TRH Analog, Taltirelin Protects Dopaminergic Neurons From Neurotoxicity of MPTP and Rotenone - PubMed (original) (raw)
doi: 10.3389/fncel.2018.00485. eCollection 2018.
Guiqin Chen 2, Yang Tan 1, Weiqi Zeng 1, Qiwei Peng 1, Ji Wang 1, Chi Cheng 1, Xiaoman Yang 1, Shuke Nie 2, Yan Xu 1, Zhentao Zhang 2, Stella M Papa 3 4, Keqiang Ye 5, Xuebing Cao 1
Affiliations
- PMID: 30618632
- PMCID: PMC6306470
- DOI: 10.3389/fncel.2018.00485
TRH Analog, Taltirelin Protects Dopaminergic Neurons From Neurotoxicity of MPTP and Rotenone
Cong Zheng et al. Front Cell Neurosci. 2018.
Abstract
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.
Keywords: AEP; MPTP; Parkinson’s disease; TRH; rotenone; taltirelin; tau; α-synuclein.
Figures
Figure 1
Taltirelin protected dopaminergic neurons from toxicity of MPP+ and rotenone. SH-SY5Y cells were pre-treated with Taltirelin (5 μM) for 2 h and incubated another 24 h with MPP+ (1, 10, 50 or 100 μM) or rotenone (0.5, 5, 25 or 100 μM; (A) phase contrast micrograph of cultured SH-SY5Y cells exposed to rotenone (50 μM) for 24 h. (B) Hoechst 33342 staining of SH-SY5Y cells. (C) The percentage of nuclear condensation in total cell population as measured by Hoechst 33342 staining. (D–F) SH-SY5Y Cell viability was measured by MTT assay after intervention. (G) Lactate dehydrogenase (LDH) release assay was used to determine the cell death rate of SH-SY5Y cells. (H) Viability of primary midbrain neurons was assessed by MTT assay. #p < 0.05 vs. Control; *p < 0.05; **p < 0.01. N = 6. Error bars represent SEM.
Figure 2
Taltirelin down-regulating tau and α-synuclein-related pathology in vitro. (A) Western blot analysis showed the expression of cleaved caspase-3 in SH-SY5Y cells exposed to MPP+ (50 or 100 μM) or rotenone (25 or 50 μM), with or without 2 h pre-treatment of Taltirelin (5 μM). (B) Quantification of cleaved caspase-3 in different groups. (C) Western blot analysis showed the expression of p-tau (S396), tau N368 and α-synuclein N103 in SH-SY5Y cells exposed to MPP+ (500 or 1,000 μM) or rotenone (25 or 50 μM), with or without 2 h pre-treatment of Taltirelin (5 μM). (D–F) Analysis of p-tau (S396), tau N368 and α-synuclein N103 vs. β-tubulin in different groups. #p < 0.05 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001. N = 3. Error bars represent SEM.
Figure 3
Taltirelin protected locomotor functions of Parkinson’s disease (PD) model mice. Schematic representation of the subacute MPTP-induced (A) and the chronic rotenone-induced (B) PD mice model experimental design. (C) Weight measurements on the first, 7th and 14th day of mice with MPTP (30 mg/kg, i.p.), with or without Taltirelin (Tal 0.2, 1 and 5 mg/kg). (D) Rotarod tests on the first, 7th and 14th day of MPTP modeling. (E) Weight measurements on the first, 30th and 55th day of mice with rotenone (30 mg/kg, IG), with or without Taltirelin (0.2 and 1 mg/kg). (F) Rotarod tests on the first and 55th day of rotenone modeling. (G,H) Pole tests on the 55th day of rotenone modeling. #p < 0.05 vs. normal; *p < 0.05; △p < 0.05 vs. other groups. N = 4. Error bars represent SEM.
Figure 4
Taltirelin protected dopaminergic neurons and reduced PD-related pathologies in MPTP-induced PD mice model. (A) Tyrosine hydroxylase (TH) immunostaining of the substantia nigra (SN) of mice treated with MPTP (30 mg/kg, i.p.), with or without Taltirelin (0.2 and 1 mg/kg). (B) Analysis of TH positive cells counts in each group. (C) Western blot analysis showed the levels of TH in the striatum of each group. (D) Quantification of the levels of TH. (E) WB showed the levels of p-tau (S396), tau N368 and α-synuclein in the SN of each group. (F–H) Quantification of WB of p-tau (S396), tau N368 or α-synuclein vs. GAPDH. #p < 0.05 vs. other groups; *p < 0.05. N = 3. Error bars represent SEM.
Figure 5
Taltirelin down-regulated tau and α-synuclein-related pathology in rotenone-induced PD model mice. (A) Double-labeled immunofluorescence images showed co-localization of α-synuclein and tau N368 in the SN of each group (normal, rotenone, 0.2 or 1 mg/kg Taltirelin). (B) Immunostaining of p-tau (S396) in the SN. Red arrows indicated the positive cells. (C) The enlarged graph of indicated field. Black arrowheads indicated intracellular p-tau (S396) aggregation. (D) Analysis of p-tau (S396) positive cells counts in each group. (E) Western blot analysis of p-ERK1/2, tau N368, p-tau (S396) and p-α-synuclein (S129) in the SN of each group. (F–I) Quantification of WB showed the levels of p-ERK1/2, tau N368, p-tau (S396) and p-α-synuclein (S129). #p < 0.05 vs. other groups; *p < 0.05; **p < 0.01. N = 3. Error bars represent SEM.
Figure 6
Flow cytometry analysis of reactive oxygen species (ROS) generation and monoamine oxidase-B (MAO-B enzymatic assay. SH-SY5Y cells were pre-treated with PBS or Taltirelin (5 μM, 2 h), then incubated with MPP+ (1,000 μM, 24 h) or rotenone (50 μM, 24 h). (A) One typical experiments of Flow cytometry analysis. Geometric mean (Geo Mean) was used to calculate the total intensity of 2′,7′-dichlorofluorescein (DCF) fluorescence of cells. (B) Quantitative analysis of DCF fluorescence intensity in each treatment group. (C) MAO-B activity was assessed by Amplex® red monoamine oxidase assay kit. #p < 0.05 vs. control; *p < 0.05. N = 3. Error bars represent SEM.
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