A Study On Neural Immune Interaction After Injury Induced By Specific Neurotoxins

Parkinson’s disease(PD) is the second largest neurodegenerative diseases in the world. The main pathological features of PD are the degeneration of dopaminergic neurons in the substantia nigra, excessive activation of neuroinflammation and adaptive immune system and the formation of Lewy bodies consisted with aggregrated alpha-synuclein(α-syn) proteins. Recently, many studies focus on the effect of interaction between neuroinflammation and development of PD. Meanwhile, it was found that peripheral lymphocyte T, B cells could pass through blood brain barrier(BBB) into central nervous system in PD patients; it was also found that the total amount of peripheral blood T lymphocyte has been changed in PD patients. These indicated peripheral immune system plays an important role in the occurrence and development of PD. PD is a progressive and multi-system complexive disease, the pathogenesis of it was not clearly. The generally accepted hypothesis is that PD is the result of combination of environmental factors and gene mutations. Neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP) and its metabolite 1-methyl-4-phenyl-2,3-dihydropyridine(1-methyl-4-phenyl-2,3-dihydropyridiumion, MPP+) is one of the environmental factors which could cause PD. Endogenous neurotoxin 1-methyl-4-phenyl-1,2,3,4-tetrahydro-isoquinoline(1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline salsolinol, Sal), an analogue of MPTP, and its metabolite 6,7-dihydroxy-1,2-dimethyl-1,2,3,4-tetrahydro-isoquinoline(1,2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-salsolinol, NMSal) are now considered to be neurotoxins causing Parkinson’s disease.In our lab, we have done lots of resreaches focusing on the relationship between endogenous neurotoxins Sal/NMSal and the occurrence and development of PD. We suggested that endogenous neurotoxin could induce the onset and progression of PD, and proposed “endogenous neurotoxin vicious circle hypothesis of PD”. Previous studies in our lab have found that endogenous neurotoxins Sal and NMSal caused elevation of oxidative stress levels of neuroblastoma cells SH-SY5 Y, changes of mitochondrial membrane potential, aggregation of over-expressed α-syn and finally lead to cell apoptosis. We also found that the level of Sal N-methyl transferase enzyme increased in 6-OHDA substantial nigra injected PD rat compared to the normal one. These studies suggested that endogenous neurotoxin Sal and NMSal play very important roles in the pathogenesis of PD. In this study, we focused on the mechanism of the interaction between neurotoxins MPP+, Sal and NMSal damaged dopamine neurons and peripheral blood lymphocyte and monocytes.1. In this study, we used SH-SY5 Y, U87 and Jurkat cell co-culture system to explore the interaction of neurotoxins damaged dopaminergic neurons and peripheral blood lymphocyte.In this section we used endogenous neurotoxion Sal and NMSal treated SH-SY5Y-EGFP, U87 and Jurkat cells mixed culture system to study the effect of Jurkat cells to damaged SH-SY5Y-EGFP cells. We found endogenous α-syn aggregated in SH-SY5Y-EGFP cells in three cells mix co-culture system treated by Sal or NMSal for 24 hours. No evidence of apoptosis and necrosis observed in α-syn aggregated SH-SY5Y-EGFP cells. In contrast, LC3 expression level increased in α-syn aggregated SH-SY5Y-EGFPcells. In addition, selective initiation of the autophagy by rapamycin resulted in the decreasing aggregation of α-syn, indicating that autolysosomes could be involved in degradation of misfolded α-syn. Our data indicated that peripheral lymphocyte Jurkat cells accelerated the speed and progress of endogenous neurotoxin Sal and NMSal induced aggregation of endogenous α-syn in SH-SY5Y-EGFP cells, and degradation of misfolded α-syn could involve activation of autophagy signal pathway.Endogenous neurotoxin Sal and NMSal could also induce endogenous α-syn aggregation in SH-SY5Y-EGFP cells in SH-SY5Y-EGFP, U87 and HPBL(human peripheral blood lymphocyte) three cells co-culture system, and there was no apoptosis and necrosis of SH-SY5Y-EGFP; while LC3 expression level of SH-SY5Y-EGFP was increased in Sal or NMSal treated SH-SY5Y-EGFP, U87 and Jurkat three cells mixed co-culture system.2. In this section, we examined the