 1 depicting activation of hiNSC. (B) Quantitation of p62 expression as shown in (A). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0011; Veh vs BMP4, **p = 0.0023; BMP4 vs BMP4 + TC-G 1005 100 nM, ***p = 0.0001; BMP4 vs BMP4 + TC-G 1005 1 µM, ****p < 0.0001; BMP4 vs BMP4 + TC-G 1005 10 µM) n = 7 - 9 /group. (C) Quantitation of Beclin1 expression as shown in (A). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0050; Veh vs BMP4, ***p = 0.0007; BMP4 vs BMP4 + TC-G 1005 100 nM, ***p = 0.0003; BMP4 vs BMP4 + TC-G 1005 1 µM, ***p < 0.0003; BMP4 vs BMP4 + TC-G 1005 10 µM) n = 7 - 11 /group. (D) Quantitation of LC3 expression as shown in (A). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (***p = 0.0003; Veh vs BMP4, p = ns; BMP4 vs BMP4 + TC-G 1005 100 nM, ***p = 0.0004; BMP4 vs TC-G 1005 1 µM, **p = 0.0072; BMP4 vs BMP4 + TC-G 1005 10 µM) n = 12 - 13 /group. (E) Immunostaining of LAMP 2 in BMP4 & TC-G 1005 treated hiNSC showing its decrease upon treatment (scale bar 8 µm). (F) Quantitation of expression of LAMP2 upon treatment as shown in (E). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (****p < 0.0001; Veh vs BMP4, ***p = 0.0001 BMP4 vs BMP4 + TC-G 1005 1 µM) n = 3 independent experiment.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Western Blot, Activation Assay, Quantitation Assay, Expressing, Immunostaining

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Diagrammatic representation of IL6 induced inflammation and TC-G 1005 treatment paradigm. (B) Representative western blots of p53, p16 & PCNA in IL6 & TC-G 1005 treated hi NSC showing dose dependent decrease in levels of p53 & p16 depicting decrease in inflammation and increase in PCNA levels suggesting resumption of hi NSC proliferation and rescue from senescence. (C) Quantitation of p53 expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0066; Veh vs IL6, p = ns; IL6 vs IL6 + TC-G 1005 100 nM, *p = 0.0156; IL6 vs IL6 + TC-G 1005 1 µM, **p = 0.0036; IL6 vs IL6 + TC-G 1005 10 µM) n = 4 /group. (D) Quantitation of p16 expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0039; Veh vs IL6, p = ns; IL6 vs IL6 + TC-G 1005 100 nM, *p = 0.0159; IL6 vs IL6 + TC-G 1005 1 µM, ***p = 0.0002; IL6 vs IL6 + TC-G 1005 10 µM) n = 6 /group. (E) Quantitation of PCNA expression as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0035; Veh vs IL6, *p = 0.0140; IL6 vs IL6 + TC-G 1005 100 nM, **p = 0.0032; IL6 vs IL6 + TC-G 1005 1 µM, **p = 0.0020; IL6 vs IL6 + TC-G 1005 10 µM) n = 4 -5 /group. (F) Immunostaining of p16 in IL6 & TC-G 1005 treated hi NSC showing its decrease upon treatment (scale bar 50 µm). (G) Quantitation of expression of p16 upon treatment as shown in (F). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (****p < 0.0001; Veh vs IL6, **p = 0.0095; IL6 vs IL6 + TC-G 1005 100 nM, ***p = 0.0003; IL6 vs IL6 + TC-G 1005 1 µM, ****p < 0.0001; IL6 vs IL6 + TC-G 1005 10 µM) n = 3 independent experiment.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Western Blot, Quantitation Assay, Expressing, Immunostaining

