Manganese chloride (MnCl₂, Cat. # 244589) was obtained from MilliporeSigma (St. Louis, MO). Pellets of tamoxifen citrate (TX, Cat. # E-351) and its placebo (Cat. # C-111) were obtained from Innovative Research of America (Sarasota, FL). Antibodies for TH (sc-25269), Bax (sc-7480), CAT (sc-271803), OPA1 (sc-393296), CREB-binding protein (CBP)/p300 (sc-32244), ER-α (sc-542), ER-β (sc-6822), interleukin (IL)-1β (sc-52012), tumor necrosis factor (TNF)-α (sc-52746), glutamate transporter-1 (GLT-1, sc-365634), and β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Dallas, TX). Antibodies for acetylated H3K27 (H3K27ac, 4353T), acetylated H3K9/14 (H3K9/14ac, 9677S), and tri-methylated H3K36 (H3K36me3, 4909T) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies for REST (07-579) used for immunoblotting, and ChIP-grade REST Ab (17-641) for the ChIP assay were obtained from MilliporeSigma. Antibodies for histone H3 (ab1791), glutamate/aspartate transporter 1 (GLAST, ab416), rabbit anti-mouse (ab6728), goat anti-rabbit (ab97051) conjugated with horseradish peroxidase, and goat anti-mouse conjugated with Alexa Fluor 568 (ab175473) were obtained from Abcam (Waltham, MA). The Fluoro-Jade^® C (FJC) ready-to-dilute staining kit (TR-100-FJ) was purchased from Biosensis (Thebarton, Australia).

All experimental procedures were approved by the Florida A&M University Institutional Animal Care and Use Committee (Protocol #023-05, Tallahassee, FL). We used dopaminergic neuron-specific REST KO mice and REST loxP (floxed) mice generated in our previous study (Pajarillo et al., 2024). Mice were kept in groups of five per cage, maintaining a 12-h light/dark cycle at a constant temperature of 22 °C ± 2 °C with ad libitum access to food and water. Eight-week-old female dopaminergic REST cKO, totaling 48 mice, and WT/REST loxP mice, totaling 30 mice, were used in the experiments. WT mice were used as a positive control for dopaminergic REST cKO mice. Sample sizes (n = 12 for REST cKO groups; n = 10 for WT groups) were determined by power analyses for behavioral outcomes (α = 0.05, power = 0.8–0.9), and tissue samples from three mice per group were used for molecular assays.

Dopaminergic REST cKO mice (n = 12/group) were separated randomly into four groups as follows: (1) placebo plus vehicle, (2) placebo plus Mn, (3) TX plus vehicle, and (4) TX plus Mn. WT mice (n = 10/group) were arranged into three groups as follows: (5) placebo, (6) placebo plus Mn, and (7) TX plus Mn. TX pellets (25 mg/pellet, 21 days release) and their placebo pellets were used and implanted as previously reported (Pajarillo et al., 2018). All pellets were implanted subcutaneously at the back of the neck of the anesthetized mice through a 0.5 cm incision. The pellets were inserted with tweezers into a small pocket formed by bluntly dissecting caudolaterally. The incision and the closed suture (made with wound clips) were performed using aseptic techniques to minimize the risk of infection.

Twenty-four hours after the implantation, mice received Mn daily (330 μg as Mn; 30 mg/kg as MnCl₂; 1 μL per nostril in both sides) for 3 weeks (Pajarillo et al., 2020a). This dose increases brain Mn levels by up to 2-folds in C57BL/6 mice, closely mimicking the 3-fold increase observed in Mn-exposed nonhuman primates (Bowman and Aschner, 2014). Although REST regulates various genes (Sun et al., 2005; Bruce et al., 2009), there’s no evidence showing that REST modulates genes linked to Mn transport or accumulation. Moreover, even mutations of genes that affect and are risk factors for dopaminergic neurodegeneration, such as BTBD9, did not change Mn concentrations (Chen et al., 2022), rationalizing to use the same paradigm for Mn exposure in REST cKO mice. Distilled water served as the vehicle. To prevent Mn loss from the nostrils, mice were sedated with isoflurane for 3 min before and after administration.

