TRH Analog, Taltirelin Protects Dopaminergic Neurons From Neurotoxicity of MPTP and Rotenone

Dopaminergic neurons loss is one of the main pathological characters of Parkinson’s disease (PD), while no suitable neuroprotective agents have been in clinical use. Thyrotropin-releasing hormone (TRH) and its analogs protect neurons from ischemia and various cytotoxins, but whether the effect also applies in PD models remain unclear. Here, we showed that Taltirelin, a long-acting TRH analog, exhibited the neuroprotective effect in both cellular and animal models of PD. The in vitro study demonstrated that Taltirelin (5 μM) reduced the generation of reactive oxygen species (ROS) induced by MPP+ or rotenone, alleviated apoptosis and rescued the viability of SH-SY5Y cells and rat primary midbrain neurons. Interestingly, SH-SY5Y cells treated with Taltirelin also displayed lower level of p-tau (S396) and asparagine endopeptidase (AEP) cleavage products, tau N368 and α-synuclein N103 fragments, accompanied by a lower intracellular monoamine oxidase-B (MAO-B) activity. In the subacute MPTP-induced and chronic rotenone-induced PD mice models, we found Taltirelin (1 mg/kg) significantly improved the locomotor function and preserved dopaminergic neurons in the substantia nigra (SN). In accordance with the in vitro study, Taltirelin down-regulated the levels of p-tau (S396), p-α-synuclein (S129) tau N368 and α-synuclein N103 fragments in SN and striatum. Together, this study demonstrates that Taltirelin may exert neuroprotective effect via inhibiting MAO-B and reducing the oxidative stress and apoptosis, preventing AEP activation and its subsequent pathological cleavage of tau and α-synuclein, thus provides evidence for Taltirelin in protective treatment of PD.

Dopaminergic neurons loss is one of the main pathological characters of Parkinson's disease (PD), while no suitable neuroprotective agents have been in clinical use. Thyrotropin-releasing hormone (TRH) and its analogs protect neurons from ischemia and various cytotoxins, but whether the effect also applies in PD models remain unclear. Here, we showed that Taltirelin, a long-acting TRH analog, exhibited the neuroprotective effect in both cellular and animal models of PD. The in vitro study demonstrated that Taltirelin (5 µM) reduced the generation of reactive oxygen species (ROS) induced by MPP+ or rotenone, alleviated apoptosis and rescued the viability of SH-SY5Y cells and rat primary midbrain neurons. Interestingly, SH-SY5Y cells treated with Taltirelin also displayed lower level of p-tau (S396) and asparagine endopeptidase (AEP) cleavage products, tau N368 and α-synuclein N103 fragments, accompanied by a lower intracellular monoamine oxidase-B (MAO-B) activity. In the subacute MPTP-induced and chronic rotenone-induced PD mice models, we found Taltirelin (1 mg/kg) significantly improved the locomotor function and preserved dopaminergic neurons in the substantia nigra (SN). In accordance with the in vitro study, Taltirelin down-regulated the levels of p-tau (S396), p-α-synuclein (S129) tau N368 and α-synuclein N103 fragments in SN and striatum. Together, this study demonstrates that Taltirelin may exert neuroprotective effect via inhibiting MAO-B and reducing the oxidative stress and apoptosis, preventing AEP activation and its subsequent pathological cleavage of tau and α-synuclein, thus provides evidence for Taltirelin in protective treatment of PD.