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A, B) Volcano plots of differentially expressed genes (DEGs) identified by DESeq2, showing IL-6 versus vehicle (A) and IL-6 versus IL-6 + TC-G 1005 (B). Red and blue denote significantly up- and down-regulated genes, respectively (Benjamini-Hochberg adjusted p ≤ 0.05); grey denotes non-significant genes. Vertical and horizontal lines mark fold-change and significance thresholds. (C) Principal component analysis of DESeq2-normalised counts. Each symbol represents one biological replicate, colour coded by treatment group; ellipses indicate group clustering. PC1 and PC2 together account for >85% of total variance. (D) Heatmap of row-scaled normalised counts for senescence- and proliferation-associated genes across vehicle, IL-6, and IL-6 + TC-G 1005 groups. Colour scale represents scaled counts (red-higher, blue-lower). Benjamini-Hochberg adjusted p-values are shown in parentheses for genes reaching p < 0.05.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Analysis

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Diagrammatic representation of drug treatment regimen and behavioral assay paradigm in 18-month-old C57BL6/J mice. (B) Representative images of dentate gyrus showing increase BrdU incorporation and increase in SOX2 population in old mice treated with TC-G 1005 suggesting proliferation of hi NSC and increase in stem cell pool (scale bar 49 µm). (C) Bar graph depicting quantitation of BrdU+SOX2+ cells as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (****p < 0.0001; Young vs Old, *p = 0.0194 Old vs Old + TCG 1005); n = 8/ group. (D) Bar graph depicting quantitation of SOX2+ cells as shown in (B). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (***p < 0.0008; Young vs Old, *p = 0.0240; Old vs Old + TCG 1005); n = 8/ group. (E) Representative images of dentate gyrus showing decrease in p16 expression in old mice treated with TC-G 1005 and increase in SOX2+ population (scale bar 49 µm). (F) Bar graph depicting quantitation of p16 as shown in (E). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (****p < 0.0001; Young vs Old, *p = 0.0398 Old vs Old + TCG 1005); n = 10-11/ group. (G) Bar graph depicting quantitation of SOX2+ cells as shown in (E). Data were plotted as mean ± SEM, Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, (**p = 0.0019; Young vs Old, *p = 0.0186 Old vs Old + TCG 1005); n = 8-10/ group.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Behavioral Assay, BrdU Incorporation Assay, Stem Cell, Quantitation Assay, Expressing

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Experimental timeline. (B) Representative images confirming dentate gyrus injection track by mCherry fluorescence (red) from AAV9-Scr-mCherry or AAV9-mTGR5-mCherry with DAPI (blue) (scale bar 2 inches). (C) Y-maze novel-arm discrimination index showing TC-G 1005 improved discrimination in Scr-shRNA aged mice but not in TGR5-shRNA aged mice. Data were plotted as Mean ± SEM; one-way ANOVA followed by Dunnett’s multiple comparisons test, ***p = 0.0009; young vs Scr-shRNA aged, *p = 0.0140 Scr-shRNA aged vs Scr-shRNA aged + TC-G 1005, p = ns TGR5-shRNA aged vs TGR5-shRNA aged + TC-G 1005; n = 8–10 mice/ group. (D) Social recognition discrimination index between a novel and a familiar conspecific mouse. TC-G 1005 restored social memory in Scr-shRNA mice. Data were plotted as Mean ± SEM; one-way ANOVA followed by Dunnett’s multiple test, **p = 0.0045 (young vs Scr-shRNA aged), *p = 0.0225 (Scr-shRNA aged vs Scr-shRNA aged + TC-G 1005), p = ns (TGR5-shRNA aged vs TGR5-shRNA aged + TC-G 1005); n = 7–8 mice/ group. ( E ) Cumulative distance travelled in the open field, recorded for 60 min in 5-min bins. All aged groups were hypoactive relative to young mice, but neither TC-G 1005 nor TGR5 knockdown altered locomotion. Data were plotted as Mean ± SEM; one-way ANOVA followed by Dunnett’s test, p = 0.0088, p = 0.0027, p = 0.0048 and p = 0.0035 for young versus Scr-shRNA aged, Scr-shRNA aged + TC-G 1005, TGR5-shRNA aged and TGR5-shRNA aged + TC-G 1005, respectively; ns = not significant, n = 7–10 mice / group. (F) Representative confocal images of the dentate gyrus immunostained for SOX2 (green), BrdU (red) and DAPI (blue) in Scr-shRNA and TGR5-shRNA aged mice treated with vehicle or TC-G 1005. Boxed regions are shown at higher magnification in the adjacent panels; arrowheads mark SOX2⁺BrdU⁺ double-positive cells (scale bar 49 µm). (G) Representative confocal images of the dentate gyrus immunostained for SOX2 (green), p16 (red) and DAPI (blue) across the same four groups, with magnified insets and arrowheads marking SOX2⁺p16⁺ senescent stem cells (scale bar 49 µm). (H) Quantification of BrdU⁺SOX2⁺ cell density from (F). TC-G 1005 increased proliferating NSCs in Scr-shRNA mice but not in TGR5-shRNA mice. Data were plotted as Mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test, *p = 0.0464 (Scr-shRNA aged versus Scr-shRNA aged + TC-G 1005), p = ns (TGR5-shRNA aged versus TGR5-shRNA aged + TC-G 1005); n = 8–10 mice /group. (I) Quantification of p16⁺SOX2⁺ cell density from (H). TC-G 1005 reduced the senescent stem cell burden in Scr-shRNA mice, with no effect after TGR5 knockdown. Data were plotted as Mean ± SEM; one-way ANOVA with Sidak’s multiple comparisons test, *p = 0.0178 (Scr-shRNA aged versus Scr-shRNA aged + TC-G 1005), p = ns (TGR5-shRNA aged versus TGR5-shRNA aged + TC-G 1005); n = 6–8 mice/ group.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Injection, Fluorescence, shRNA, Knockdown, Confocal, Stem Cell, Immunohistochemistry

