NR expression profiling in ER stress–induced differentiated N2a cells

Owing to the relevance of NRs in the modulation of ER stress and its subsequent bearing on various disease conditions including neurodegenerative diseases, it is plausible to identify all those NRs having altered expression during ER stress in neuronal cells. To address this, differentiated neuro-2a (N2a) cells were treated with three classic ER stress inducers: thapsigargin (TG, inhibitor of sarcoplasmic/ER Ca2+ ATPase), tunicamycin (TM, inhibitor of N-linked glycosylation), and brefeldin A (BFA, perturbs ER–Golgi protein trafficking) for 24 h. Standard markers for ER stress were analyzed by Western blotting to examine the effect of ER stress inducers on mouse neuroblastoma cells. There was a significant increase in BiP and CHOP at 2.5 μg/ml TM, 0.5 μg/ml TG, and 2.5 μg/ml BFA (Fig S1). Accordingly, these concentrations were selected to induce ER stress and further identification of NR expression using a PCR array. We found that of 49 murine NRs, 37 found expression in differentiated N2a cells (Fig 1A). A complete analysis of all NRs showed that the expression levels of some of the NRs were altered upon induction of ER stress, which is depicted in the heatmap in Fig 1B. 13 NRs were having more than twofold change in their expression pattern as compared to untreated controls, and they are being uniquely or commonly modulated in TM-, TG-, and BFA-induced ER stress conditions (Fig 1C). Thrb, Nr1h4, and Pparg were commonly modulated in all three stress conditions. The expression of endocrine receptor thyroid hormone receptor-α (Thra) and estrogen receptor-α (Esr1) was down-regulated in the presence of TM and TG. Also, up-regulation of estrogen-related receptor-γ (Esrrg) and repression of Ppard were observed in the presence of TG and BFA. There were some receptors that were having opposite expression patterns in different stress conditions. Nr1h2/LXRβ was repressed in TM-treated cells, whereas its expression was increased in BFA-treated cells; on the contrary, RAR-related orphan receptor-α (Rora) was down-regulated in the presence of TM with elevated expression in TG-induced stress conditions. Few NRs were uniquely regulated in either of the stress conditions. Among these, Nr4a1 was only modulated in cells treated with TM, whereas Vdr, Nr3c1/GR (glucocorticoid receptor), and retinoid X receptor-α (Rxra) were modulated in BFA-treated cells (Fig 1D). We then looked for the protein expression of those NRs, which were commonly modulated in all three stress conditions, and in consistent with their mRNA expression, the protein levels of all three NRs were up-regulated (Fig 1E). Collectively, our results ascertained the expression pattern of all the NRs in mouse neuronal cells. Although some of these receptors have already been shown to modulate ER stress including Pparg, Ppard, Nr1h2, Vdr, few NRs were not previously known to have any implications in ER stress–related pathological conditions (Chiang et al, 2015; Toral et al, 2016; Gavini et al, 2018; Zhou et al, 2020). This calls for further detailed studies to decipher their unidentified functions in modulating ER stress biology in neurons.

Nr1h4 and Thrb activation ameliorates TM-induced ER stress in differentiated N2a cells

Nr1h4 is a bile acid–sensing NR that has a predominant role in the liver where it regulates triglyceride levels, cholesterol, bile acids, and glucose metabolism (Panzitt & Wagner, 2021). However, recent studies have shown its additional functional roles in different tissues and several disease conditions. There are few reports where activation of Nr1h4 has been shown to alleviate ER stress and hence provide protection in various pathological conditions such as diabetic tubulopathy and non-alcoholic fatty liver disease, and in liver injury caused by ER stress (Gai et al, 2017). Thyroid receptors belong to a class of endocrine receptors having cardinal roles in metabolism, and the development and dysregulation of these receptors has ramifications on human health (Torres-Manzo et al, 2018). Despite the availability of a large body of literature stating the importance of these two receptors in other tissues, little is known about their relevance in the brain. The common modulation, higher fold change, and unrecognized role of Nr1h4 and Thrb in the modulation of ER stress in neuronal cells instigated us to investigate their functional relevance. As the effect of ER stress inducers on the mRNA and protein expression of both the receptors was similar, we chose to use TM for induction of ER stress in all further experiments.

