Parkinson's disease (PD), a progressive neurodegenerative disorder, is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to reduced striatal dopamine levels. PD is more prevalent in men, a sex bias whose underlying mechanisms remain unclear. While the neuroprotective effects of estradiol in women have been suggested, the role of central aromatase (responsible for local estradiol synthesis) and its regulation by sex hormones and RORA have not been fully explored. RORA, a gene known to regulate aromatase transcription and protect neurons against oxidative stress (a key factor in PD pathogenesis), is a potential link. This study hypothesized that sex differences in SNpc RORA expression contribute to the observed sex bias in PD prevalence and pathogenesis.

The literature extensively documents the sex bias in PD, with males exhibiting earlier onset, different clinical courses, and varied treatment responses compared to females. Estradiol's neuroprotective role has been highlighted, particularly its local, centrally-synthesized form, rather than peripherally circulating estradiol. Central aromatase inhibition studies revealed that intra-nigrostriatal oestradiol generation was neuroprotective in both sexes. RORA, a regulator of aromatase transcription, is also known for its neuroprotective functions, including protection against oxidative stress. However, its role in PD, especially concerning sex differences, has been unexplored. Previous research has linked RORA to several neurological and psychological disorders, further supporting the investigation of its potential role in PD.

This study employed a case-control design using post-mortem brain samples (14 late-stage PD patients and 11 age- and sex-matched controls). Quantitative PCR (qPCR) and Western blot analyses were used to quantify RORA gene and protein expression in the SNpc and the anterior cingulate cortex (CgCx, a control region). An in vitro PD model was established using the N27 dopaminergic cell line challenged with 6-OHDA (6-hydroxydopamine), a dopaminergic neurotoxin. The effects of RORA agonists (SR1078) and antagonists (SR1001) on cell viability, mitochondrial function, and apoptosis were assessed using MTS, LDH, caspase 3/7 activity assays, MitoSOX Red staining for mitochondrial ROS, and Annexin V/propidium iodide flow cytometry. Western blotting was also used to analyze proteins involved in intracellular redox signaling pathways (aromatase, 17βHSD10, NADPH oxidases (Nox1, Nox2, Nox4), active MMP-2 (actMMP-2), and PKC). Statistical analyses included the Mann-Whitney U test for post-mortem data and one-way ANOVA with post-hoc Tukey's tests for cell culture data.

In control subjects, significantly higher RORA gene and protein expression were observed in the SNpc of females compared to males. In PD patients, male subjects displayed significantly higher SNpc RORA expression than females. The CgCx showed no significant RORA differences between sexes or PD status. In the in vitro model, 6-OHDA exposure dose-dependently reduced N27 cell viability and increased cell death, with significant increases in caspase 3/7 activity (apoptosis indicator) and mitochondrial ROS production. Treatment with SR1078 (RORA agonist) significantly attenuated 6-OHDA-induced toxicity, reducing cell death, caspase 3/7 activity, and mitochondrial ROS. SR1001 (RORA antagonist) exacerbated 6-OHDA-induced cell death. Western blot analysis revealed that SR1078 pre-treatment significantly reduced Nox1 and Nox2 levels induced by 6-OHDA and decreased cleaved PKC levels, indicating modulation of oxidative stress pathways. A significant reduction in actMMP-3 was also observed after SR1078 pre-treatment.

This study's findings demonstrate, for the first time, sex differences in human SNpc RORA expression and its differential alteration in male and female PD subjects. The increased RORA expression in male PD patients, but not females, suggests a potential compensatory neuroprotective response. In contrast, higher baseline RORA levels in female control brains might contribute to their relative resilience to PD. The in vitro data directly confirm the neuroprotective properties of RORA, showing that a RORA agonist protects dopaminergic neurons against 6-OHDA-induced toxicity via mechanisms involving reduced oxidative stress and apoptosis. The lack of significant changes in aromatase and 17βHSD10 suggests alternative neuroprotective mechanisms for RORA beyond its regulation of these enzymes.

This study provides compelling evidence for a sex-specific role for RORA in PD pathogenesis and highlights the therapeutic potential of RORA agonists. Future research should focus on elucidating the precise mechanisms of RORA's neuroprotection, exploring the interplay between sex hormones and RORA expression, and developing RORA-targeted therapies for PD.

The study's limitations include the relatively small sample size of the post-mortem brain study and the use of an immortalized cell line (N27) in the in vitro model, which may not fully capture the complexity of human dopaminergic neurons. Further studies with larger sample sizes and more sophisticated in vitro and in vivo models are needed to validate these findings and explore the clinical implications of RORA modulation in PD.