INTRODUCTION
Thyrotropin-releasing hormone (TRH) is widely known as a metabolism-regulating endocrine hormone acting through the hypothalamus-pituitary-thyroid axis (HPT axis). However, another role of TRH as a neuropeptide is underestimated (Gary et al., 2003;Khomane et al., 2011). TRH has long ago been proved useful in multiple animal models of diseases such as brain/spinal cord injuries (Faden et al., 1981;Pitts et al., 1995), depression (Szuba et al., 1995;Marangell et al., 1997), epilepsy (Jaworska-Feil et al., 2001;Kubek et al., 2009), schizophrenia (Wilson et al., 1973), amyotrophic lateral sclerosis (ALS; Hawley et al., 1987;Brooks, 1989), Alzheimer's disease (AD; Mellow et al., 1989;Molchan et al., 1990) and Parkinson's disease (PD; Ogata et al., 1998). However, TRH has the intrinsic shortcomings such as short halflife, poor lipophilicity and strong HPT axis stimulating effect, which severely restrict its application (Kinoshita et al., 1998). Taltirelin (TA-0910, Ceredist ), an oral-effective TRH analog, has 10-100 times more potent central nervous system (CNS) stimulant activity and eight times longer duration than TRH. More importantly, Taltirelin has been approved in the treatment of spinocerebellar degeneration (SCD), thus is highly promising in investigating other possible applications of TRH (Kinoshita et al., 1994(Kinoshita et al., , 1998. PD is a neurodegenerative disease with the second high incidence among elderly over the age of 65 (Lee and Gilbert, 2016). Motor (such as bradykinesia, tremor and rigidity) and non-motor (such as depression and constipation) symptoms of PD are directly related to the significant loss of dopaminergic neurons in the substantia nigra (SN) and accordingly dopamine (DA) deficiency within the CNS. Although these symptoms are managed by pharmacological DA substitution, lack of the disease-modifying interventions, which could prevent further neuron loss thus slow or halt the progression of the disease, PD remains an incurable disease that causes severe disability and high mortality (Poewe et al., 2017). The death of dopaminergic neurons are caused by several factors, the most prominent one among which is the pathologically elevated intracellular α-synuclein, whose toxic forms (oligomers or fibrils) or deposition induces and exacerbates other neurotoxic events, such as mitochondrial function, oxidative stress and neuroinflammation, ultimately driving the neuron death in PD (Kalia and Lang, 2015). Nevertheless, growing evidence showed that α-synuclein and another pathologic protein tau could form hybrid oligomers and enhance their aggregation in the brains of PD and AD patients (Sengupta et al., 2015). The overlap of these proteinopathies not only emphasizes a similar structural characteristics of α-synuclein and tau (Moussaud et al., 2014), but also suggests the existence of a common upstream pathological trigger. Asparagine endopeptidase (AEP, or legumain), a pH-dependent endo-lysosomal cysteine protease, is found abnormally up-regulated in AD and PD. Once activated, AEP is able to cleave amyloid precursor protein (APP), tau and α-synuclein, whose protein fragments are more susceptible to hyperphosphorylation and aggregation, thus resulting in neurotoxicity (Zhang et al., 2014(Zhang et al., , 2016(Zhang et al., , 2017. Moreover, we have recently reported that the crosstalk among α-synuclein, monoamine oxidase-B (MAO-B), 3,4-dihydroxyphenylacetaldehyde (DOPAL) and AEP plays a central role in the pathogenesis of PD (Kang et al., 2018).
TRH and its analogs reveal neurotrophic and anti-apoptotic effects by counteracting the toxic effects of various cytotoxins in PD cellular models (Jaworska-Feil et al., 2010) via antiapoptosis, inhibiting the activity of glycosynthase kinase-3β (GSK-3β), decreasing the phosphorylation level of tau (Luo and Stopa, 2004), protecting against Aβ toxicity and reducing the production of inflammatory cytokines TNF-α and IL-6 in vitro (Faden et al., 2005). However, the underlying mechanisms were still unclear. In this study, we showed that Taltirelin significantly reduced the toxicity of MPP+ and rotenone in SH-SY5Y cells and rat primary SN neurons, ameliorated the behavioral disturbance induced by MPTP or rotenone in PD mice via inhibiting MAO-B activity and reactive oxygen species (ROS) generation, reducing the abnormal phosphorylation of α-synuclein and tau, inhibiting the activation of AEP and its specific cleavage effect, thus provides evidence for Taltirelin in protective treatment of PD.

Animals
Male C57/BL6 mice, 8-9 weeks old (Beijing HFK Bioscience Co., Ltd., Beijing, China) weighing 20-25 g were used in this experiment. The animals were housed with food and water provided ad libitum, 12 h light/dark cycle, constant temperature and humidity. This study was carried out in accordance with the recommendations of the Guidelines of Laboratory Animals Ethics of Tongji Medical College, Huazhong University of Science and Technology. The protocol was approved by the Ethics Committee of Huazhong University of Science and Technology.