Behavioral tests were conducted 24 h after the last Mn exposure to evaluate the effects of Mn as previously described (Pajarillo et al., 2024). The open-field, rotarod, and NOR tests assessed locomotor activity, motor coordination, and cognition, respectively. The open-field test was conducted in a Plexiglas arena, and the movement of each mouse was tracked using Fusion SuperFlex software v6.25. Prior to testing, mice were acclimated to the arena for three consecutive days. The distance traveled, vertical activity, and stereotypy activity for each mouse were recorded for 30 min, and these parameters were compared between groups using statistical analysis.

The AccuRotor rotarod system was used to assess motor coordination (Pajarillo et al., 2024). Mice were trained for three consecutive days with three trials per session, and the latency to fall was recorded using Fusion Software v6.3. The measurements of motor coordination were taken for 650 s in mice that remained on the rod throughout the test, and the average duration for each group was used for comparisons.

For the NOR test (Pajarillo et al., 2022a), mice were acclimated to the open-field arena for three consecutive days before testing. During the trial, mice underwent a familiarization phase followed by the NOR test. In the familiarization phase, each mouse was exposed to two identical objects (familiar objects, FO) placed in the back corners of the arena for 10 min. After this, one FO was replaced with a novel object (NO), differing in texture, shape, and color. Mice were then placed in the center of the arena with one FO and one NO for 10 min, and the time spent exploring both objects was recorded using Fusion software. Mice remained in the chamber until they accumulated at least 30 s of object exploration (Clark et al., 2000). NO difference scores and discrimination ratios were calculated to compare groups. A positive NO difference score and discrimination ratio above 0.5 indicated normal memory and object retention, while negative scores and ratios below 0.5 suggested memory deficits (Bevins and Besheer, 2006).

After behavior tests, three animals per group were perfused transcardially with 4% (w/v) paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) at pH 7.4 under anesthesia with ketamine/xylazine. Brains were removed and prepared for cryosectioning and immunohistochemistry. For cryosectioning, brains were post-fixed in a fixative overnight and transferred into a 30% w/v sucrose solution for cryoprotection. Serial coronal sections of 25-µm thickness were made with an HM525 NX Cryostat (Thermo Fisher Scientific, Waltham, MA). Coronal sections of substantia nigra (SN), spanning from Bregma −2.90 to −3.65 mm, were prepared for immunohistochemistry.

All processes for brain dissection were completed within a 2-h window (4:00 p.m.–6:00 p.m.). The time from decapitation to storing brain samples in dry ice was under 4 min. The striatum and midbrain were selected for study due to their relevance as primary sites of Mn accumulation and their critical roles in regulating movement and memory (Jay, 2003; Mizumori and Jo, 2013; Puig et al., 2014; Bradbury et al., 1985). After extracting the brain, these regions were dissected as described in previous protocols (Meyerhoff et al., 2021), then quickly frozen in dry ice and stored at −80 °C until further processing.

TH expression was analyzed in coronal brain sections from placebo/TX plus vehicle/Mn-treated dopaminergic REST cKO mice. Frozen sections (25 µm) were washed 3 times with PBS for 5 min. After washing, sections were incubated with a blocking solution (PBS with 0.01% Triton X-100 and 2% normal goat serum) for 1 h at room temperature. The primary TH antibody was applied overnight at 4 °C at a 1:200 dilution. The sections were washed 3 times with PBS, followed by incubation with a goat anti-mouse IgG Alexa Fluor 568 secondary antibody in a blocking solution for 1 h at room temperature. After washing, the sections were processed for FJC staining of degenerated neurons according to the manufacturer’s instructions. The sections were then washed with PBS, followed by air-drying and mounting onto glass slides with mounting medium. High-resolution imaging was performed using a Nikon Eclipse Ts2R-FL microscope with a DS-Qi2 high-definition camera.

