Role Of T Cell-mediated Immunity And Autoantibody Against Substantia Nigra In The Pathogenesis Of Parkinson's Disease

Parkinson's disease (PD) is a disabling neurodegenerative disorder which was known by uncontrollable resting tremor, rigidity, gait imbalance and slowness of movement. It is one of the common diseases which damaged the life quality of older persons. Parkinson's disease is characterized by a progressive degeneration of dopamine (DA) neurons projecting from the substantia nigra pars compacta (SNpc) to striatal motor loci. But till now, the pathogenesis of Parkinson's disease is still unclear.Selective injury to dopamine neurons was focused on in the past years. Nowadays, many factors were thought taking part in this selective injury such as oxidative stress, excitatory amino acids, calcium overloading and specific protein expressed on domanine neurons in area nine(A9). However the selective injury to A9 could not be diminished obviously by removing those factors. So there must be some other factors affectting the selective injury. And an immunity cause for PD has been discussed for years.Immune abnormalities have been described in PD during the past 30 years. Disturbed cellular and humoral immune functions have been reported in patients with PD.In cellular immunity: Changes of lymphocyte subpopulations in cerebrospinal fluid and peripheral blood has been known for many years in Parkinson's disease. There was an decrease of the CD3+CD4+/CD3+CD8+ ratio. Researchers thought it was the increase of CD3+CD8+ cell that leaded the decrease of the CD3+CD4+/CD3+CD8+ ratio. In further studies, T helper cell analysis revealed a decreased percentage of CD45RA+ "naive" and an increased percentage of CD45RO+ "memory" T cell subset from CD4+ T cells in peripheral blood of patients with Parkinson's disease. An elevated gamma-delta(+) T cell population was also found in PD. Although there were lots of reports on cellular immunity, the role of CD4 and CD8 was still unclear.In humoral immunity: The studies on the serum autoantibody against substantia nigra neuron were always debatable. Though there were some negative results, researchers still believed the serum from PD patients containing some kinds of autoantibody, which could react with the substantia nigra neuron and damage the dopaminergic neurons. Identification of the autoantibody and antigen could provide a new way to postpone the development of PD.Based on the former research on cellular and humoral immune functions in PD, we prepared for our study. Firstly we investigated the injury of dopamine neurons induced by MPTP after being pretreated by different Low dose irradiation, and analysed the relationship between peripheral blood lymphocyte and injury of dopamine neurons. Then the role of CD4 and CD8 in the PD development was investigated by using CD4 knock-out mice and CD8 knock-out mice. At last, we tried to identify the target antigen by using the serum from PD mice and PD patients. So this study contains three parts as following.Part 1: After pretreated with a single low dose(0.5Gy, 2.0Gy or 3.5Gy) total-bodyγ-irradiation (TBI), C57BL/6 mice were administered with MPTP(15 mg/kg, four times, 2 h apart) intraperitoneally (i.p.). The numbers of blood lymphocytes were counted with haemacytometer manually before TBI and on the first day, third day and sixth day after TBI respectively.Then the animals were sacrificed by cervical dislocation on the seventh day. The brains were rapidly removed. Then the striatum and midbrain were dissected and processed for High Performance Liquid Chromatography (HPLC) and immunohistochemistry with antibodies against tyrosine hydroxylase (TH), CD11b or GFAP to show dopamine neuron, microglia and astrocyte respectively.As a result, in the group pretreated with 2.0Gy TBI, neuroprotection was found by HPLC determination of the striatal dopamine, with lower lymphocyte number. Contrarily, in the group pretreated with 0.5Gy TBI, with higher lymphocyte number, dopaminergic neuron toxicity was enhanced. So it was probably the decrease of lymphocyte, not the radiation hormesis, which rendered the potential neuroprotection. And it was the balance between radiation injury and lymphocytopenia neuroprotection that decided the effect of low doseγ-irradiation on MPTP induced dopaminergic neurotoxicity.Part 2: The dopaminergic neurotoxicity induced by MPTP was compared among widetype, CD4 knockout (CD4-Ko) and CD8 knockout (CD8-Ko) C57 mice. MPTP were freshly prepared in saline and administered in mice intraperitoneally (i.p. 30mg/Kg.d×5d). The striatum were dissected and processed for HPLC and TH Western-Blotting. The midbrain were processed for immunohistochemistry with antibodies against tyrosine hydroxylase (TH), CD11b or GFAP to show dopamine neuron, microglia and astrocyte respectively.As a result, there was no evidently difference in DA and its metabolism level detected by HPLC among control saline groups of widetype group, CD4 knockout group and CD8 knockout group (P>0.05). DA and its metabolism decreased obviously on the 10th days after MPTP administration in each group. DA in striatal tissues was higher in CD4 knockout group and CD8 knockout group than that of WT group (P<0.05). Astrocyte and microglia were activated in all groups after MPTP injection. Microglia was more active in CD4 knockout mice slightly and CD8 knockout mice notably than that of WT mice. Astrocyte was more active in CD8 knockout mice, yet less active in CD4 knockout mice than that of WT mice.We found the knockout of CD4 or CD8 could provide neuroprotection in MPTP induced PD. But the neuroprotection was slightly and could not affect the number of dopaminergic neurons obviously. And the neuroprotection was maybe related to the activatation of the astrocyte and microglia which could change the level of cytokine and neurotrophic factors .Part 3: The studies on the serum autoantibody against substantia nigra neuron was proceeded in our experiments with immunohistochemistry, enzyme-linked immunosorbentassay (ELISA) and Western-Blotting. Two-dimensional gel electrophoresis (2-DE) was carried out to screen the antigen from substantia nigra.Autoserum from mice of all groups could react with nerve cells in brain slice widespreadly. But the autoserum from PD mice could react with nerve cell of substantia nigra more actively than autoserum from normal mice. Similar results were found in ELISA and Western-Blotting. And a 35kDa protein from substantia nigra was found reacting with autoserum. It suggested that the serum from PD mice induced by MPTP could react with nerve cell of substantia nigra. And a 35kDa protein from substantia nigra was probably the target.In conclusion:1. Low dose TBI could change MPTP induced dopaminergic neuron toxicity slightly. It was probably the balance among radiation injury, neuroprotection of lymphocytopenia and radiation hormesis that decided the effect of low dose TBI on MPTP induced dopaminergic neuron toxicity. The number of lymphocyte in peripheral blood, which was probably the most important role, was related to dopaminergic neuron injury induced by MPTP. And the effect of radiation hormesis was quite weak in the dopaminergic neuron injury induced by MPTP.2. CD4 and CD8 may take part in the pathogenesis of PD. The knockout of CD4 or CD8 could provide slight neuroprotection in MPTP induced dopaminergic neurons toxicity without affecting the number of dopaminergic neurons obviously. The neuroprotection was maybe related to the activatation of the astrocyte and microglia which could change the level of cytokine and neurotrophic factors .3. There is probably some kind of autoantibody against nerve cell of substantia nigra, which may take part in the pathogenesis of PD. A 35kDa protein in substantia nigra is probably the target of autoantibody.