SH-SY5Y Neuroblastoma Cells Culture
SH-SY5Y neuroblastoma cells (Cell Resources Center of Shanghai Institutes for Biological Sciences) were grown in DMEM/F12 medium (Hyclone, South Logan, UT, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) and 100 units/ml of penicillin and streptomycin. Cultures were maintained in a humidified incubator at 37 • C/5% CO 2 with medium changed every 3 days. After reaching 80% confluence, cells were seeded onto appropriate multi-well plates at a density of 4 × 10 5 cells/ml.

Primary Neonatal Rat Midbrain Neurons Culture
Primary rat midbrain cultures were prepared as described previously (Beaudoin et al., 2012). In brief, the midbrain was dissected from P1-3 mouse pups. The isolated tissues were then chemically and mechanically dissociated into single cell suspensions. Cells were plated into 0.5 mg/ml PLL coated 6-well plates or glass coverslips at a density of 5.5-6 × 10 5 per well. Incubate the cells with plating medium (DMEM/F-12 medium with 20% FBS, 100 units/ml of penicillin and streptomycin) for 4 h and replace the medium with maintenance medium: Neurobasal medium (Gibco, Carlsbad, CA, USA) with 2% B-27 supplement (Gibco, Carlsbad, CA, USA), 2 mM glutamine (Gibco, Carlsbad, CA, USA) and 100 units/ml of penicillin and streptomycin. Cultures were maintained in a humidified incubator at 37 • C/5% CO 2 for 3 days in vitro prior to functional studies.

MTT Assay
Cell viability was quantified by colorimetric assay with MTT (Beyotime, China) according to the manufacturer's protocol. Briefly, 20 µl MTT (5 mg/ml) was added to each well (100 µl medium each) and incubated for 3 h at 37 • C, then the dye was solubilized by 150 µl DMSO and the absorbance of each sample was measured at 570 nm with 630 nm as reference in a microplate reader (BioTek, Winooski, VT, USA). Cell viability = [(drugtreated well OD − MTT-free well OD)/(vehicle-treated well OD − MTT-free well OD)] × 100%.

Measurement of Lactate Dehydrogenase (LDH) Release
In order to estimate cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into the culture media was measured after intervention. 1 h before the incubation endpoint, LDH release reagent was added to intervention-free wells as the maximum enzyme activity control. Then culture supernatants were collected and incubated with the appropriate reagent mixture according to the supplier's instructions (LDH Cytotoxicity Assay Kit, Beyotime, China) at room temperature for 30 min. Absorbance of each well was measured at a wavelength of 490 nm with 630 nm as reference. The absorbance of MTT-free controls was subtracted from each value; Cell death rate = [(drug-treated well OD − vehicle-treated control well OD)/(maximum enzyme activity control well OD − vehicletreated control well OD)] × 100%.

Identification of Apoptotic Cells
In order to visually assess DNA fragmentation in SH-SY5Y cells, Hoechst 33342 staining was applied after intervention. The cultures were washed with PBS and fixed for 20 min with 4% paraformaldehyde (PFA). After fixation, cultures were washed and exposed to Hoechst 33342 (5 µg/ml; Beyotime, Shanghai, China) for 15 min. After washing, coverslips were mounted on slides and images were recorded using inverted fluorescence microscope (Olympus, Japan) with excitation wavelength 350 nm (blue fluorescence). Cells with bright blue fragmented nuclei showing condensation of chromatin were identified as apoptotic cells and counted.

Subacute MPTP-Induced PD Mice Model and Intervention
Mice were randomly divided into five groups: normal, MPTP, Taltirelin 0.2 mg/kg, 1 mg/kg, 5 mg/kg (N = 8). The intervention (14 days) was divided into two sections (7 days each). In the first section (MPTP + drug intervention), MPTP group was injected i.p. with MPTP (Sigma, St. Louis, MO, USA) of 30 mg/kg/d and three Taltirelin groups were pre-treated with Taltirelin (i.p.) of respective doses 4 h before MPTP injection for 7 days. In the second section (drug intervention), the MPTP group was given saline (i.p.), while three Taltirelin groups continued Taltirelin administration for another 7 days. Behavioral assessments and weights measurement were conducted on day 1, day 7 and day 14 of intervention period.