Midbrain tissues (3 samples/group) were used to assess the mRNA levels of TH, REST, CAT, SOD1, SOD2, heme oxygenases 1 and 2 (HO-1 and HO-2), OPA1, peroxiredoxins 1 and 5 (PRDX1 and PRDX5), NADH:ubiquinone oxidoreductase subunit A2 (NDUFA2), IL-1β, TNF-α, GLAST, and GLT-1. Total RNA was extracted from the midbrain tissues using RNeasy^® Mini Kit (Qiagen, Valencia, CA), and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For real-time qPCR, the following primers for mouse were used: TH, 5′-CAC TAT GCC CAC CCC CAG-3′ (forward) and 5′-CGC CGT CCA ATG AAC CTT-3′ (reverse); REST, 5′-ACT TTG TCC TTA CTC AAG CTC-3′ (forward) and 5′-CAT TTA AAT GGC TTC TCA CCT G-3′ (reverse); IL-1β, 5′-GAG GAC ATG AGC ACC TTC TTT-3′ (forward) and 5′-GCC TGT AGT GCA GTT GTC TAA-3′ (reverse); TNF-α, 5′-GAT GAG AAG TTC CCA AAT GGC-3′ (forward) and 5′-ACT TGG TGG TTT GCT ACG ACG-3′ (reverse); GLT-1, 5′-CTC ACT GAC TGT GTT TGG TG-3′ (forward) and 5′-GAG GTG CCA CCA GAA CTT TC-3′ (reverse); GLAST, 5′-GAT CGG AAA CAT GAA GGA GC-3′ (forward) and 5′-CAA GAA GAG GAT GCC CAG AG-3′ (reverse); CAT, 5′-ACC AGA TAC TCC AAG GCA AAG G-3′ (forward) and 5′-CCA GTG ACT GTG GAG AAT CGA A-3′ (reverse); SOD1, 5′-GGG GAC AAT ACA CAA GGC TGT A-3′ (forward) and 5′-GTC TCC AAC ATG CCT CTC TCT TCA-3′ (reverse); SOD2, 5′-CAG ACC TGC CTT ACG ACT ATG G-3′ (forward) and 5′-CTC GGT GGC GTT GAG ATT GTT-3′ (reverse); HO-1, 5′-AAG CGA GAA TGC TGA GTT CA-3′ (forward) and 5′-GCC GTG TAG ATA TGG TAC AAG GA-3′ (reverse); HO-2, 5′-TCG GAG GGG GTA GAT GAG TC-3′ (forward) and 5′-GCT TCC TTG GTC CCT TCC TT-3′ (reverse); OPA1, 5′-TGG AAA ATG GTT CGA GAG AGT CAG-3′ (forward) and 5′-CAT TCC GTC TCT AGG TTA AAG CG-3′ (reverse); PRDX1, 5′-AAT GCA AAA ATT GGG TAT CCT GC-3′ (forward) and 5′-CGT GGG ACA CAC AAA AGT AAA GT-3′ (reverse); PRDX5, 5′-TTC TGT GCT CCG TGC ATC G-3′ (forward) and 5′-TCC TGG TCC CCA GTT TCT GAT-3′ (reverse); NDUFA2, 5′-TTG CGT GAG ATT CGC GTT CA-3′ (forward) and 5′-ATT CGC GGA TCA GAA TGG GC-3′ (reverse); GAPDH, 5′-CTC ATG ACC ACA GTC CAT GC-3′ (forward) and 5′-CAC ATT GGG GGT AGG AAC AC-3′ (reverse). The total reaction volume (25 µL) contained 1 µL of cDNA template of each sample, 0.4 µM of primers, and iQ™ SYBR^® Green Supermix from Bio-Rad Laboratories, Inc. (Hercules, CA). The qPCR parameters were set for one cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min in the Bio-Rad CFX96 real-time PCR system. GAPDH was used to normalize all samples. Data analysis was performed using the Bio-Rad CFX Manager version 3.1.

The harvested tissues from the striatum and midbrain were homogenized and used for protein extraction and further analysis. A 3:1 ratio of RIPA buffer with protease inhibitors to each tissue was used, and the protein concentration was determined by bicinchoninic acid assay. Ten µg of total protein per lane was run on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane for immunoblotting with relevant antibodies. All blots were developed using the SuperSignal™ West Pico PLUS chemiluminescent substrate kit, followed by blot imaging and quantification with the Molecular Imager ChemiDoc™ XRS+ System (Bio-Rad).

