Sex-dependent immune activation shapes disease progression in a model of Parkinson's disease

Abstract

Background: While it is clear that inflammation contributes to Parkinson's disease (PD) and prevalence is higher in males, sex remains an underexplored determinant of immune responses in PD.

Methods: Using the 3KL transgenic mouse model, which expresses three E to K α-synuclein mutations, we investigated how sex and age shape peripheral and central immunity and behavior in synucleinopathy. Male and female 3KL mice were aged to 8- and 14-months. At these ages animals underwent motor and cognitive assessment, followed by assessment of the peripheral immune response using flow cytometry and analysis of microglial transcriptional profiles by bulk RNA sequencing.

Results: Male 3KL mice exhibited earlier onset and greater severity of motor and cognitive impairments, which was linked to a pro-inflammatory peripheral immune profile marked by increased cytotoxic CD8⁺ T cells and IFNγ-producing CD4 Th1 cells. In contrast, female mice displayed delayed symptom onset, preserved cognition, along with early elevations in regulatory IL-10⁺ CD4 and γδ T cells. RNA sequencing of microglia revealed broad sex differences at 8 months. Males demonstrated early upregulation of microglia neurodegenerative signatures, MHC class I/II signaling, ceramide signaling, and pronounced lipid dysregulation, while females showed upregulation of microglial pathways related to protein, metabolic, and neuronal maintenance, including phagosome formation, docosahexaenoic acid signaling, and synaptogenesis pathways. Microglial transcriptional differences were nearly absent by 14 months, suggesting sex-specific trajectories converge during late-stage disease, which is concurrent with a decrease in estrogen in aged female mice.

Conclusions: Together, these findings reveal distinct immune signaling in male and female 3KL mice and identify coordinated changes in T cell and microglial responses that may contribute to sex differences in PD vulnerability and progression. This work underscores the importance of incorporating sex as a biological variable in neurodegeneration research and provides mechanistic insight into immune-mediated modulation of synucleinopathy.

Keywords: 3KL; Aging; Alpha synuclein; Cognition; Microglia; Parkinson’s disease; Sex; T cells.

Fig. 1

Male 3KL mice display accelerated motor and cognitive decline compared to females. (a) Latency to fall on the accelerating rotarod, (b) time to complete the pole test, (c) distance travelled in the blocked arm Y-maze, and (d) duration of time spent exploring the novel arm (NA) and familiar arm (FA) in the blocked arm Y maze. (e) Mouse serum estrogen levels (pg/mg protein). N = 6–7 per group. Results are presented as a box and whisker plot, with whiskers representing minimum and maximum values. Rota rod, pole test, distance travelled on the Y maze and estrogen levels were analyzed using linear mixed-effects models with sex, age, and their interaction as fixed effects. Post hoc comparisons were performed using uncorrected Fisher’s Least Significant Difference test following significant main or interaction effects. Novel arm exploration on the Y maze was compared to an average of time spent exploring the two previously explored arms (familiar arms) and was analyzed using a two-way ANOVA with Tukey’s multiple comparison test. P < 0.05 was considered significant. *P < 0.05, ****P < 0.0001. F: female, FA: familiar arms, M: male, NA: novel arm. Overall, these findings indicate that male 3KL mice develop earlier and more severe motor impairments and age-related cognitive decline, whereas females are relatively protected until later in life, in part aligning with differences in estrogen levels

Fig. 2

Age and sex-dependent T cell alterations in 3KL mice. (a) γδ, CD4⁺, and CD8⁺ T cell sub-sets in the cervical lymph nodes. (b) γδ, CD4⁺, and CD8⁺ T cell sub-sets in the spleen. (c) CD4⁺ T cell sub-types of Tregs, Th17, and Th1 in the cervical lymph nodes. (d) CD4⁺ T cell sub-types of Tregs, Th17, and Th1 in the spleen. (e) Tbet⁺ γδ T cells from cervical lymph node and spleen. (f) Tbet⁺ CD8⁺ T cells from cervical lymph node and spleen. (g) Gating strategy to isolate γδ, CD4⁺, and CD8⁺ T cells. (h) Representative pseudocolor plots from 14 mo CLN samples for females (left) and males (right) of Treg, Th17, and Th1 T cells. Whiskers of box and whisker plot represent minimum and maximum. Grey boxes are female data; blue boxes are male data. Data were analyzed by mixed effect analysis with post-hoc Fisher’s LSD. P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001

Fig. 3

Age and sex-dependent cytokine-producing T cell alterations in 3KL mice. (a) IFN-producing, (b) IL-17-producing, (c) GM-CSF-producing, and (d) IL-10-producing and CD4⁺ T cells from cervical lymph nodes and spleen. (e) Representative plot IFN fluorescence minus one (FMO) and IFNγ-producing CD4⁺ T cells from cervical lymph node. Whiskers of box and whisker plot represent minimum and maximum. Grey boxes are female data; blue boxes are male data. Data were analyzed by mixed effect analysis with post-hoc Fisher’s LSD. P < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001

Fig. 4

Microglia from male 3KL mice show an early inflammatory profile at 8 months of age. (a) Volcano plots of the differentially expressed genes of FACS sorted microglia at 8- and 14-months old. (b) Principal coordinate analysis (PCoA) plots showing the variations between male and female microglial transcriptional profiles. (c) Gene expression fold change of homeostatic, Stage 1 DAM and Stage 2 DAM microglia. (d) Activated pathways from isolated of FACS sorted microglia at 8- and 14-months old determined by Ingenuity Pathway Analysis. Blue represented activation in females compared to males, red represents activation in males compared to females. Differentially expressed gene significant differences detected by DESeq2. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001