Chronic Rotenone-Induced Mice Model and Intervention
Mice were randomly divided into four groups: normal, rotenone, Taltirelin 0.2 mg/kg, 1 mg/kg (N = 8). Rotenone (4% Tween-20, 0.5% CMC; Sigma, St. Louis, MO, USA) was administered orally once a day at a dose of 30 mg/kg for 56 days. The Taltirelin groups were given Taltirelin i.p. of respective doses before gavage. Behavioral assessments and weights measurement were conducted on day 1 and day 56 of the intervention period.

Rotarod Test
Mice were placed on a rotarod system (YLS-4C, Jinan Yiyan Technology Development Co., Ltd., China) that accelerated from 5 rpm to 40 rpm over a period of 90 s and 40 rpm was maintained for maximal 300 s. The length of time that animal was able to stay on the rod (latency time) was recorded. Test of each animal was repeated three times with at least 30 min interval between each experiment and the longest latency time was adopted. Before the formal experiments, adaptational trainings were conducted for 3 days and the animals were evenly divided into different groups based on their performances.

Pole Test
Mice in each group were placed head-up on the top of a vertical pole (50 cm tall and 1 cm in diameter). The base of the pole was placed in the home cage. The mice would turn its head downward and climb along the pole to the home cage. Time of the mouse head orient downward (time-turn) and total time from the top of the pole to home cage (time-down) as an indication of the locomotion activity was recorded. Each mouse received three successive trials and the average time was calculated. Before the formal experiments, mice in each group received training for 3 days.

Serum Thyroid Hormone Measurement
Blood was collected at 2 h post-administration of drugs and placed in 4 • C overnight. Then the blood was centrifugated at 3,000 rpm for 15 min and serum was collected for further analysis. The levels of thyroid stimulating hormone (TSH), free and total thyroxine (T4) or triiodothyronine (T3) were measured using ELISA kit respectively (ALPCO) according to the manufacturer's protocol. Briefly, 50 µL of serum samples or reference was pipetted into the assigned wells, followed by T3/T4 enzyme-conjugated solution with 1 h of incubation. The mixture was removed, and the plate was washed several times with water; then, working substrate solution was added to each well for reaction, and after 20 min in the dark, the reaction was stopped by adding 3 M HCl. Absorbance was read at 450 nm. The corresponding concentrations were calculated based on the standard curve.

Immunohistochemistry Staining
Animals were deeply anesthetized and transcardially perfused with saline, followed by 4% PFA in 0.1 M PBS (pH 7.4). Brain were dissected and fixed in 4% PFA overnight at 4 • C. After fixation, tissues were transferred to 70% ethanol and processed for paraffin sectioning (4 µm/section). The sections were mounted on glass slides, deparaffinized by xylene, dehydrated in graded ethanol solutions, baked in the basic antigen retrieval buffer (pH = 6.0) and washed with phosphate buffer (pH 7.4). After washing, sections were blocked with 3% bovine serum albumin (BSA) and then incubated with diluted primary antibody in a humidified chamber overnight at 4 • C. Primary antibodies against following proteins were used: TH (1:1,000, Proteintech, 25859-1-AP), phospho-tau (S396; 1:10,000, Abcam, ab109390). The sections were washed and subsequently incubated with biotinylated goat anti-rabbit IgG, then HRP labeled streptavidin fluid, followed by DAB solutions, counterstained with Harris hematoxylin, dehydrated in graded ethanol solutions and eventually cover slipped. Images were collected through an Olympus camera connected to the microscope at the same light intensity, and analyzed using Image-Pro Plus software.

Immunofluorescent Staining
For brain slices, immunofluorescent staining shared a same procedure with immunohistochemistry staining before secondary antibodies incubation. For cellular cultures, sterile coverslips were placed in 24-well plates and coated with PLL before cells were plated on the coverslips. After intervention, cells were first washed with PBS and fixed with 4% PFA. Then cells were permeabilized with 0.2% Triton X-100 and blocked with 5% BSA. Incubate brain slices or cell slides with following primary antibodies overnight at 4 • C: α-synuclein (1:1,000, Abcam, ab85862), tau N368 (1:1,000, provided by Professor Keqiang Ye of Emory University). After being washed, sections were incubated in dark with appropriately diluted Alexa 488-or Cyanine 3-coupled secondary antibodies followed by DAPI staining nucleus for 10 min. To stain nuclei, incubate with DAPI for 10 min. Coverslips were mounted on slides with one drop per coverslip of antifade mounting medium (Beyotime, China). Images were collected using fluorescence microscopy with image manipulation software, and analyzed using Image-Pro Plus software.