To characterize their role in the modulation of ER stress, gain-of-function and loss-of-function experiments were done. Control or Nr1h4 knockdown N2a cells were treated with TM, either with or without GW4064, a pharmacological activator of Nr1h4. Similarly, we treated control and Thrb knockdown cells with TM in the presence or absence of T3 (ligand for Thrb). We then looked for the expression of several genes majorly involved in three arms of UPR pathways such as BiP, CHOP, spliced X-box binding protein 1 (sXBP1), activating transcription factor 4 (ATF4), and ATF6α. TM treatment increased the expression of all five ER stress response genes, whereas treatment with either GW4064 or T3 abrogated the TM-induced gene expression of BiP, CHOP, and ATF4 significantly (Fig 2A and B). GW4064 treatment did not affect the expression of sXBP1, whereas it abrogated TM-induced ATF6α expression (Fig 2A). On the contrary, treatment with T3 led to the reduced expression of sXBP1, whereas there was little to no effect on the expression of ATF6α (Fig 2B). The absence of either Nr1h4 or Thrb led to the induction of ER stress upon TM treatment, whereas treatment with their cognate ligands (GW4064 or T3) did not rescue the TM-induced stress (Fig 2A and B). Furthermore, to ascertain the individual effect of receptor–ligand pair, we treated Nr1h4 knockdown cells with T3 and Thrb knockdown cells with GW4064 in the presence of TM. There was down-regulation of BiP, CHOP, and ATF4 in either case. However, ATF6α was only modulated by GW4064, and sXBP1 only by T3 (Fig S2A). To scrutinize the effect of the combination of ligands on the ER stress genes, cells were treated with TM in the presence or absence of GW4064 and T3. We observed that the expression of BiP, CHOP, sXBP1, ATF4, and ATF6α was significantly down-regulated in combination treatment as compared to either ligand alone. However, the absence of both receptors failed to repress UPR genes (Fig 2C). We also looked for the expression of all these genes in the presence of individual ligands and in cells treated with the combination of ligands where no significant difference in gene expression was observed (Fig S2B).

To further verify the above results, the effect of activation of these receptors on UPR pathway proteins was investigated. We looked for the protein levels of p-IRE1α, IRE-1α, sXBP1, ATF6α, p-PERK, PERK, phosphor-eukaryotic initiation factor 2 alpha (p-eIF2α), eIF2α, ATF4, BiP, and CHOP in control, Nr1h4 knockdown, Thrb knockdown, and Nr1h4 and Thrb double-knockdown N2a cells. Consistent with mRNA data, similar results were obtained at protein levels (Fig 2D–F). GW4064 was able to ameliorate TM-induced ER stress by suppressing the p-eIF2α/ATF4/CHOP and ATF6α axis of UPR pathways (Fig 2D), whereas T3 is exerting its protective effect by impeding the p-eIF2α/ATF4/CHOP and IRE-1α/XBP1/sXBP1 axis (Fig 2E) in differentiated N2a cells. As a result, the GW4064 and T3 combination was more efficient in mitigating TM-induced ER stress as compared to treatment with either ligand alone (Fig 2F).

Treatment with Nr1h4 and Thrb agonists prevented differentiated N2a from ER stress–mediated cell death

Elevated ER stress leads to cell death (Sano & Reed, 2013). We next sought to look for the effects of the combination of ligands on cell survival and death employing both 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay. Treatment with TM leads to reduced cell viability (Fig 3A) and more LDH release (Fig 3B), which were rescued upon the addition of GW4064 or T3. Furthermore, an increase in cell viability (up to 85%) and a decrease in LDH release were noted when the cells were incubated with both ligands, indicating that TM-induced cytotoxicity could be remarkably attenuated by the combination of ligands (Fig 3A and B). To further discern their effects on ER stress–induced apoptosis, cells were treated with TM alone or in the presence of GW4064 or T3 or both for 24 h. Apoptosis was determined by an annexin-V/propidium iodide (PI) assay. Results revealed that the presence of TM significantly elevated the number of apoptotic cells, which was reduced upon individual ligand treatment as detected by flow cytometry analysis, whereas the presence of both the ligands further decreased the number of cells in apoptosis (Fig 3C). Furthermore, the expression of ER stress–related apoptotic proteins such as pro-apoptotic cleaved PARP and Bax and anti-apoptotic Bcl-2, Mcl-1, and pro-caspase-3 was detected by Western blot. We observed that pro-apoptotic protein levels were increased in the presence of TM, which were reduced in the presence of GW4064 or T3, whereas a significant reduction was observed in combination treatment (Fig 3D). Cleaved caspase-3 levels were detected by a fluorescent assay where similar results were obtained (Fig 3E). These findings substantiate the neuroprotective role of Nr1h4 and Thrb in ER stress–induced cell death.

