Neuroprotection of recombinant arginine deiminase (rADI) in a neurons and microglia co-culture system

一氧化氮 (nitric oxide, NO●) 在人體中具有雙重功能，既是重要的訊息調控因子，也涉及許多病理機轉。特別是誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 過度表現所產生大量的NO● 與許多神經疾病有關，例如：帕金森氏症、阿茲海默症、和腦缺氧所導致的神經損害等。 精氨酸脫亞氨酶 (arginine deiminase, ADI) 是一種具有催化精氨酸 (L-arginine, L-arg) 分解為瓜氨酸 (L-citrulline, L-cit) 能力的蛋白質，此一蛋白僅表現於多種微生物內，而未見於哺乳類細胞中，且ADI已經被證實可以抑制iNOS所產生的NO●。因此，為了解ADI在iNOS所造成的神經毒性中所扮演的角色，我們利用BV2 (大鼠小神經膠質細胞株) 和SHSY5Y (人類神經母細胞瘤) 建立了小神經膠質細胞和神經細胞的共同培養系統，而在此共同培養系統中，加入2 μg/mL 的脂多醣 (lipopolysaccharide, LPS) 和1 ng/mL的γ-干擾素 (interferon-γ, IFN-γ) 兩天，成功地引起神經細胞死亡。利用此實驗模式，加入1 mU/mL的以基因合成的ADI (recombinant ADI, rADI) 於共同培養系統中持續二天到三天後，分別使用MTT assay和Griess method測定該培養系統中細胞存活率和NO● 產量。另外，進行神經細胞免疫染色和同位素標定多巴胺攝取實驗，分別作為專一地測定神經細胞存活率和神經功能。 結果發現在第二天時，和起始細胞的MTT數值相比，在只有投與LPS/IFN-γ的細胞中，細胞存活率僅剩21.1±4.1 %；而合併使用rADI後，細胞存活率則維持在89.2±2.2 %。 並且合併使用rADI也讓LPS/IFN-γ刺激所產生的NO● 從67.0±1.3 μM減少為19.5±5.5 μM。神經細胞免疫染色的結果清楚顯示，rADI確實具有神經細胞保護作用。在多巴胺的攝取功能上，與起始細胞相比，在只有投與LPS/IFN-γ的細胞中減少到35.7±11.4%，合併使用rADI的細胞則維持在103.0±12.6 %。另一方面，我們發現在第四天時，連續投與三天的rADI比只投與二天具有更好的保護效果，從MTT assay的數值上來看，投與三天的rADI可以保持76.8±11.7 %的細胞存活率，而投與二天的只有42.7±2.0 %；從多巴胺的攝取功能來看，連續投與三天與二天的rADI分別為75.1±10.8%和34.5±10.5 %。 為了解rADI的神經保護效果機轉，在rADI加入共同培養系統後，補充L-arg到培養液中；結果顯示L-arg的補充，可使rADI的神經保護效果完全消失。接著，我們將1400W (iNOS選擇性抑制劑) 和vinyl-L-NIO (nNOS選擇性抑制劑) 分別加入共同培養系統中，發現1400W可以有效的增加細胞存活率和抑制NO●產生，其EC50及IC50分別為2.3 μM 和5.7 μM，但是加入vinyl-L-NIO卻沒有這樣的現象。從以上結果來看，在此共同培養系統中，rADI對神經細胞之保護機轉可能是抑制過多由iNOS所產生的NO● 而非nNOS。 綜合上述結果，本研究之結論為：在此共培養系統中，rADI可以保護神經細胞免於LPS/IFN-γ所引起的神經毒性，不只是增加細胞的存活率，也維持神經細胞的功能。並且，rADI的保護機轉可能是透過消耗L-arg後，抑制iNOS產生NO●，達到神經保護的效果。為評估rADI在iNOS所導致的神經疾病是否具有臨床治療之可能性，未來可利用初始細胞 (primary cell) 及人工腦缺氧小鼠(ischemic mice)實驗，證實rADI是否在其他細胞及動物中具有相似的效果。Nitric oxide (NO●) plays double-edged roles in human, not only important in physiological functions but also involved in many pathological pathways. Particularly, the overproduction of NO● generated by inducible nitric oxide synthase (iNOS) is associated with many neuronal disorders such as Parkinson’s disease, Alzheimer’s disease, and cerebral ischemic neuronal damage, etc. Arginine deiminase (rADI), expressed in some microorganisms but not in mammalian cells, can catalyze L-arginine (L-arg) to L-citrulline (L-cit). ADI has been reported the inhibitory activity of iNOS-mediated NO●production. A co-culture of BV2 (a murine microglial cell line) and SHSY5Y (a human neuroblastoma cell line) was established to understand the role of rADI in iNOS-mediated neurotoxicity. The co-culture was treated with the combination of 2 μg/mL of lipopolysaccharide (LPS) and 1 ng/mL of interferon-γ (IFN-γ) for 2 days, and successfully induced iNOS-mediated neuronal death. In the co-culture system, 1 mU/mL of purified recombinant ADI (rADI) was administrated for 2 and 3 days into the co-culture with LPS/IFN-γ treatment. The cell viability and NO● production were measured by MTT assay and Griess method, respectively. In addition, the specific neuronal viability and functionality were analyzed by neuron-specific immunostaining and 3H-dopamine uptake assay, respectively. The results showed that rADI treatment significantly preserved the cell viability (89.2±2.2 % of the initial cells) and decreased the NO● production (19.5±5.5 μM) on day 2, compared with the cells with LPS/IFN-γ treatment only (21.1±4.1 % and 67.0±1.3 μM, respectively) by MTT assay and Griess assay. Furthermore, the results of immunostaining showed that rADI treatment substantially preserved the neurons, and the dopamine uptake function was also maintained on day 2 (from 35.7±11.4% to 103.0±12.6 % of the initial cells) by rADI treatment. In addition, the neuroprotection of 3-day rADI treatment was observed better than 2-day treatment on day 4 by MTT assay (76.8±11.7 and 42.7±2.0 % of the