Flow Cytometric Analysis of Reactive Oxygen Species (ROS)
After pre-treatment of PBS or Taltirelin (5 µM) for 0.5 h, SH-SY5Y cells were incubated with MPP+ (1,000 µM) or rotenone (50 µM) for 24 h. Then cells were stained with 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime, China) according to the protocol. Briefly, the culture medium was removed and cells were washed twice with serum-free culture medium. Next, the cells were loaded with DCFH-DA (10 µM) in serum-free culture medium for 20 min in 37 • C. After being washed twice with PBS, cells were digested with trypsin, then washed once with PBS and finally collected. The samples were analyzed for the fluorescence (488 nm excitation and 525 nm emission) of DCF by flow cytometry (FACS LSR Fortessa System, BD Biosciences, San Jose, CA, USA).

Monoamine Oxidase-B (MAO-B) Activity Assay
After pre-treatment of PBS or Taltirelin (5 µM) for 0.5 h, SH-SY5Y cells were incubated with MPP+ (1,000 µM) or rotenone (50 µM) for 24 h. Next, cells were scraped down by a cell scraper and homogenized in PBS with cocktail of protease inhibitors. Large debris was then removed by weak centrifugation (1,000 g, 10 min). Next, the supernatant was collected and incubated with the working solution of 100 µl containing 400 µM Amplex Red reagent, 2 U/ml HRP and 2 mM benzylamine substrate (Molecular Probes, Eugene, OR, USA). The fluorescence of MAO-B activity was measured in a fluorescence plate reader (530-560 nm excitation and 590 nm emission).

Statistic Analysis
Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey HSD or LSD post hoc tests for multiple comparisons between groups, and Student's t-test for comparing two groups. All statistical analyses were performed in SPSS 21.0 software. The significance level was set at p < 0.05. Data are presented as Mean ± SEM for all results.

Taltirelin Protected Dopaminergic Neurons Against Apoptosis in vitro
The neuroblastoma SH-SY5Y cells are a well-established and widely-used in vitro model of dopaminergic neuron origin. We found 5 µM Taltirelin induced the highest proliferation (109.70 ± 2.41% of control, p < 0.05) of cells after 24-h-incubation, while higher concentration (10 µM) caused a decrease of cell viability (91.64 ± 1.90% of control, p < 0.05; Figure 1D). Thus, we adopted 5 µM for our following experiments.

Taltirelin Protected Locomotor Functions of PD Model Mice
We further assessed the neuroprotective effects of Taltirelin in the subacute MPTP-induced PD mice model ( Figure 3A). In the pilot test, we found that the mice injected with MPTP (30 mg/kg, i.p.) 2 h after Taltirelin (5 mg/kg, i.p.) would develop irritation, spasm and finally death, which may be due to the metabolism increased by Taltirelin aggravated the toxicity of MPTP. Therefore, the injection interval was adjusted to 4 h and similar death was prevented. Possibly due to the same reason, after 7 days of intervention with MPTP, the body weights of the mice injected with 5 mg/kg Taltirelin (23.04 ± 0.90 g) was lower than other groups (24.20 ± 1.31 g), but gradually recovered (25.47 ± 1.09 g) after following Taltirelin protective treatment (7 days), while the body weight of the MPTP group (23.43 ± 0.83 g), obviously decreased on the 14th day. However, there was no significance difference between MPTP and Taltirelin groups ( Figure 3C). On the 7th day of the MPTP modeling, no obvious performance differences in the rotarod test were observed among groups, but on the 14th day, the latency times in the rotarod test in the MPTP group (179.33 ± 94.62 s, p < 0.05) significantly decreased, indicating impaired locomotor functions. Taltirelin 1 mg/kg (287.20 ± 28.62 s) successfully alleviated the impairment induced by MPTP. Besides, there was a trend of better performance of Taltirelin 0.5 mg/kg group (245.5 ± 77.25 s) or 5 mg/kg (239.20 ± 75.31 s) group compared with MPTP group (Figure 3D).
In addition, we also examined the motor improving effect of Taltirelin immediately after injections and found a time-and dose-dependent locomotor improvement of both MPTP and rotenone mice, which could maintain for at least 6 h (p < 0.05). This result was in consistency with our findings that Taltirelin could improve motor function of 6-OHDA lesioned PD rats (unpublished data).

