Taurine protects dopaminergic neurons in a mouse Parkinson's disease model through inhibition of microglial M1 polarization

Abstract

Microglia-mediated neuroinflammation is implicated in multiple neurodegenerative disorders, including Parkinson's disease (PD). Hence, the modulatioein of sustained microglial activation may have therapeutic potential. This study is designed to test the neuroprotective efficacy of taurine, a major intracellular free β-amino acid in mammalian tissues, by using paraquat and maneb-induced PD model. Results showed that mice intoxicated with paraquat and maneb displayed progressive dopaminergic neurodegeneration and motor deficits, which was significantly ameliorated by taurine. Taurine also attenuated the aggregation of α-synuclein in paraquat and maneb-intoxicated mice. Mechanistically, taurine suppressed paraquat and maneb-induced microglial activation. Moreover, depletion of microglia abrogated the dopaminergic neuroprotective effects of taurine, revealing the role of microglial activation in taurine-afforded neuroprotection. Subsequently, we found that taurine suppressed paraquat and maneb-induced microglial M1 polarization and gene expression levels of proinflammatory factors. Furthermore, taurine was shown to be able to inhibit the activation of NADPH oxidase (NOX2) by interfering with membrane translocation of cytosolic subunit, p47^phox and nuclear factor-kappa B (NF-κB) pathway, two key factors for the initiation and maintenance of M1 microglial inflammatory response. Altogether, our results showed that taurine exerted dopaminergic neuroprotection through inactivation of microglia-mediated neuroinflammation, providing a promising avenue and candidate for the potential therapy for patients suffering from PD.

Fig. 1. P + M induces progressive dopaminergic neurodegeneration and α-synuclein aggregation.

a Mice were treated with P + M. After 2, 4 and 6 weeks of initial treatment, dopaminergic neurons were immunostained with anti-TH antibody and the representative images were shown. b The number of TH⁺ neurons in the SNpc was quantified. c After 6 weeks of treatment, the expression of α-synuclein in midbrain was detected by using western blot and the representative blots were shown. GAPDH was used as an internal control. d The band density of blots was quantified. *p< 0.05, **p < 0.01; Scale bar = 200 μm

Fig. 2. P + M induces gait abnormality in mice.

a Schematic illustration of gait analysis measurements of stride length and stride distance was shown. b–e After 2, 4 and 6 weeks of initial P + M treatment, the distance between subsequent limb placements (stride length) was measured. f, g The stride distance between limb placements of the mice was detected. *p < 0.05, **p < 0.01

Fig. 3. Taurine attenuates P + M-induced dopaminergic neurodegeneration and α-synuclein aggregation.

a Taurine was administrated to mice prior to 30 min of P + M exposure. After 6 weeks of P + M treatment, dopaminergic neurons were immunostained with antibody against TH and the representative images were shown. b The number of TH⁺ neurons in the SNpc was quantified. c After 6 weeks of treatment, the expression of α-synuclein in midbrain was detected by using western blot and the representative blots were shown. GAPDH was used as an internal control. d The band density of blots was quantified. *p < 0.05, **p < 0.01; Scale bar = 200 μm

Fig. 4. Taurine ameliorates P + M-induced motor deficits.

a, b After 6 weeks of initial P + M treatment, the distance between subsequent limb placements (stride length) was measured in mice with or without taurine pre-treatment. c The stride distance between limb placements in P + M-intoxicated mice with or without taurine pre-treatment was detected. *p < 0.05

Fig. 5. Microglia are essential for taurine-afforded dopaminergic neuroprotection.

a After 6 weeks of initial P + M intoxication, microglial cells were immunostained with two markers, Iba-1 and CD11b, in mice with or without taurine pre-treatment and the representative images were shown. Activated microglia are characterized by enlarged cell bodies and high staining density. b Microglial activation was quantified by calculating the density of expression of Iba-1 and CD11b in the SN. c Primary midbrain neuron-glia or microglia-deleted cultures were treated with taurine (25 or 50 μM) 30 min prior to P + M lesion. After 2 days of P + M treatment, cultures were stained with antibody against TH and the representative images were shown. d The number of TH⁺ neurons was counted in both neuron-glia and microglia-deleted cultures. Results were expressed as a percentage of controls from three independent experiments.*p < 0.05, **p < 0.01; Scale bar = 50 μm in a and 100 μm in c

Fig. 6. Taurine attenuates microglial M1 polarization in mice treated with P + M.

a, b After 6 weeks of initial P + M intoxication, the gene expression levels of microglial M1 (iNOS, TNFα and IL-1β) and M2 (Arg-1, Ym-1 and CD206) markers were determined in midbrain of mice with or without taurine by using RT-PCR. *p < 0.05. **p < 0.01

Fig. 7. Taurine attenuates expression and activation of NOX2 induced by P + M.

aAfter 6 weeks of intoxication, the levels of NOX2 subunits p47^phox and gp91^phox were determined in the midbrain of P + M-treated mice with or without taurine by western blot using specific antibodies and the representative blots were shown. b The band density of blots was quantified. c After 6 weeks of intoxication, the levels of 4-HNE were determined in the midbrain of P + M-treated mice with or without taurine by western blot and the density of blots was quantified. d The membrane translocation of NOX2 cytosolic subunit, p47^phox was detected in microglial cells using western blot and the density of blots was quantified. Gp91phox and GAPDH were used as internal membrane and cytosolic control, respectively. e Microglial cells were treated with P + M with or without taurine pre-treatment. The production of superoxide was assessed by DHE and the density of red fluorescence of DHE oxidation was quantified. *p<0.05, **p < 0.01

Fig. 8. Taurine attenuates P + M-induced activation of NF-κB pathway in mice.

a After 6 weeks of initial P + M intoxication, the levels of phosphorylated and nonphosphorylated p65, IκBα and IKKα in the midbrain of P + M-treated mice with or without taurine were determined by western blot using specific antibodies and the representative blots were shown. GAPDH was used as an internal control. b, c The density of p-p65 and total p65 blots was quantified. d The density of p-IKKα blots was quantified. e, f The density of p-IκBα and total IκBα blots was quantified.*p < 0.05. **p < 0.01