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1α,25-Dihydroxyvitamin D3 Protects Dopaminergic Neurons in Rodent Models of Parkinson's Disease through Inhibition of Microglial Activation

Background Recent studies have demonstrated the molecular basis of the immunomodulatory and anti-inflammatory activities of 1,25-dihydroxyvitamin D3(1,25-(OH)2D3). This hormone improves behavioral deficits and normalizes the nigral dopamine levels in animal models of Parkinson's disease (PD). Methods We studied whether the administration of 1,25-(OH)2D3 would protect against 6-hydroxydopa (6-OHDA)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury, and its potential regulatory effect on microglia activation. Results We found that 1,25-(OH)2D3 pretreatment significantly decreased 6-OHDA- and MPTP-induced dopaminergic neuronal loss in the substantia nigra pars compacta by preventing the activation of microglia. This observed neuroprotective effect in MPTP-treated mice that were given 1,25-(OH)2D3 may be attributable to inhibition of proinflammatory cytokine expression. Conclusion These results suggest that 1,25-(OH)2D3 is a potentially valuable neuroprotective agent; it may therefore be considered for the treatment of pathologic conditions of the central nervous system, such as PD, where inflammation-induced neurodegeneration occurs.


INTRODUCTION
The etiology of sporadic Parkinson's disease (PD) remains unknown, but there is increasing evidence of a role of inflammation in the brain as a factor in its pathogenesis. 1 Microglia, the resident immune cells in the brain, play a role in immune surveillance under normal conditions, and become readily activated in response to injuries in the brain and immunologic challenges. Activated microglia produce a wide array of cytotoxic factors, including tumor necrosis factor-α (TNF α), interleukin (IL)-1β, eicosanoids, nitric oxide, and reactive oxygen species that impact neurons and induce neurodegeneration. 1 Evidence of microglial activation has been derived mostly from postmortem analyses of the substantia nigra of PD patients. 2 The active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3(1,25-(OH)2D3), has been reported to exert profound anti-inflammatory and immunosuppressive effects. This hormone inhibits B-and T-lymphocyte proliferation, downregulates the production of IL-2 and interferon (IFN)γ by T lymphocytes, and prevents immunoglobulin secretion by B lymphocytes. 3 Moreover, 1,25-(OH) 2 D 3 enhances endogenous glial cell-linederived neurotrophic factor (GDNF) expression in vitro and in vivo, 4,5 which is believed to play an important role in microglial activation.
Locomotor activity was tested with the aid of apomorphine (0.1 mg/kg injected subcutaneously) 4 weeks after inflicting unilateral 6-OHDA lesions. Rats were killed 4 weeks after surgery, and their brains were fixed and processed for immunohistochemical staining as described previously. 6 The primary antibodies used in this study were as follows: rabbit polyclonal anti-tyrosine-hydroxylase (TH) antibody (diluted 1:1000; Santa Cruz Biotechnology, CA, USA) and mouse monoclonal anti-rat-CD11b antibody (diluted 1:500; OX-42, Serotec, Oxford, UK). Immunostaining was visualized using the Envision TM detection kit (K5007, Dako, Carpinteria, CA, USA) and 3,3diaminobenzidine (Sigma, St Louis, MO, USA) containing 0.01% H 2 O 2 . Coronal 30-μm-thick sections of the substantia nigra were made (coordinates: bregma -5.30 mm, interaural 3.70 mm), and measurements were made on pairs of sections separated from each other by 300 μm. 7 The numbers of TH-immunoreactive neurons and activated microglia (CD11b-immunoreactive cells) were counted in each section from normal and lesioned substantia nigra pars compacta (SNpc), while viewing at a magnification of x100. Average numbers of immunoreactive cells per section were calculated. The ANOVA with two variables test was used for statistical analysis.
The mice were sacrificed, transcardial perfusion fixation was performed at 24 or 48 h after MPTP injection, and the brains were fixed and processed for immunohistochemical staining as described above. Measurements were made only for those cells that fell clearly within the confines of the substantia nigra (coordinates: bregma -3.16 mm, interaural 0.64 mm) in three coronal sections that were 30-μm thick and separated from each other by 300 μm. 8 To investigate the temporal pattern of expression of the key cytokines involved in inflammation and immune responses (IFNγ and TNFα), gene expression was quantified using a modification of a previously described reverse transcriptase (RT)-polymerase chain reaction (PCR) method. 9 Animals were killed by spinal cord dislocation and then decapitated at 4, 12, 24, or 48 h after MPTP intoxication (3 animals for each time point). RNA was prepared from the substantia nigra, as     Fig. 1D).
RT-PCR analysis revealed a similar pattern in the kinetics of INFγ and TNFα mRNA expression following MPTP injection (Fig. 2). A rapid increase in the amount of mRNA for INFγ and TNFα was observed between 4 and 24 h after MPTP injection. Comparison with the vehicle-treated group revealed that pretreatment with 1,25-(OH) 2 D 3 protected against the overexpression of proinflammatory cytokines.

DISCUSSION
Our experiments employed two representative pharmacologically generated rodent models of PD. The 6-OHDA-lesioned rat is a well-validated model for motor alteration related to the advanced phases of PD. Moreover, the systematic injection of MPTP in mice may be considered a good model of acute-phase PD, which can cause sustained nigral degeneration, biochemical changes, and motor abnormalities. 10,11 The main finding of this study is that inhibition of microglial activation by pretreatment with 1,25-(OH) 2 D 3 protects against nigrostriatal degeneration in a rodent model of PD. In rats that received 1,25-(OH) 2 D 3 , protective effects against 6-OHDA were confirmed by histopathological findings. These effects were also identified in the MPTP-induced acute mouse model of PD.
In mice that received 1,25-(OH)2D3, the neuroprotective effect of 1,25-(OH) 2 D 3 might be related to a decrease in proinflammatory cytokines. The detection of elevated levels of proinflammatory cytokines in postmortem PD brains provides strong evidence that microglia are involved in the degenerative process. 12 The important role of activated microglia in inflammationmediated neurodegeneration and potentially in the pathogenesis of PD, prompted us to speculate that the inhibition of microglial activation by 1,25-(OH)2D3 is neuroprotective. The result of this experiment, that 1,25-(OH) 2 D 3 appeared to attenuate the neurotoxininduced microglial activation and expression of proinflammatory cytokines, raises the possibility that 1,25-(OH)2D3 is neuroprotective in experimental models of inflammation-mediated neurodegenerative disease.
Other mechanisms may also be involved in the neuroprotective effects of 1,25-(OH)2D3 in preventing nigral degeneration. First, 1,25-(OH) 2 D 3 treatment might enhance GDNF mRNA and protein expression in the striatum. 5 A previous study has shown that the exogenous administration of GDNF reduces the toxicity of MPTP-and 6-OHDA-induced nigral dopaminergic cell injury. 13 Second, the influence on the antioxidant defense mechanisms of 1,25-(OH) 2 D 3 may play a role in the pathogenesis of nigral cell damage. 14,15 Our results suggest that 1,25-(OH)2D3 is a potentially valuable neuroprotective agent that may be used for the treatment of pathologic conditions of the central nervous system, such as PD, where inflammation-induced neurodegeneration occurs. Further study is planned to evaluated the efficacy of 1,25-(OH) 2 D 3 for the treatment of PD and associated neurodegenerative disorders.