Taltirelin Protected Dopaminergic Neurons and Reduced PD-Related Pathologies in MPTP-Induced PD Mice Model
In order to quantify the protective effect of Taltirelin, we stained the brain slices of SN with TH, the rate-limiting enzyme in DA synthesis which mainly expressed by dopaminergic neurons. We found that the numbers of TH positive neurons were much fewer in MPTP treated mice (36.66 ± 2.08% of normal) than that in 0.2 mg/kg (64.47 ± 3.91%) or 1 mg/kg (68.29 ± 4.45%) Taltirelin groups (Figures 4A,B, p < 0.05). We also quantified the level of TH in the striatum using western blot and the results validated the immunostaining observation (Figures 4C,D, p < 0.05).
We further examined the level of toxic forms of tau and αsynuclein in the SN and found that compared with the normal group, the levels of p-tau (S396), tau N368 and α-synuclein of the MPTP group were significantly higher, whereas the levels of the above proteins were reduced by Taltirelin of three doses by at least 55.43% (Figures 4E-H). Therefore, consistently with in vitro results, Taltirelin may have a protective effect against MPTP-induced neurotoxicity on dopaminergic neurons by decreasing the phosphorylation of tau and α-synuclein and down-regulating activation of AEP.

Taltirelin Down-Regulated Tau and α-Synuclein-Related Pathology in Rotenone-Induced PD Mice Model
Our previous studies have shown co-localization and increase of α-synuclein and tau N368 in the rotenone PD mice model (Nie et al., 2015). Thus, we also performed immunofluorescence double-labeling of the above two proteins in the SN, striatum, hippocampus and cortex of rotenone mice. The results showed that normal mice also had α-synuclein and tau N368 co-localized and evenly distributed within the neurons, whereas in the rotenone group, intracellular α-synuclein and co-localized tau N368 exhibited higher and denser fluorescence with wider distribution in the cytoplasm, indicating increased and aggregation of α-synuclein and tau N368. In contrast, Taltirelin, especially 1 mg/kg group had much fewer and more dispersive distribution of α-synuclein and tau N368 in the SN ( Figure 5A) and striatum (Supplementary Figure S1). However, similar difference among groups in the expression of two proteins was not significant in the hippocampus and cortex (Supplementary Figures S2, S3). Besides, we also stained p-tau (S396) in the SN and found an elevation of the number of p-tau (S396) positive cells in the rotenone group (626.57 ± 66.31% of normal), in which immunostaining positive dense particles of aggregating p-tau (S396) could be easily spotted. In the 1 mg/kg Taltirelin group (175.44 ± 25.06%), both the number of p-tau (S396) positive cells and intracellular dense particles significantly decreased (Figures 5B-D, p < 0.01).

Taltirelin Reduced the Generation of ROS and the Activity of MAO-B
It is reported that oxidative stress facilitates accumulation (Conway et al., 2001) and oligomerization (Norris et al., 2003) of α-synuclein, which could be promoted by age-dependent activation of MAO-B (Kang et al., 2018). Thus, in order to get a further insight of the protective mechanisms of Taltirelin (Figures 6A,B). MPP+ (1,000 µM) and rotenone (50 µM) induced obvious increase of MAO-B activity (363 ± 30.58%, 238.41 ± 35.97%, respectively, p < 0.05) in SH-SY5Y cells. Cells pre-treated with Taltirelin (5 µM) exhibited a not significant but consistent decrease of the MAO-B activity elevated by MPP+ (by 36.15 ± 10.79%, p > 0.05) and rotenone (by 33.02 ± 6.71%, p > 0.05) ( Figure 6C). Thus, these results suggested that Taltirelin may alleviate the neurotoxicity of MPTP and rotenone via reducing the generation of ROS and decreasing the activity of MAO-B.