effect of Sal and NMSal damaged neuroblastoma SH-SY5 Y cells on Jurkat/ THP1 cells in a SH-SY5 Y, U87 and Jurkat three cells co-culture system. The results showed that conditioned medium from Sal treated co-cultured SH-SY5 Y and U87 cells, but not the conditioned medium without U87 cells, induced proliferation of both Jurkat and THP1 cells indicating soluble mediators underlie the observed proliferation with cytokine arrays indicating IL-6 and CCL2 as the likely candidates; while the conditioned medium from NMSal treated co-cultured SH-SY5 Y and U87 cells, but not the conditioned medium without U87 cells, also induced proliferation of Jurkat cells and protected THP1 cells from the toxicity of NMSal indicating soluble mediators underlie the observed protection and proliferation with cytokine arrays indicating CCL2 and CXCL12 as the likely candidates.3. In this section, SH-SY5 Y co-cultured with U87 cells were treated with neurotoxin 1-methyl-4-phenylpyridinium(MPP+) for 24 hours, after that conditioned medium were used to culture T-cell leukaemia Jurkat cells for another 24 hours, and we investigated cell number, cell cycle and necrosis of Jurkat. The results showed MPP+ treated co-culture medium of SH-SY5 Y and U87 cells inhibited the proliferation of Jurkat cells compared to control medium without MPP+. Furthermore, co-culture medium with 100 μM MPP+ slowed Jurkat cells growth by G2/M cell cycle checkpoint through increased CDC2 expression level in a Cyclin B1 independent pathway; while co-culture medium with 500 μM MPP+ slowed Jurkat cells growth by necrosis through cellular swelling and membrane broken. Our data suggested that damaged dopamine neuron inhibit proliferation of Jurkat cell impled the reduced peripheral immune function in Parkinson’s disease, which could aggravate Parkinson’s disease progression.Here, we used an in vitro cells co-culture system to study the effect of conditioned medium from Sal and NMSal damaged co-cultures of SH-SY5 Y and U87 cells on Jurkat cells. We found conditioned medium from NMSal treated co-cultures of SH-SY5 Y and U87 cells activated m TOR signal pathway with elevated m RNA levels of IL-6R, CXCR4 and m TOR, as well as phospholated PI3 K, 4E-BP1, AKT, PI3 K and m TOR expression level. Furthermore, pharmacological inhibition of m TOR by rapamycin illustrated endogenous neurotoxin induced neruodegeneration caused proliferation of lymphocyte involving m TOR signal cascade. Similarly, conditioned medium from Sal treated co-cultured SH-SY5 Y and U87 cells induced lymphocyte proliferation with elevated m RNA levels of IL-6R, CCR2 and m TOR accompanied with activated m TOR signal pathway involved.4. In this paper, we studid the effect of endogenous neurotoxin Sal and NMSal damaged co-cultures of SH-SY5 Y and U87 cells on the human peripheral blood lymphocyte. And we found that Sal and NMSal treated co-cultures of SH-SY5 Y and U87 cells could induce proliferation of human peripheral blood lymphocyte through m TOR signal pathway.5. Here, we used an in vitro cells co-culture system to study the effect of conditioned medium from Sal and NMSal damaged co-cultures of SH-SY5 Y and U87 cells on THP1 cells. For this purpose, SH-SY5 Y and U87 cells co-cultures treated by Sal, the conditioned media containing mediators indicating CCL2 as the likely candidate were isolated and applied to THP1 cells. Such treatment resulted in approximately 19 % cell proliferation as well as activation of m TOR and induction of phosphorylated PI3 K and Akt proteins. Treatment of the THP1 cells with m TOR inhibitor rapamycin attenuated proliferation induced by the conditioned medium. A role for glioma U87 cells was established as conditioned medium without it had no effect on THP1 cells. These results suggested positive effects of THP1 cells in endogenous neurotoxin Sal induced toxicity in a cellular model of PD that is likely mediated by enhancement of cell proliferation markers through activation of m TOR signal pathway. Hence, PBMC and its m TOR signal pathway could be of therapeutic benefit in endogenous neurotoxin induced neuroinflammation in PD.SH-SY5 Y and U87 cells co-cultures treated by Sal, the conditioned media were isolated and applied to THP1 cells. The results showed that co-cultures of SH-SY5 Y and U87 cells decreased apoptosis of THP1 cells mediated