Nr1h4 and Thrb activation is protective in an in vitro PD model

Emerging data suggest a plausible implication of ER stress in the pathogenesis of PD (Mou et al, 2020). MPP⁺, a dopaminergic neuron-specific neurotoxin that causes symptoms of PD, has been frequently used as a model for studying the pathways of cell death in PD (Hutter-Saunders et al, 2012). We first looked for the expression of Nr1h4 and Thrb upon MPP⁺ exposure where we found the increased expression of both these receptors at mRNA and protein levels (Fig 4A). We used a 500 μM MPP⁺ concentration as it is the lowest concentration that decreased the cell viability significantly as observed by us and reported by other groups (Mazzio & Soliman, 2012; Su et al, 2021; Mahmoudian Esfahani et al, 2023).

To elucidate the effect of MPP⁺ treatment on ER stress in differentiated N2a cells, we treated the cells with 500 μM MPP⁺ for 24 h along with either ligand alone or in combination. Ligand treatment attenuated the MPP⁺-induced increase in BiP and CHOP expression, whereas a significant reduction was observed in combination treatment. These results indicate that activation of Nr1h4 and Thrb is abrogating ER stress in an in vitro PD model (Fig 4B).

Mitochondrial dysfunction is one of the hallmarks of PD (Helley et al, 2017). To scrutinize the role of mitochondria in MPP⁺-induced neuronal cell death, cells were treated with MPP⁺ to monitor mitochondrial membrane potential (Δψm). MPP⁺ treatment for 24 h caused dissipation of Δψm, as evidenced by a decrease in the red-to-green fluorescence ratio of JC-1, as compared to untreated controls. However, administration of either GW4064 or T3 attenuated MPP⁺-induced Δψm loss as indicated by an increase in fluorescence intensity. Simultaneous administration of both ligands abolished the effect of MPP⁺ on Δψm suggesting their neuroprotective relevance (Fig 4C). Exaggerated generation of ROS has been proposed to be a crucial mechanism underlying the cytotoxicity of MPP⁺ (Dias et al, 2013). We measured total ROS production using the fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) by both flow cytometry (Fig 4D) and confocal imaging (Fig S3). ROS levels were markedly augmented in MPP⁺-exposed cells, whereas treatment with either GW4064 or T3 rescued these elevated levels, which were further decreased significantly in GW4064 and T3 combination treatment.

These results demonstrate that the combination of GW4064 and T3 protected mitochondrial function and suppressed MPP⁺-induced ROS production in differentiated N2a cells.

Co-administration of GW4064 and T3 protected mice from MPTP intoxication

To validate the in vitro findings, in an in vivo setting, we used the MPTP mouse model of PD. MPTP is extensively employed as a model for investigating the molecular and cellular mechanisms, as well as neuropathological events in PD (Mustapha & Mat Taib, 2021). Animals were divided into three groups: control group, MPTP group, and MPTP along with GW4064 and T3 group. A schematic representation of the MPTP mouse model of the PD experimental design is depicted in Fig 5A. We first measured the protein levels of BiP and CHOP (ER stress markers) in the striatum by immunoblotting, and we found that elevated levels were found in MPTP-treated mice as compared to control mice. BiP and CHOP were significantly reduced in the striatum of ligand-treated mice (Fig 5B). The rate-limiting enzyme tyrosine hydroxylase (TH) is known to produce dopamine in the brain of mice. We measured the amount of TH by Western blot (Fig 5B) and immunohistochemistry (IHC) (Fig 5C). A significant reduction in TH immunoreactivity was observed in the striatum of MPTP-treated animals as compared to the equivalent regions of control animals. Interestingly, there was an increase in TH expression and TH-positive neurons in the SNpc of animals that were subjected to GW4064 and T3 treatment. These data suggest that pretreatment of the combination of GW4064 and T3 in mice is protective not only in curtailing ER stress but also in safeguarding dopaminergic neurons from cell death.