DISCUSSION
TRH is stored in the releasable pool in the synaptic terminals and widely distributed in anatomically distinct pathways throughout the neuroaxis (Gary et al., 2003), regulating a variety of physiological activities in the nervous system. The neuroprotective effects of TRH and its analogs have been showed in animal models of cerebral ischemia (Urayama et al., 2002), cellular models of PD (Jaworska-Feil et al., 2010) and AD (Luo and Stopa, 2004). Here, we report that Taltirelin, a TRH analog had neuroprotective effect in both cellular and animal models of PD. MPP+ (metabolite of MPTP) and rotenone, highly lipophilic insecticide, both are mitochondrial complex I inhibitors which impair the mitochondrial respiration chain, increase ROS production and trigger apoptosis of dopaminergic neurons (Giordano et al., 2012;Xicoy et al., 2017). MTT assay and the LDH release assay showed that Taltirelin (5 µM) co-incubation for 24 h alleviated cytotoxicity induced by MPP+ or rotenone and rescued the viability of both SH-SY5Y cells and primary midbrain neurons. Hoechst 33342 staining and the level of cleaved caspase-3 further suggested that Taltirelin reduced the apoptosis caused by MPP+ and rotenone. Activation of ERK1/2 is found to be able to initiate a series of anti-apoptosis processes (Roskoski, 2012). We have demonstrated that the actions of Taltirelin both in vitro and in vivo were companied by an elevation of p-ERK1/2, which may mediate the resistance to apoptosis of Taltirelin. We further established the subacute MPTP-induced and chronic rotenone-induced PD mice models and found that Taltirelin significantly rescued the locomotor functions of these animals. However, long-term and high-dose administration of Taltirelin led to the loss of weight in animals, which is most likely due to an over-activation of HPT axis. We also measured the level of TSH and thyroid hormones in the serum of mice and found a significant increase of total T4 when the dose of Taltirelin exceeded 0.2 mg/kg, while free T4 or T3 had no significant alteration (Supplementary Figure S4). Since the total T4 is of the highest concentration among thyroid hormones and also the effector hormones, the results suggested that the thyroid function of patients should be thoroughly evaluated and adjusted accordingly before administration of Taltirelin if possible, in the future.
AEP, also known as legumain, is an endo-lysosomal cysteine protease activated under acidic conditions. The pH within the brain decreased with aging, while pH in the brains of patients of AD or PD is generally low and AEP is significantly elevated (Basurto-Islas et al., 2013). The cleaved products of AEP, tau N368 or α-synuclein N103 fragments are themselves highly neurotoxic and are prone to hyperphosphorylation and aggregation. We thus examined the influence of Taltirelin on these AEP-related pathologies both in vitro and in vivo. MPTP and rotenone caused an elevation of p-tau (S396), p-α-synuclein (S129), tau N368 and α-synuclein N103 fragments in SH-SY5Y cells and in the SN of PD mouse models, while Taltirelin successfully reduced the levels and aggregation of tau or α-synuclein in toxic forms. We recently reported that MPP+ could increase endogenous MAO-B, α-synuclein and AEP protein levels in SH-SY5Y cells in a time-dependent manner (Kang et al., 2018), while MAO-B activation is closely related with increase in free radical damage and ROS (Kang et al., 2018). In addition, rotenone administration may interfere with DA distribution and metabolism, leading to DA accumulated in the cytoplasm of PC12 cells, which may contribute to the ROS formation and cell death (Sai et al., 2008). Our results showed that Taltirelin decreased the generation of ROS induced by rotenone in SH-SY5Y cells though no significance was with MPP+. Taltirelin also moderately reduced activity of MAO-B, which was elevated by MPP+ and rotenone though the reduction was not significant. As for possible explanations for the insignificance: First, MAO-B mainly exists in glial cells, while SH-SY5Y cells as a neuron-origin species may contain low expression levels of MAO-B (Maruyama et al., 2003) to demonstrate the influence of Taltirelin. Second, previous studies showed that the amount of DA that SH-SY5Y cells could synthesize and release is barely detectable (Xicoy et al., 2017). However, MAO-B-induced DA metabolite, DOPAL, is neurotoxic per se and is able to activate and raise protein levels of both AEP and MAO-B (Kang et al., 2018). Since Taltirelin is a potent and sustained DA-releasing agent, which decreases the intracellular DA, this part of contribution to reducing MAO-B activity could not be elucidated under this condition. Thus, it is predictable that in vivo assessment of MAO-B activity or elevating MAO-B level with genetic manipulations could provide more insight into the mechanisms.