Following the manufacturer’s protocol, the ChIP assay was conducted using the EZ-ChIP kit (MilliporeSigma). Briefly, midbrain tissues were crosslinked with formaldehyde for 10 min at room temperature and then washed with ice-cold PBS. Tissues were lysed in SDS lysis buffer with protease inhibitors, sonicated, and centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant was diluted and incubated with protein G agarose beads for 1 h at 4 °C. After incubation, 1% of the supernatant was saved as input, and the remainder was incubated overnight with H3K27ac, REST, and rabbit IgG (negative control) antibodies at 4 °C. Protein G agarose beads were added and incubated for 1 h, followed by washing with low and high salt buffers. Following reverse crosslinking, immunoprecipitated DNA was purified and analyzed by qPCR using promoter-specific primers for mouse: TH, 5′-CCC ATA TGC CCT GGT TTG AT-3′ (forward) and 5′-AGG CCT CCG TCC CAT TAG AT-3′ (reverse); REST, 5′-CCA ACT TTT CCC CGC TCT-3′ (forward) and 5′-GCG TCC TAC CCT CTG AAC TG-3′ (reverse); CAT, 5′-AAA TAA GCT GCA AAG CCA CCA A-3′ (forward) and 5′-CAT AGC TCC TTT GAG ACC AGA C-3′ (reverse); SOD2, 5′-GAT GAA CAC ACG CAA ACC TG-3′ (forward) and 5′-CTG GGA AAC CCT GGA GAC TT-3′ (reverse); OPA1, 5′-CTT GTT GCT GAG GGC CTC TT-3′ (forward) and 5′-CCA GAG CAG ACC ACA CAC AG-3′ (reverse). DNA was quantified using Bio-Rad CFX Manager 3.1 software, and results were expressed as enrichment of H3K27ac or REST binding at the selected gene promoters.

All data were expressed as the mean ± standard deviation (SD). Multiple comparison analyses were performed by a two-way analysis of variance (ANOVA) followed by Fisher LSD post hoc tests using the GraphPad Prism Software version 10.0 (San Diego, CA). A p-value of <0.05 was considered statistically significant.

Since TX exerted neuroprotection against Mn toxicity in ovariectomized WT female mice (Pajarillo et al., 2018) and dopaminergic REST is critical for neuroprotection against Mn-induced toxicity in mice (Pajarillo et al., 2024), first, we tested whether dopaminergic REST is critical for the neuroprotective effects of TX against Mn toxicity.

REST cKO and WT female mice were used for molecular biology experiments. Mice were not ovariectomized in the present study, as they undergo estrous cycles every 4 to 5 days during the 21-day experimental period. This Mn exposure paradigm is different from our previous studies in which a single dose of Mn was given to the ovariectomized mice (Pajarillo et al., 2018). REST cKO mice were divided into four groups (two vehicle groups and two Mn-exposed groups), with one group in each receiving a TX pellet implant 1 day prior to Mn exposure. Mn was administered daily via intranasal instillation (330 μg as Mn, 30 mg/kg as MnCl₂) for 3 weeks, while control mice received distilled water.

To assess TX effects on behavioral changes in Mn-exposed mice, 24 h after the last Mn exposure, locomotor activities and motor coordination were assessed by open-field and rotarod tests, respectively, in WT and REST cKO mice. Results revealed that Mn decreased locomotor activities in the open field arena, while TX attenuated several parameters of Mn-induced motor deficits in WT and REST cKO mice (Figure 1). Mn decreased total distance traveled, vertical activity, and stereotypy activity (Figure 1A). Importantly, TX did not attenuate the Mn-induced decrease in total distance traveled in REST cKO mice, unlike its protective effects in WT mice (Figure 1A). However, TX attenuated Mn-decreased vertical activity and Mn-increased stereotypy activity in REST cKO mice, as well as in WT mice (Figures 1A,C). In addition, Mn decreased the latency to fall, corroborating impairment of motor coordination, while TX mitigated this Mn-induced effect in both WT and REST cKO mice (Figures 1B,C).

The NOR test was performed to assess the effects of TX on Mn-induced cognitive impairment in REST cKO mice. Given that TX attenuated Mn-induced memory and learning deficits in rats (Owoyemi et al., 2024), we tested if dopaminergic REST plays a role in TX’s attenuation of Mn-induced cognitive impairment in mice. Results revealed that Mn significantly decreased the recognition and discrimination indices, corroborating impaired cognitive function, while TX treatment mitigated these Mn-induced cognitive deficits in WT and REST cKO mice (Figures 2A–D), indicating that TX attenuated Mn-induced impairment in cognition in the absence of dopaminergic REST.