Next, to determine the functional deficits caused by MPTP, we performed various behavioral tests such as open field test (OFT) for general locomotor activity, Morris water maze for cognitive decline, pole test, narrow beam walk, and rotarod for coordination, and an elevated plus maze for determining anxiety-related behavior. In OFT, the movement of mice in the open field was monitored using ANY-maze video tracking software. Various parameters such as the overall distance traveled, the number of entries, and the amount of time spent in the center zone during the 15-min test duration were recorded. MPTP intoxication caused a marked decrease in the number of entries and time spent in the center zone as compared to the control group indicating anxiety-like behavior. Also, MPTP-treated animals were less active and traveled smaller distances compared with animals that did not receive MPTP. Interestingly, GW4064 and T3 significantly improved MPTP-induced anxiety and hypolocomotion (Fig 6A).

Given that non-motor symptoms such as anxiety are frequently reported in PD patients, we performed an elevated plus-maze test (EPMT). MPTP treatment resulted in a considerable increase in the amount of time spent in the closed arms, as well as the frequency of entries into the closed arms, an effect that was not observed in control mice. In the ligand-treated group, the length of time and the number of entries into closed arms were reduced (Fig S4A). We next performed a pole test to monitor coordination. In the MPTP group, the overall time required by mice to reach the bottom of the pole and T-turn time were significantly prolonged to 18.1 and 4.40 s, respectively, compared with the control group, whereas in the ligand-treated group, they were shortened to 13.2 and 3.33 s, respectively (Fig 6B). Coordination was also monitored by a narrow beam walk test where mice injected with MPTP took longer time to traverse the beam with enhanced foot slip errors, which were reverted in the ligand-treated group (Fig S4B). We next performed a rotarod test where the foot movement of the mice was observed at different speeds. Compared with the poor performance of the MPTP group, the ligand-treated group had a longer time to fall off the rotating bar showing improved performance at all speeds (Fig 6C). To assess the neuromuscular strength, a wire hanging test was performed. While performing this test, animals were directed to grab the wire with their paws for 180 s or until they fell. It was observed that the ligand-treated group displayed better grip strength as compared to the MPTP group as evidenced by more latency to fall (Fig 6D).

Furthermore, to examine the effect of GW4064 and T3 on cognitive impairment caused by MPTP, we performed the Morris water maze test. The escape latency was recorded as an index of spatial learning. The time taken by MPTP-injected mice to get to the hidden platform was comparatively longer than the control group. However, the beneficial effect of GW4064 and T3 on learning was noticed during the 5-d experimental period (Fig 6E). In the probe trial experiment, impaired memory deficits were observed in the MPTP group, spending much less time in the target quadrant and shorter platform crossing duration compared with the control group. The ligand-treated group reached levels of performance equal to those of the controls suggesting their ability to memorize the platform position (Fig 6F).

GW4064 and T3 effectively reduced MPP⁺-induced ER stress and ROS production in differentiated human neuronal SH-SY5Y cells

We next monitored the effect of MPP⁺-induced ER stress in differentiated human neuronal SH-SY5Y cells. Treatment with GW4064 and T3 reduced ER stress, which was observed in MPP⁺-treated cells. Levels of BiP and CHOP were significantly reduced in combination ligand treatment signifying the relevance of activation of Nr1h4 and Thrb in human neuronal cells (Fig 7A). We next examined the TM-induced apoptosis by flow cytometry, and as expected, there was increased apoptosis in the differentiated SH-SY5Y cells after TM treatment, whereas ligand treatment prevented TM-induced cell death (Fig 7B). Next, to examine whether GW4064 and T3 suppressed MPP⁺-induced ROS production, we evaluated ROS generation in differentiated SH-SY5Y by both flow cytometry (Fig 7C) and confocal analysis (Fig S5). It was observed that ROS production in MPP⁺-treated cells was significantly reduced in both GW4064- and T3-treated cells. This reduction was more pronounced in combination with ligand treatment indicating the protective effects of activation of Nr1h4 and Thrb in human neuronal cells. Several studies have demonstrated that α-syn aggregate formation can elicit ER stress response. Elevated ER stress, in turn, promotes α-syn aggregation (Cóppola-Segovia et al, 2017). To investigate the impact of GW4064 and T3 on α-syn aggregation, cells were subjected to α-syn aggregates, active proteins existing in preformed fibrils. In addition, cells were treated with pre-incubated α-syn aggregates and active monomers. The α-syn aggregates serve as templates, or “seeds,” by recruiting active monomers into fibrils. Furthermore, treatment of TM, an ER stress inducer, leads to the increased punctation of phosphorylated α-syn aggregates. This pathology was decreased in cells treated with GW4064 and T3. These results suggested that the ligands are ameliorating TM-induced α-syn aggregate formation in differentiated SH-SY5Y cells (Fig 7D). To assess whether the ligands exert a direct effect on aggregation or act through ER stress pathways, we conducted a thioflavin-T (ThT) assay in a cell-free state. There was an increase in the fluorescence intensity when active α-syn aggregates were incubated with active monomers as compared to active α-syn aggregates alone. Interestingly, the fluorescence intensity of aggregates remained constant upon treatment with TM alone or in the presence of ligands (T3 and GW4064). This indicates that ligands did not have a direct effect on the aggregates (Fig S6). Ligands are able to reduce the aggregation in the cellular milieu by reducing the ER stress. These results substantiate the neuroprotective effects of GW4064 and T3 in human neuronal cells.