by endogenous neurotoxin NMSal,. We found co-cultures of SH-SY5 Y and U87 cells inhibit the swelling and rupture of mitochondrial, deduced the production of malondialdehyde(MDA) and H2O2 which play critical roles in mitochondrial dysfunction and free radical mediated cell death and decreased expression level of apoptosis related proteins of caspase 3, Bax and FADD of THP1 cells induced by NMSal. Collectively, these studies suggested that human monocyte THP1 cells were sensitive to endogenous neurotoxin NMSal, while co-cultures of SH-SY5 Y and U87 cells could enhance immune ability of THP1 cells to resistant to the toxicity of NMSal. All of the results showed the necessity and importance of U87 cells in the protection of THP1 cells in co-cultures system; furthermore, the interaction of SH-SY5 Y and U87 cells could recover the immune function of THP1 cells and in turn to protect the neuroblastoma SH-SY5 Y cells from endogenous neurotoxin NMSal. These indicated that the interaction of neurons with astrocyte and peripheral blood mononuclear cells(PBMC) play a very important role in the pathogenesis of PD.6. In this study, we used endogenous neurotoxins NMSal right side nigra position injected Wistar male rats PD model to study the effect of the damaged dopaminergic neurons to the peripheral blood lymphocyte in vivo. Firstly, we checked whether NMSal injected rat model could induce the symptom of PD, and the results showed that NMSal induced apoptosis of dopaminergic neurons and aggregation of α-syn in SNpc in rat; and Appleton morphine could induce NMSal injected rat rotation. All of these results indicated that NMSal could induce PD symptom of Wistar male rat.Secondly, we investigated the peripheral blood lymphocyte of PD rat model induced by NMSal. And we found that CXCL12, IL-6 and TNF-α cytokines in peripheral blood serum of NMSal injected rat was increased compared to PBS injected rat; and we also found that the percent of CD3+CD4+, lymphocyte and the percent of Th2 and CD3+TCRvβ8.2/8.4 T-lymphocyte was increased in NMSal injected rat compared to PBS injected rat, while the percent of CD3+CD8+ T-lymphocyte was not changed; furthermore, we found that both the CD3+CD4+ and CD3+CD8+ T-peripheral blood lymphocyte proliferated through m TOR signal pathway in NMSal injected rat compared to PBS injected rat.In this study, we found that the neurotoxin damaged dopaminergic neurons is closely related to the function of peripheral immune cells, endogenous neurotoxin Sal and NMSal can induce aggregation of alpha-syn protein in SH-SY5Y-EGFP cells in SH-SY5Y-EGFP, U87 and Jurkat/HPBL mixed cells co-culture system. Endogenous neurotoxin Sal damage SH-SY5 Y and U87 cells co-culture system released inflammatory factor of IL-6 and the CCL2 to cells co-cultures conditioned medium, which induced proliferation of THP1(human monocytes cell line)/Jurkat(human CD4+ T lymphocyte cell line)/HPBL(human peripheral blood lymphocyte); Endogenous neurotoxin NMSal damage SH-SY5 Y and U87 cells co-culture system released inflammatory factor of IL-6 and CXCL12 to cells co-culture conditioned medium, thereby induced proliferation of Jurkat(human CD4+ T lymphocyte cell line)/HPBL(human peripheral blood lymphocyte), at the same time it could reduce toxicity of NMSal on THP1(human monocytes cell line); While neurotoxin MPP+ damaged SH-SY5 Y and U87 cells co-culture conditioned medium had different effect on Jurkat cells compared to Sal and NMSal, high concentration of neurotoxin MPP+(500 μM) damaged SH SY5 Y and U87 cell co-culture conditioned medium can lead to necrosis of Jurkat, low concentration MPP+(100 μM) damaged SH-SY5 Y and U87 cells co-culture conditioned medium can lead to Jurkat G2/M phase of the cell cycle block. Experimental results suggested Sal and NMSal treated conditioned co-culture medium could enhance immune function of mononuclear cells and lymphocytes, thus to induce the degeneration of dopaminergic neurons and accelarate the onset and development of PD; And MPP+ damaged SH-SY5 Y and U87 cells co-culture of conditioned medium could reduce the immune function of Jurkat cells(human CD4+ T lymphocyte cell line), and further accelerate the progression of PD.This study provides a way to study the interaction of neuron toxins damaged dopaminergic neurons and peripheral blood immune system.