Journal: bioRxiv

Article Title: THE BILE ACID RECEPTOR TGR5 MODULATES HIPPOCAMPAL NEURAL STEM CELL STATE AND AGEING

doi: 10.64898/2026.10.08.757807

Figure Lengend Snippet: (A) Experimental workflow: hippocampal tissue was collected from young, aged, and TC-G 1005-treated aged mice, digested, and analysed using LC-MS/MS. (B) Partial least-squares discriminant analysis (PLS-DA) of hippocampal proteomes, showing separation of young (Y), aged (O), and treated-aged (OT) groups along the first two components (34% and 13% explained variance, respectively). (C) Venn diagram of differentially abundant proteins (fold change ≥ 1.2, Proteome Discoverer-adjusted p < 0.05) in the ageing (O vs Y) and treatment (OT vs O) comparisons, showing 185 ageing-specific, 152 treatment-specific, and 86 shared differential proteins. (D) Differential proteins and rescue trajectories: (a, b) volcano plots for the ageing and treatment comparisons, respectively (red, increased abundance; blue, decreased abundance; grey, not significant; open circles, values at the Proteome Discoverer ratio-reporting floor/ceiling); selected proteins are labelled. Scatter plot of each protein’s ageing effect (log2 fold change, O vs Y) against its treatment effect (log2 fold change, OT vs O), classified as rescued , partially rescued , showing a directional trend (73), not rescued (91), or treatment-specific (129); the dotted line indicates exact reversal of the ageing effect. (E) Heatmap of the top 45 (of 93) treatment-rescued proteins, showing relative group profiles across Y, O, and OT, annotated by pathway association and rescue classification. (F) Pathway-enrichment analysis across four query groups shows that treatment with TC-G1005 reversed/restored pathways (green labels), increased in O vs Y (light blue), decreased in O vs Y (dark blue), and ageing-associated but not rescued (black) plotted by enrichment ratio, with point size reflecting the number of contributing genes and colour indicating −log10(BH-adjusted FDR). (G) Module-level analysis of four biologically defined protein sets (mitochondrial OXPHOS/import/mitoribosome, astroglia reactivity, complement C1q, myelin/oligodendrocyte) tested for a treatment effect against all other quantified proteins (Mann-Whitney); heatmap shows individual protein log2 fold changes for ageing and treatment, with asterisks marking |fold change| ≥ 1.2 and adjusted p < 0.05.

Article Snippet: TGR5 agonist TC-G 1005 (Medchem Express; HY-110173) was administered subcutaneously (s.c.) at a dose of 5 mg/kg for 28 days twice a day.

Techniques: Tissue, Liquid Chromatography with Mass Spectroscopy, Analysis, MANN-WHITNEY, Significance Assay