Animals & ethics statement

C57BL/6 male mice (6–8 wk old) were obtained from the Jackson Laboratory and were kept in a disease-free environment with adequate food and water in the institute’s animal house facility. Mouse experiments were approved by the Institutional Animal Ethics Committee and were conducted in accordance with the National Regulatory Guidelines stated by the Committee for the Purpose of Supervision of Experiments on Animals (No.55/1999/CPCSEA), Ministry of Environment and Forest, Government of India.

Cell culture and reagents

Mouse N2a cells and human SH-SY5Y (ATCC) were grown in DMEM and DMEM/F12 media, respectively, supplemented with 100 U/ml penicillin–streptomycin and 10% FBS, and maintained at 37°C and 5% CO₂. For differentiation of N2a into dopaminergic neurons, media were changed to DMEM+ 0.5% FBS with 1 mM dbcAMP for 24–48 h (Tremblay et al, 2010). To differentiate SH-SY5Y cells, 10 μM retinoic acid was added for 3 d, and after 3 d, media were changed and fresh media containing 16 nM phorbol 12-myristate 13-acetate were added for another 3 d. For every experiment in N2a and SH-SY5Y cells, 5 μM GW4064 and 10 nM T3 were used.

PCR array

RNA was isolated from cells using RNeasy Mini Kit (QIAGEN). cDNA was prepared from 1 μg of total RNA using RT² First Strand Kit (QIAGEN) followed by qRT–PCR using RT² SYBR Green ROX qRT-PCR Mastermix (QIAGEN). Customized PCR array plates (QIAGEN) were used to ascertain the expression profile of all the murine NRs and associated co-regulators as per the manufacturer’s instructions. Relative fold regulation was determined by the 2^−ΔΔCt method. The NRs with a Ct value less than 35 were only included in the analysis (Saini et al, 2018).

Measurement of mitochondrial membrane potential (Δψm)

A membrane-permeant JC-1 dye was used to measure Δψm in MPP⁺-treated differentiated N2a cells using flow cytometry analysis as per the manufacturer’s instructions. Briefly, cells at a density of 5 × 10⁵ cells per well were seeded in a 12-well plate and were treated with GW4064 or T3 or both for 3 h followed by MPP⁺ treatment for 24 h. The cells were then suspended in 1 ml PBS. For a positive control, carbonyl cyanide m-chlorophenyl hydrazone at 50 μM concentration was used. Cells were treated with 2 μM JC-1 and kept for 30 min at 37°C and 5% CO₂, cells were then washed with PBS twice, and the pellet was resuspended in 300 μl PBS. Samples were acquired on a flow cytometer where standard compensation was performed using a carbonyl cyanide m-chlorophenyl hydrazone–treated sample. The lower red (JC-1 aggregates)/green (JC-1 monomers) fluorescence intensity ratio is suggestive of mitochondrial depolarization (Sivandzade et al, 2019).

