Fig. 4: α-synuclein preformed fibril (PFF)-induced mTORC1 activation, α-synuclein aggregation, dopaminergic neurotoxicity, and motor impairment promoted by gut-colonized S. mutans.

a Representative immunofluorescence images showing the α-synuclein (α-Syn) aggregation marker S129 phosphorylated α-Syn (pS129-α-Syn) and neuronal marker microtubule-associated protein 2 (MAP2) in mouse primary cultured cortical neurons treated with the indicated combinations of ImP (1, 10, 100, 1000 nM every three days for six days) and α-Syn PFF (0.1 μg/mL treated once and incubated for seven days), counterstained with DAPI. Scale bar = 20 μm. b Quantification of relative immunofluorescence intensities of pS129-α-Syn in the indicated experimental groups (n = 4 per vehicle (DMSO) group, n = 3 per ImP group). c Assessment of neuronal viability in each experimental group, determined by counting MAP2-positive neuritic beads with a pathological phenotype of fragmentation (n = 4 per vehicle (DMSO) group, n = 3 per ImP group). d Schematic illustration of the experimental procedure showing antibiotic cocktail treatment to sterilize the gut, cessation of Abx treatment, and subsequent intranigral stereotaxic injections (inj.) of α-Syn PFF (10 μg in 2 μL) to model sporadic PD in mice followed by S. mutans colonization. Created in BioRender. Kim, J. (https://BioRender.com/x59j418). e Representative immunofluorescence images of pS6 and TH in the ventral midbrain coronal sections from the PBS-injected control or nigral α-Syn PFF-injected mice colonized with S. mutans or vehicle. Original red pS6 immunofluorescence signals are pseudocolored in white. Nuclei were counterstained with DAPI. Quantification of relative pS6 signal intensities in TH-positive dopamine neurons of the SNpc is shown in the right panel (n = 8 mice per group). Scale bar = 100 μm. f Lewy-like inclusions in the ventral midbrains of the PBS-injected control or nigral α-Syn PFF-injected mice colonized with S. mutans or vehicle, determined by anti-pS129-α-Syn (Lewy body marker) immunohistochemistry. Quantified % area fraction of pS129-α-Syn signals in the SNpc subregion is shown in the bottom panel (n = 6 mice per group). Scale bar = 100 μm, and 30 μm for enlarged images, respectively. g (Upper panel) Representative anti-TH immunohistochemistry of ventral midbrains from the indicated experimental mouse groups. Magnified images showing dopaminergic processes in SNr are also presented on the right. (Bottom panel) Stereological counts of TH-stained and Nissl-stained dopamine neurons in the lesioned hemisphere of the SNpc. Nissl-stained neuronal counts and TH fiber density in the SNr of the indicated experimental mouse group (n = 6 mice per group). Scale bar = 100 μm, and 25 μm for enlarged images, respectively. h Assessment of bradykinesia in the PBS-injected control or nigral α-Syn PFF-injected mice colonized with S. mutans or vehicle monitored by measuring latency to reach the base in the pole test (n = 8 mice per PBS-injected and PFF-injected group, n = 9 mice per PFF + S. mutans group). i Assessment of motor coordination in the indicated experimental mouse groups monitored by measuring latency to fall off in the accelerating rotarod test (n = 8 mice per PBS-injected and PFF-injected group, n = 9 mice per PFF + S. mutans 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. Source data and test statistics are provided as a Source Data file. See also Supplementary Fig. 4.