Mn caused neuronal damage as evidenced by increased FJC staining and decreased TH protein levels in the SN where dopaminergic cell bodies are located in the nigrostriatal pathway of REST cKO mice (Figure 3A). This Mn-induced decrease in TH immunofluorescence in the SN paralleled the decrease in TH and REST mRNA and protein levels in the midbrain and striatum of REST cKO mice (Figures 3B–D). TX alone increased REST protein levels in the striatum of REST cKO (Figure 3D), indicating that TX exerts an agonistic effect to increase REST, possibly in non-dopaminergic and other neural cell types.

To further determine if TX induces neuroprotection against Mn toxicity in the dopaminergic REST-deleted mice, we examined TX’s modulatory effects on Mn-induced apoptosis by measuring the protein levels of proapoptotic Bax in both WT and REST cKO mice. The results revealed that Mn increased Bax levels in both midbrain and striatum in WT as well as REST cKO mice, with a further increase in cKO, while TX significantly attenuated this Mn-induced effect in both WT and REST cKO (Figures 4A,B).

Since Mn caused oxidative stress in dopaminergic neuronal cultures and the nigrostriatal regions of mice, and TX, as well as REST, attenuated these Mn-induced effects (Pajarillo et al., 2018; Pajarillo et al., 2024; Pajarillo et al., 2020b), we investigated whether dopaminergic REST plays a role in TX’s protective effects against Mn-induced oxidative stress. Our analyses focused on the substantia nigra pars compacta in the midbrain region, where dopaminergic cell bodies are localized and dopaminergic REST was selectively deleted. This REST deletion will likely exert greater transcriptional and epigenetic effects than the striatum, which may be less affected. Several antioxidant genes, including CAT, SOD1/2, HO-1/2, and PRDX1/5, were assessed. In addition, since Mn causes mitochondrial damage (Liccione and Maines, 1988), we assessed the mitochondrial protein NDUFA2, a subunit of NADH dehydrogenase located in the mitochondrial inner membrane, and mitochondrial fusion protein OPA1. The results demonstrated that Mn exposure led to reduced mRNA expression of antioxidant genes (CAT, SOD2, HO-1/2, and PRDX1/5) and mitochondrial genes (OPA1 and NDUFA2) in the midbrain of both WT and REST cKO mice, with more pronounced decreases observed in REST cKO mice for most of the genes examined (Figure 5A). Importantly, TX’s attenuation of Mn-dysregulated antioxidant and mitochondria-related genes was differentially regulated in WT and REST cKO mice. TX attenuated Mn-induced dysregulation of all genes tested above in WT mice (Figures 5A–C). In REST cKO mice, on the other hand, TX significantly restored the mRNA levels of antioxidant PRDX1, PRDX5, and mitochondrial NDUFA2, but failed to attenuate the Mn-induced decreases in antioxidants CAT, SOD1/2, HO-1/2, and mitochondrial OPA1 (Figure 5A). TX was also unable to attenuate Mn-decreased protein levels of CAT and OPA1 in the midbrain of REST cKO mice, similar to its effects on mRNA levels of those (Figure 5B).

Since inflammation plays a critical role in neurotoxicity, we tested if TX attenuates Mn-induced inflammation and the role of dopaminergic REST in these TX effects. The expression levels of proinflammatory cytokines IL-1β and TNF-α in the nigrostriatal regions of WT and REST cKO mice were assessed. These brain regions contain other types of neural cells, not just dopaminergic neurons, as well as non-dopaminergic neurons, and glial cells. Thus, inflammatory effects could be derived from all neural cell types. Results showed that Mn increased mRNA and protein levels of IL-1β and TNF-α in both the midbrain and striatum of WT and REST cKO mice, with further increases in REST cKO mice (Figures 6A–C). However, TX attenuated these Mn-induced effects regardless of genotype, indicating that dopaminergic REST did not play a critical role in TX’s anti-inflammatory effects against Mn.