Cell viability assay

The cell viability was assessed using an MTT dye. Differentiated N2a cells at a density of 5 × 10³ cells/well were seeded and pretreated with either GW4064 or T3 or both for 3 h and then incubated with or without TM for 24 h. Cells were then stained with an MTT dye (5 mg/ml, 37°C, 4 h), and a solubilization solution was added to dissolve the formazan crystals. Absorbance at 570 nm was measured using a microplate reader. The cell cytotoxicity assay was performed using BioVision’s LDH Cytotoxicity Assay Kit II (#K313-500) as per the manufacturer’s protocol. Briefly, cells were seeded in a 96-well plate and the reaction was performed in triplicate. 100 μl culture medium with no cells was taken as the background control, 100 μl cells only were taken as low control, 100 μl cells with 10 μl cell lysis solution added were taken as high control, and 100 μl cells with TM alone or TM along with GW4064 or T3 or both were taken as test samples. 10 μl of each sample medium was transferred to a 96-well plate, and 100 μl LDH reaction mix was added to each well, mixed, and incubated for 30 min at room temperature. The absorbance of all the controls and samples was measured with a plate reader at 450 nm, with a reference wavelength of 650 nm. The % cytotoxicity was determined using the following formula:Cytotoxicity (%)=(Test sample−Low control)/(High control−Low control)×100

Measurement of ROS production

Briefly, 5 × 10⁵ cells/well were plated for 24 h in a 12-well plate with or without coverslips followed by treatment with GW4064 or T3 or both for 3 h, and later incubated with MPP⁺ for 24 h. Intracellular ROS was measured by labeling cells with 2.5 μM of H₂DCF-DA (D399; Molecular Probes) for 30 min at 37°C. Fluorescence intensity was monitored by flow cytometer using 488-nm lasers and by confocal microscopy.

Caspase-3 assay

Activity of caspase-3, a critical executioner of apoptosis, was detected by Caspase-3 Activity Assay Kit (#5723; CST) according to the manufacturer’s instructions. It is a fluorescent assay that contains a fluorogenic substrate for caspase-3 where activated caspase-3 cleaves this substrate generating high fluorescence. After treatment according to the experimental design, the cell lysate was prepared, and 40 μg/ml lysate was diluted in assay buffer. 200 μl of substrate solution B and 25 μl of lysate solution were mixed in a 96-well black plate followed by incubation at 37°C in dark for 2–4 h. Relative fluorescent units were determined with excitation at 380 nm and emission at 420–460 nm on a fluorescence plate reader.

Annexin-V and PI staining

Briefly, 5 × 10⁵ cells per well were plated onto 12-well plates and treatments were given as mentioned in the Results section. Cells were harvested after 24 h, washed with cold 1X PBS twice, and stained with annexin-V and PI using BD Biosciences Kit (#556547) according to the manufacturer’s instructions. Cells were resuspended in 100 μl 1X binding buffer with 5 μl annexin-V and PI followed by 15-min incubation in dark at RT. The cells were acquired on a BD Accuri C6 flow cytometer, and data were analyzed by FlowJo software (Bhagyaraj et al, 2019). Apoptotic cells comprising both late apoptotic (annexin-V–positive and PI-positive) and early apoptotic (annexin-V–positive and PI-negative) cells were quantified.

RNA isolation and quantitative real-time PCR (qRT–PCR)

Using TRIzol reagent, total RNA was extracted from the cells and cDNA was prepared from 1 μg of total RNA using a verso cDNA synthesis kit. The expression of various ER stress genes was determined by qRT–PCR using gene-specific primers and DyNAmo ColorFlash SYBR Green qPCR Kit. For normalization, β-actin was used as a housekeeping gene. The 2^−ΔΔCt method was used to calculate the fold change.

PD animal model

C57BL/6 male mice (8 wk old) were procured from the animal facility at CSIR-IMTECH, and all experiments received approval from the Institutional Animal Ethics Committee of IMTECH as stated earlier. Mice were categorized into three groups (15 mice per group): Group I, normal control, received saline; Group II, received MPTP (30 mg/kg, i. p.); and Group III, received MPTP treatment (30 mg/kg, i. p.) + GW4064 (30 mg/kg, i. p.) and T3 (250 μg/kg, i. p.). The MPTP-treated groups received five injections of MPTP-HCl (30 mg/kg, i. p.) in saline every day for a consecutive 5 d. Group III received ligand treatment 4 h before MPTP administration. 2 d after the last injection, behavioral assessments were performed, then all the animals were euthanized, and brains were isolated for further analysis (Meredith & Rademacher, 2011).

siRNA knockdown

For transient silencing of Nr1h4 and Thrb in differentiated N2a cells, a final dose of 60 nM of either a scrambled control siRNA or gene-specific siRNA was used to transfect cells at 70% confluency using TurboFect Transfection Reagent for 48 h according to the manufacturer’s instructions.