Besides, other possible explanations for the protective mechanism of Taltirelin may exist. First, Taltirelin mainly interacts with G-protein coupled receptors, TRH type 1 receptor (TRH-R1). As with many other rhodopsin-like GPCRs, the mitogen-activated protein kinase (MAPK; or Ras-Raf-MEK-ERK1/2) pathway has been found to be a downstream effector of TRH-R1 signaling (Smith et al., 2001;Oride et al., 2008). We also observed that Taltirelin caused a rise of p-ERK1/2 in the striatum and SN of mice after protective treatment. p-ERK1/2 participates in many signaling pathways and may therefore mediate the protective effects of Taltirelin. Second, TRH and its analogs are able to increase cerebral blood flow and metabolism which often decline with aging and in PD or AD (Farkas et al., 2000;Borghammer et al., 2010;Austin et al., 2011), thus achieve the protection by accelerating the elimination of toxins in the CNS and reducing the formation of anoxia or toxins-induced acidic micro-environment, which is a trigger in AEP activation. Finally, multiple studies have suggested that TRH acted as a regulator of homeostasis (Gary et al., 2003) involving immune and endocrine system, whereas imbalance of inflammation (Zheng et al., 2016) or diabetes (Arvanitakis et al., 2004) plays a recognized role in neurodegenerative diseases like PD or AD. Therefore, it will be interesting to investigate the interaction of TRH or Taltirelin with internal environment in PD or AD.
It is widely accepted that thyroid function has great impact on cognition. Deficiency of thyroid hormone in the critical period of brain development leads to severely stunted physical and mental growth, such as Cretinism. No surprise, functions of the HPT axis have been shown to undergo numerous changes with aging (Roelfsema and Veldhuis, 2013). Although there are still many controversies regarding the relationship between thyroid function and neurodegenerative disease (Villanueva et al., 2013), study showed that the free T3 level was significantly lower in PD patients especially with akinetic-rigid motor subtype, while TSH level was lower in patients with tremor-dominant type or mixed type, suggesting a relation of the thyroid hormone level with PD motor symptoms (Umehara et al., 2015). TRH is decreased in the hippocampus of AD patients (Luo et al., 2002) and alterations in TSH (van Osch et al., 2004) or thyrotropin (Tan et al., 2008) have also been implicated as a risk factor for AD and dementia. Rat models of hypothyroidism exhibited increases of APP protein (Contreras-Jurado and Pascual, 2012), tau hyperphosphorylation, proinflammatory cytokines and spatial memory impairments (Chaalal et al., 2014), indicating that hypothyroidism represents an important risk factor of developing sporadic forms of AD. Since the regulatory functions in the nervous system of TRH are achieved in trace amount acting as a neuromodulator rather than endocrine hormones, even if the synthesis of TRH in the hypothalamus is disturbed or compensated, whether the total level of TRH in the brain is sufficient or excessive in maintaining its normal regulation functions is hard to assess. Therefore, the relationship between TRH and PD still needs further discussion. In conclusion, this study suggests TRH analog, Taltirelin possesses a protective effect on SH-SY5Y cells and rat primary midbrain neurons, as well as MPTP-induced subacute and rotenone-induced chronic PD mice models and this neuroprotective effect is associated with inhibition of apoptosis, ROS generation, phosphorylation of tau and α-synuclein, inhibition of AEP, thus providing evidence for applying a promising neuroprotective agent for treatment of PD.

AUTHOR CONTRIBUTIONS
CZ, GC, YX, ZZ, SP, KY and XC conceived and designed the study. CZ, GC, YT, WZ, QP, JW, CC, XY and SN performed the experiments. CZ and GC analyzed the data. CZ, GC, YX, SP, KY and XC wrote the manuscript. All authors read and approved the final manuscript.