Neuroprotective role for RORA in Parkinson’s disease revealed by analysis of post-mortem brain and a dopaminergic cell line

Parkinson’s disease (PD) is a progressive neurodegenerative movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and consequent striatal dopamine deficiency. It is the second most common age-related neurodegenerative disease and exhibits a male sex bias in incidence (male-to-female ratio ~1.3–3.7). Sex differences extend to age of onset, symptomatology, disease course, and treatment responses. Experimental and clinical evidence suggests gonadal sex hormones, particularly the neuroprotective effects of oestradiol, contribute to this bias. While peripherally circulating oestradiol shows sex-dependent protection, locally synthesized brain oestradiol via aromatase is neuroprotective in both sexes, indicating therapeutic potential via central aromatase regulation. RORA regulates aromatase transcription and has direct neuronal functions including protection against oxidative stress, a key driver in PD pathogenesis. RORA has been implicated in multiple neuropsychiatric disorders and is regulated by sex hormones, but its involvement in PD remains unexplored. The study tests the hypothesis that RORA expression in the human SNpc shows sex differences and that RORA activation confers neuroprotection in a dopaminergic model of PD.

Prior work shows: (1) PD exhibits a consistent male predominance, with sex differences in clinical features and treatment responses; (2) Oestradiol confers neuroprotection in females peripherally, whereas brain aromatase-derived oestradiol is protective in both sexes, suggesting centrally mediated estrogenic mechanisms are beneficial; (3) RORA regulates aromatase transcription and is itself regulated by sex hormones, forming a potential link between sex steroid signaling and neuroprotection; (4) RORA protects neurons from oxidative stress and hypoxic injury and has been associated with Alzheimer’s disease, depression, autism, ADHD, PTSD, bipolar disorder, and sex-biased conditions such as autism; (5) Despite these connections, RORA’s role in PD had not been directly investigated prior to this study.

Human post-mortem study: Case-control design using SNpc and anterior cingulate cortex (CgCx) samples from brains donated via the Parkinson’s UK Brain Bank. Late-stage pathology-confirmed PD cases (Braak ≥3 to ≤6; n=14) and age- and sex-matched controls (n=11), average age ~81 years in both groups. Tissue was harvested within 24 h post-mortem, hemisected; one hemisphere snap-frozen for molecular analyses, the other fixed in paraformaldehyde. SNpc and CgCx were dissected. qPCR quantified RORA mRNA using specific primers/probe and the comparative Ct method normalized to GAPDH and β-actin. Western blotting quantified RORA protein normalized to β-actin. Statistics: Mann–Whitney U tests for between-group comparisons. Cell culture model: Rat mesencephalic dopaminergic N27 neuronal cell line was used as a PD model. Oxidative dopaminergic injury was induced by 6-hydroxydopamine (6-OHDA). The RORα/γ agonist SR1078 and antagonist SR1001 were applied to test neuroprotective or deleterious effects, respectively. Experimental paradigm typically involved: 24 h pre-incubation, followed by 24 h with SR1078 or SR1001 (various concentrations; commonly 3 µM SR1078), then 24 h exposure to 10 µM 6-OHDA (dose selected from prior dose-response tests). Assays: (1) Viability (MTS) and cytotoxicity (LDH release); (2) Apoptosis (caspase 3/7 activity); (3) Mitochondrial ROS (MitoSOX Red by flow cytometry), with Antimycin A as positive control; (4) Annexin-V/PI flow cytometry to categorize live, early apoptotic, late apoptotic, and necrotic cells; (5) Western blot of proteins implicated in oxidative stress and apoptosis (aromatase, 17β-HSD10, NOX1/2/4, active MMP-3, PKCδ native and cleaved), normalized to β-actin. Statistics: one-way or two-way ANOVA with appropriate post hoc tests (Tukey) or non-parametric equivalents (Kruskal–Wallis; Mann–Whitney) as indicated; significance at p<0.05. All culture experiments run as three independent replicates, typically in duplicate wells.