Western blot analysis

Immunoblot analysis was performed on differentiated N2a and SH-SY5Y cell extracts, and brain extracts of experimental mice. Protein concentrations in the samples were assessed using Bradford’s reagent. SDS–PAGE was used to separate equal amounts of proteins, which were then transferred to a polyvinylidene difluoride membrane (Immobilon-P, IPVH00010; Millipore) for 2 h. The membrane was blocked with blocking buffer (5% BSA in Tris-buffered saline/Tween-20) for 2 h at RT. Blots were then probed with primary antibodies overnight at 4°C. After washing (three times for 5 min each with Tris-buffered saline/Tween-20 buffer), the membrane was then probed with a secondary antibody conjugated with horseradish peroxidase and visualized by chemiluminescent HRP substrate Luminata Forte (WBLUF0500; Millipore). Primary antibodies used were as follows: BiP (C50B12) (#3177; CST), CHOP (L63F7) (#2895; CST), PERK (#5683 D11A8; CST), phospho-PERK (Thr980) (16F8) (#3179; CST), eIF2α (#9722; CST), phospho-eIF2α (Ser51) (#9721; CST), ATF-4 (D4B8) (#11815; CST), ATF6 (70B1413.1) (#NBP1-40256SS; Novus Biologicals), IRE1α (14C10) (#3294; CST), phospho-IRE1α (pSer724) (#NB100-2323SS; Novus Biologicals), XBP1 (#NBP1-77681SS; Novus Biologicals), tyrosine hydroxylase (E2L6M) (#58844; CST), Bax (D2E11) (#5023; CST), cleaved PARP1 (E51) (#ab32064; Abcam), Mcl-1 (D35A5) (#5453; CST), Bcl-2 (50E3; CST), farnesoid X receptor (D-3) (#sc-25309; SCBT), Thrb (#ab5622; Abcam), PPARγ (E−8) (#sc-7273; SCBT), β-actin (C4) (#sc-47778; SCBT).

IHC

Brains were removed carefully from three mice per group and were dehydrated with a graded series of ethanol after being fixed in 4% PFA for 24 h. They were then embedded in paraffin blocks, and 4-μm-thick sections were mounted on the slide followed by staining with anti-TH antibody.

Brain sections and tissue preparation

After performing behavioral tests, three mice per group were used for the preparation of brain sections for IHC. The brains were removed from the remaining mice and quickly frozen in liquid nitrogen. The two striata from each hemisphere were isolated and used for Western blot analysis.

Confocal imaging

Differentiated SH-SY5Y cells were treated with human α-syn protein aggregates (active) (ab218819, 4 μg/ml; Abcam) and human α-syn protein monomer (active) (ab218818; Abcam). Immunostainings were performed as described previously. Differentiated SH-SY5Y cells were fixed with 4% PFA and subsequently incubated with primary antibodies against phosphorylated S129 α-syn (α-syn-P; 1:1,000; ab184674) at 4°C for 24 h. After washing 3× with PBS, the cells were incubated with Alexa Fluor 647 anti-mouse secondary antibody (1/500) for 1 h at RT, counterstained with Hoechst (blue) nuclear stain (1/4,000), and mounted on slides with antifade reagent. Images were acquired by a Nikon AIR confocal microscope using a 60× objective.

ThT assay

A ThT assay was performed as described previously (Xue et al, 2017). Following the manufacturer’s protocol, a 25 μM ThT solution was prepared in PBS and incubated with human α-syn protein aggregates (active) (10 μM) and monomers (active) (100 μM) at a 1:10 ratio in a 96-well black plate. Subsequently, the mixture of active α-syn aggregates with monomers and ThT was incubated with TM in the presence or absence of T3 (10 nM) and GW4064 (5 μM). Fluorescence measurements were taken at 450-nm excitation and 485-nm emission using a plate reader set at 37°C with linear shaking at 30-s intervals.