Mn-impaired astrocytic glutamate transporters, GLAST and GLT-1, which TX attenuated in rat astrocyte cultures (Lee et al., 2009) as well as ovariectomized mice (Pajarillo et al., 2018). Thus, we further tested if dopaminergic REST plays a role in TX’s attenuation against Mn-induced dysregulation of GLT-1 and GLAST in the nigrostriatal region of WT and REST cKO mice, possibly via indirect mechanisms such as elevated glutamate levels (Kim et al., 2025) and oxidative stress. Results showed that TX was able to attenuate the Mn-induced decrease in mRNA and protein levels of the GLAST and GLT-1 similarly in both WT and REST cKO mice (Figures 7A–C), indicating that dopaminergic REST may not contribute to TX’s protective effects against Mn-induced reduction in GLT-1 and GLAST.

Since TX attenuated Mn-dysregulated expression of several genes involved in neuroprotection, including TH, REST, and antioxidant genes at the transcriptional levels, we further investigated whether epigenetic alteration via histone modification contributed to those transcriptional changes induced by Mn and/or TX and potentially by dopaminergic REST deletion. Midbrain tissues were used since transcriptional regulations mostly occur in this region, where the cell bodies of dopaminergic neurons are localized. We assessed acetylation of histone marks, such as histones H3 lysine 27 (H3K27ac), lysine 9 and 14 (H3K9/14ac), and tri-methylation of histone H3 lysine 36 (H3K36me3) in the midbrain of WT and REST cKO mice. Results showed that the basal levels of H3K27ac are lower in REST cKO mice compared to WT mice, but Mn decreased H3K27ac in the midbrain of both WT and REST cKO mice (Figures 8A,B), which TX significantly attenuated in both genotypes. On the other hand, the basal levels of H3K36me3 were higher in REST cKO compared to WT, and Mn markedly decreased H3K36me3 levels, which were significantly attenuated by TX in WT, but not in REST cKO mice. Mn increased H3K9/14ac in the midbrain of both WT and REST cKO mice, which TX attenuated (Figures 8A,B).

We further tested if the histone-acetylating enzyme CREB-binding protein (CBP)/p300 plays a role in Mn and TX’s modulation of histone acetylation by measuring the protein levels of CBP/p300 in the midbrain of WT and REST cKO mice. Results showed that Mn markedly decreased CBP/p300 protein levels, which TX attenuated in both WT and REST cKO mice (Figures 8A,C).

TX’s attenuation of Mn-decreased H3K27ac, which is known to upregulate multiple genes, including antioxidant genes (Yang et al., 2022; Ao et al., 2024), could mitigate Mn-induced transcriptional repression of the antioxidant genes tested. Accordingly, we determine if H3K27ac contributes to Mn/TX-regulation of TH, REST, and genes involved in oxidative stress and mitochondrial dysfunction. We assessed the interaction of H3K27ac with the promoters of TH, REST, CAT, SOD2, and OPA1 by ChIP assays in the midbrain of WT and REST cKO mice. Results showed that TX attenuated the Mn-decreased interaction of H3K27ac with the TH as well as REST promoters in both WT and REST cKO mice (Figure 9A). TX attenuated Mn-induced decrease in interaction of H3K27ac with the REST promoter in the midbrain of REST cKO mice (Figure 9A), possibly due to the presence of the REST promoter in other neural cell types and/or presence of dopaminergic REST gene sequences may still be present in the REST cKO, which has only REST’s exon 4 deletion (Gao et al., 2011).

Importantly, TX attenuated Mn-reduced interaction of H3K27ac with the promoters of CAT, SOD2, and OPA1 in WT, not REST cKO mice (Figure 9B), indicating that dopaminergic REST plays a critical role in the TX/Mn-induced modulation of H3K27ac’s interaction with these antioxidant genes. Since REST protein is deleted in dopaminergic neurons in REST cKO mice, REST binding to the TH promoter was almost completely absent (Figure 9C).

Our previous studies have shown that Mn decreased ER-α protein levels, which TX attenuated in WT female mice (Pajarillo et al., 2018). In the present study, we determined if dopaminergic REST played a role in these TX or Mn effects on the ERs. Thus, ER-α and ER-β protein levels were analyzed in the nigrostriatal region of both WT and REST cKO mice. Mn significantly decreased ER-α and ER-β protein levels in these regions of both WT and REST cKO mice, while TX attenuated these Mn-induced reductions, indicating that dopaminergic REST was not involved in TX’s attenuation of Mn-reduced ER-α and ER-β protein levels (Figures 10A,B).