Fig. 8: Microglial Tbk1-KO leads to focal microglial activation and T cell infiltration in the substantia nigra pars reticulata.

a Representative IBA1 immunofluorescence image of microglia in brains of 4-month-old Tbk1-µG-KO mice. Filled arrowhead: Pallidum (Pa), Empty: Substantia nigra pars reticulata (SNr). Enlargements show representative images of focal microglial activation in the SNr of Tbk1-µG-KO vs control Tbk1-µG-WT mice. Scale bar = 2 mm. b Quantification of IBA1 immunofluorescence intensity in different brain regions between Tbk1-µG-KO and control mice. n = 4 mice per genotype (males only). Adjusted p values determined by repeated measures two-way ANOVA with Sidak’s multiple comparisons test. c Representative IBA1 images of microglia (green) with nuclear PU.1 (red) in the SNr of Tbk1-µG-KO and Tbk1-µG-WT mice. Scale bar = 50 µm. Enlargement, microglial morphology. Scale bar = 10 µm. d–f Quantification of microglia characteristics in the SNr: d density, e processes, and f process length. n = 4 mice per genotype (males only). Adjusted p values determined by two-tailed unpaired t-tests. g Expression profile of a top DEG (Cxcl10) from the brain microglial RNAseq (Fig. 6g) comparing Tbk1-µG-KO to Tbk1-µG-WT mice. RNAseq TPM values are shown and fold changes indicated (DESeq2, log₂FC ≥ 0.5, adjusted p values < 0.05). n = 5 (4-month-old, except Tbk1-µG-WT, n = 6) or n = 6 (22-month-old, except Tbk1-µG-WT, n = 7) mice per genotype (females only). h Representative Cxcl10 mRNA expression image (RNAscope) of 4-month-old Tbk1-µG-KO and Tbk1-µG-WT (male) mice brain sections. Scale bar = 2 mm. Enlargement showing Cxcl10 expression only in the SNr of Tbk1-µG-KO mice. Scale bar = 20 µm. i Representative images of microglia (IBA1⁺) and T cells (CD3⁺) in the SNr of 4-month-old Tbk1-µG-KO (KO) and Tbk1-µG-WT (WT) mice. Scale bar = 50 µm. j Quantification of CD3⁺ T cells density. n = 4 mice per genotype (males only). Adjusted p values determined by a two-tailed unpaired t-test. k Representative images of microglia (IBA1⁺), T cells (CD3⁺), and CD8⁺ subtype T cells in the SNr. Scale bar = 20 µm. h, k Full set of mice were stained (n = 4 mice per genotype, males only), with one representative image shown. b, d–g, j Data are shown as single data points and means ± SEM.