Behavioral tests

Narrow beam walk test

To determine the motor coordination requiring stability and balance, a narrow beam walk test was performed. Training was provided to all the animals to move on a narrow, flat immovable wooden beam (L100 cm × W1 cm) that was 50 cm above the ground and fitted with a covered escape platform (20 × 20 × 20 cm) at one end of the beam. The time taken to traverse from one end of the beam to the other and the number of foot slip errors were measured as reported earlier (Luong et al, 2011).

Wire hanging test

The wire hanging test is used to assess muscle strength and coordination, which is impaired in the mouse PD model. Mice were trained to grasp a horizontal metallic wire (55 cm long) placed 38 cm above the home cage and were gently turned upside-down along the axis of the wire. Three trials for 3 min with 30-s intervals were performed, and latency to fall was recorded (Hutter-Saunders et al, 2012).

EPMT

The comprehensive assessment of anxiety-related behavior can be provided by EPMT by examining multiple parameters. The EPM device consisted of four arms (30 × 5 cm): two opposite open arms and two opposite closed arms equipped with high black walls (15 cm), elevated to a height of 45 cm above the floor. Each mouse was kept at the central square facing one of the two open arms of the maze. Mice’s behavior was captured for 15 min. The duration and the number of entries into the open and closed arms were noted using ANY-maze software. The tendency of mice to avoid entry into the open arms is noted as an index of anxiety (Lister, 1987).

Pole test

A pole test was performed to elucidate the movement disorders. Animals were placed on a vertical iron pole (1 cm in diameter and 60 cm high) grasping the pole with their four paws and their head upward. The pole’s base was positioned inside a mouse home cage with bedding material. The time required to orient 180° downward (“T-turn”) and the time taken to reach the bottom of the pole back into the cage were recorded. The maximum time allowed was 60 s (Matsuura et al, 1997).

Rotarod test

To examine grip strength and motor coordination, a rotarod test (Orchid Scientific) was performed. Mice were trained for three consecutive days for 5 min each. Mice received five trials for which the rod was accelerated from 5 to 20 rpm over the time of 3 min during the training period. The intertrial interval was 5 min. On the experimental day, latency to fall from the rotating rod for each mouse at four different rotation speeds was recorded (Rozas et al, 1998).

Morris water maze test

The spatial learning and memory were determined by the Morris water maze test. The apparatus includes a black circular pool (40 cm in height and 120 cm in diameter), filled with water to a depth of 20 cm. The movement of mice was tracked using a camera linked to ANY-maze video tracking software. Four equally sized quadrants were created in the pool. To make the water opaque and background light, milk was added to water. The test was conducted in two phases. The first phase consisted of hidden platform training where mice were trained for five consecutive days to find a hidden platform placed 1 cm below the water surface in one of the four quadrants. Mice were gently placed in water facing the pool’s wall, and the amount of time it took to find the hidden platform was measured as escape latency (a measure of spatial learning). The maximum time was 90 s. Whenever a mouse took longer than 90 s to reach the platform, the experimenter would lead the mouse there and its escape latency was recorded as 90 s. After reaching the platform, each mouse was permitted to stay on the platform for 30 s. At the end, animal was dried and placed back in its cage. Memory was assessed in the second phase of the experiment, which consisted of a spatial probe trial that was performed on the sixth day after the 5-d training phase. The platform was taken out of the water, and mice were positioned in the quadrant diagonally opposite to the platform location with a cutoff time of 60 s. The duration of the time in the target quadrant (quadrant where the platform was positioned during training) and the number of times animals crossed the original platform location were recorded (Terry, 2009).

Statistical analysis

The statistical analysis was performed using SigmaPlot or GraphPad Prism software. The normality of the data was assessed using the D'Agostino–Pearson omnibus test. Results are presented as the mean ± SD or SEM. Statistical analysis was performed using a two-tailed t test for comparisons between two groups. To evaluate the differences between more than two groups, one-way analysis of variance (ANOVA) was employed, followed by either the Tukey post hoc analysis (in cases of homogeneous variance) or Dunnett’s T3 test (for heterogeneous variance). Significance levels are reported with symbols in the figures (*comparisons with control or as indicated and ^#comparisons with TM) where the following thresholds for statistical significance were set: *, #P < 0.05; **, ##P < 0.01; and ***, ###P < 0.001.

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