Fig. 3: mTORC1-dependent selective dopaminergic neurotoxicity, astrogliosis, and motor impairments by gut-colonized S. mutans.

a (Upper panel) Representative immunofluorescence images of S235/S236 phosphorylated S6 ribosomal protein (pS6) and TH in the ventral midbrain coronal sections from GF mice gavaged with Veh, S. mutans, or pasteurized S. mutans (P-S. mutans). pS6 signals originally captured in the red channel are pseudocolored in white for visualization. (Bottom panel) Quantification of relative pS6 signal intensities in TH-positive dopamine neurons of the SNpc (n = 7 mice per group). Scale bar = 100 μm. b (Upper panel) Representative immunofluorescence images of pS6 and TH in the ventral midbrain coronal sections from GF mice gavaged with E. coli (Con) or E. coli (urdA). Original red pS6 immunofluorescence signals are pseudocolored in white. (Bottom panel) Quantification of relative pS6 signal intensities in TH-positive dopamine neurons of the SNpc (n = 6 mice per E. coli (Con) and n = 7 mice per E. coli (urdA) group). Scale bar = 100 μm. c (Upper panel) Representative Western blots of pIRS1 (S636/S639), IRS1, pS6K1 (T389), and S6K1 in mouse primary cortical neurons treated with ImP (100 μM, 24 h) in the presence or absence of the mTORC1 inhibitor rapamycin (Rap, 20 nM, 24 h) or the p38γ inhibitor pirfenidone (Pirf, 1 mM, 24 h). (Bottom panel) Quantification of relative expression levels of pIRS1 (S636/S639) and pS6K1 (T389) (n = 4 per group). d Schematic illustration of the experimental procedure showing antibiotic cocktail treatment to sterilize the gut, cessation of antibiotics (Abx) treatment, and subsequent S. mutans colonization with or without rapamycin intraperitoneal (i.p.) injection. Fourteen days after colonization, mice were subjected to behavior tests and immunohistochemical analysis. Created in BioRender. Kim, J. (https://BioRender.com/yjm4i12). e, f Quantification of imidazole propionate (ImP) concentration in the plasma (e) and brains (f) from the antibiotic-treated mice colonized with S. mutans or Veh and treated with rapamycin (Rap, 4 mg/kg/day) or vehicle (n = 7 mice per Veh group, n = 8 mice per S. mutans group, n = 6 mice per S. mutans+Rap group). Box plots represent the median (centre line), 25%–75% quartile (bounds of box), minima and maxima (whiskers), with all individual data points shown. g (Upper panel) Representative immunofluorescence images of pS6 and TH in the ventral midbrain coronal sections from the antibiotic-treated mice colonized with S. mutans or Veh and administered with rapamycin or vehicle. Original red pS6 immunofluorescence signals are pseudocolored in white. (Bottom panel) Quantification of relative pS6 signal intensities in TH-positive dopamine neurons of the SNpc is shown below (n = 6 mice per group). Scale bar = 100 μm. SNpc and SNr subregions of the VM are indicated by the white dashed line. h (Left panel) Representative anti-TH immunohistochemistry of ventral midbrain coronal sections from the antibiotic-treated mice colonized with S. mutans or vehicle (Veh) and administered with rapamycin or vehicle. Magnified images showing dopaminergic processes in the SNr are presented on the right. (Right panel) Stereological counts of TH-stained and Nissl-stained dopamine neurons in the SNpc. Quantification of Nissl-stained neuronal counts and TH fiber density in the SNr of the indicated experimental groups (n = 5 mice per group). Scale bar = 100 μm, and 25 μm for enlarged images, respectively. i Inflammatory astrogliosis in the ventral midbrains of the indicated experimental antibiotic-treated mouse groups determined by anti-GFAP immunohistochemistry. Magnified images of the SNpc are shown below. Quantified % area fraction of GFAP signals is shown in the right panel (n = 5 mice per group). Scale bar = 100 μm. j Assessment of bradykinesia in the antibiotic-treated mice colonized with S. mutans or Veh and administered with rapamycin or vehicle monitored by measuring latency to reach the base in the pole test (n = 10 mice per Veh and S. mutans group, n = 9 mice per S. mutans+Rap group). Quantitative data are expressed as the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. P values were determined using one-way ANOVA with Tukey’s post hoc test (a, c, e–j) and unpaired two-tailed Student’s t-tests (b). Source data and test statistics are provided as a Source Data file. See also Supplementary Fig. 3.