Human post-mortem SNpc and CgCx: - RORA mRNA and protein exhibit sex-specific expression in the SNpc of controls: females show higher levels than males. Reported control SNpc RORA mRNA (geometric ratio) mean ± SEM: females 0.432 ± 0.068 vs males 0.210 ± 0.022; protein levels were approximately three-fold higher in female vs male controls (p<0.01). - In PD SNpc, RORA expression changes differentially by sex: males show increased RORA relative to male controls (approximately two-fold elevation; p<0.01), whereas females showed little change vs female controls; absolute levels in females remained higher than in males (male PD: 0.272 ± 0.291; female PD: 1.641 ± 1.697 for protein levels). - CgCx/CgC: No significant sex differences or PD effects on RORA mRNA or protein. N27 dopaminergic cell model: - 6-OHDA (2.5–10 µM) reduced viability (MTS; p<0.001) and increased cytotoxicity (LDH; p<0.001) in a dose-dependent manner; 10 µM was used for subsequent challenges. - SR1078 (ROR agonist) alone had no effect on basal measures but significantly attenuated 10 µM 6-OHDA toxicity when used as a 24 h pre-treatment (commonly 3 µM): improved MTS viability (p<0.01), reduced LDH release (p<0.001), and blocked caspase 3/7 activation (p<0.001). Microscopy confirmed morphological preservation with SR1078 pre-treatment. - SR1001 (ROR antagonist) exacerbated 6-OHDA-induced cell death (MTS), and endogenous RORA protein in N27 cells was increased by 6-OHDA exposure, supporting an intrinsic protective response. - Mitochondrial ROS: 6-OHDA increased MitoSOX signal up to ~5-fold at 10 µM; SR1078 pre-treatment (3 µM) significantly attenuated this increase (p<0.001). - Apoptosis by flow cytometry: 10 µM 6-OHDA increased late apoptotic cells and reduced live cells; SR1078 pre-treatment increased live cell proportion (p<0.05) and reduced late apoptosis (p≤0.01). - Mechanisms from protein analyses: 6-OHDA increased NOX2 and NOX4 proteins; SR1078 pre-treatment significantly reduced NOX2 and reduced NOX1 elevations induced by higher 6-OHDA doses (p<0.001), with a non-significant trend to reduce NOX4. SR1078 also reduced active MMP-3 levels compared to 6-OHDA alone and blocked 6-OHDA-induced PKCδ cleavage (p≤0.01). No consistent significant changes were observed for aromatase or 17β-HSD10 under SR1078 treatment. Collectively, RORA is sexually dimorphic in the human SNpc and ROR activation protects dopaminergic cells through attenuation of oxidative stress (NOX pathway, mitochondrial ROS), reduced activation of apoptotic mediators (caspase 3/7, PKCδ cleavage), and decreased actMMP-3.

Findings demonstrate that RORA expression in the human SNpc is sex-dependent, being higher in females, and is differentially altered in PD, with an adaptive increase in males but little change in females. This pattern suggests RORA may contribute to sex differences in vulnerability and disease progression in PD, potentially acting downstream of sex steroid signaling. The in vitro dopaminergic model establishes a direct neuroprotective role for ROR activation against oxidative toxin-induced degeneration, consistent with RORA’s known anti-oxidative functions. Mechanistically, ROR agonism limits mitochondrial ROS production, attenuates NOX-driven oxidative stress, reduces actMMP-3, and prevents PKCδ cleavage, leading to reduced apoptosis. These data support RORA as both a biological contributor to sex differences in PD and a promising therapeutic target, with translational potential for ROR agonists to enhance dopaminergic neuron resilience in PD.

This study links RORA to PD for the first time in human brain tissue and identifies a sex-specific expression pattern in the SNpc that is differentially altered in PD. In a dopaminergic cell model, ROR agonism confers robust neuroprotection against oxidative stress and apoptosis via modulation of NOX enzymes, mitochondrial ROS, actMMP-3, and PKCδ cleavage. These results suggest that targeting ROR pathways could offer disease-modifying strategies in PD and may help address sex-related disparities. Future work should validate these findings in in vivo PD models, delineate RORA’s regulation by sex hormones within the nigrostriatal system, and evaluate the efficacy and safety of ROR agonists in preclinical and clinical studies, considering sex as a biological variable.