initial cells, respectively), and dopamine uptake assay (75.1±10.8 and 34.5±10.5 % of the initial cells, respectively). To understand the possible neuroprotection mechanism of rADI treatment, replenishment of L-arg into the co-culture system with rADI treatment was conducted. Besides, the treatments of 1400W (a selective iNOS inhibitor) and vinyl-L-NIO (a selective neuronal NOS (nNOS) inhibitor) in the co-culture system were also performed. The results showed that the replenishment of L-arg abolished the neuroprotective and NO● suppressive effect of rADI in the co-culture system. In addition, the treatment 1400W preserved the cell viability (EC50=2.3 μM) and inhibited NO● production (IC50=5.7 μM), but vinyl-L-NIO did not. The results indicated that the neuroprotective mechanism of rADI is via L-arg depletion which inhibits the NO● production produced by iNOS, but not nNOS. According to the accumulative results, the conclusions are that rADI can protect the neurons from LPS/IFN-γ induced neurotoxicity in the co-culture system. rADI not only preserves the neuron viability but also maintains the functionality. The protection mechanism of rADI may be via depletion of L-arg and subsequently inhibits the iNOS mediated NO● production. To evaluate the therapeutic role of rADI in iNOS mediated neuronal disorders, further investigations in the neuroprotection of rADI in primary cell and ischemic mice model are needed in the future.致謝 I 中文摘要 II ABSTRACT IV INDEX VI LIST OF FIGURES XI LIST OF TABLES XIII LIST OF SCHEMES XIV ABBREVIATIONS XV 1 INTRODUCTION 1 1.1 Nitric oxide (NO●) 1 1.2 Different isoforms of NOS 2 1.3 The role of NO● in neuronal disease 3 1.3.1 The different roles of NOS isoforms in neuronal diseases 5 1.3.2 iNOS mediated NO● in glial cells and neuronal diseases 6 1.4 Selective NOS inhibitors and NO● mediated neuronal diseases 7 1.5 Recombinant arginine deiminase (rADI) 9 1.5.1 Anti-tumoral activity of rADI 9 1.5.2 Suppression of NO● production by rADI 10 1.5.3 rADI and iNOS mediated neurotoxicity 11 2. HYPOTHESIS AND SPECIFIC AIMS 12 3. MATERIALS AND METHODS 13 3.1 Materials 13 3.2 Cell culture 14 3.3 Preparation of rADI 15 3.3.1 Clone, Expression, refolding, and purification of rADI 15 3.3.1.1 Transformation of expression vector into E. coli 15 3.3.1.2 Expression and refolding 16 3.3.1.3 Purification 17 3.3.2 rADI enzymatic activity assay 18 3.4 Effect of rADI in SHSY5Y and BV2 cultures 19 3.4.1 Cell viability of SHSY5Y culture treated with rADI 19 3.4.2 Cell viability of BV2 treated with rADI 19 3.4.3 Effect of cultured medium refreshment in SHSY5Y and BV2 cultures treated with rADI 19 3.4.4 AS protein expressions in SHSY5Y and BV2 cells 20 3.5 Induction of iNOS expression in BV2 20 3.5.1 Cell viability and NO● production of BV2 treated with variant doses of LPS 20 3.5.2 Cell viability and NO● production of BV2 treated with IFN-γ alone or combination of LPS and IFN-γ 21 3.5.3 Cell viability and NO● production of BV2 treated with variant doses of IFN-γ combined with LPS 22 3.6 Effect of treatment of LPS/IFN-γ in the SHSY5Y-BV2 co-culture 22 3.6.1 Cell viability and NO● production of BV2 treated with LPS/IFN-γ followed with refreshing the cultured medium 22 3.6.2 Cell viability and NO● production of SHSY5Y mono-culture treated with LPS/IFN-γ 23 3.6.3 Cell viability and NO● production of co-culture of SHSY5Y and BV2 treated with LPS/IFN-γ 23 3.7 Effect of rADI in the co-culture system 24 3.7.1 Cell viability and NO● production of BV2 treated with LPS/IFN-γ combined with rADI at different time point 24 3.7.2 Cell viability and NO● production of co-culture of SHSY5Y and BV2 co-treated with LPS/IFN-γ and rADI 24 3.7.3 Cell viability and NO● production of BV2 co-treated with LPS/IFN-γ and rADI 25 3.7.4 iNOS protein expression in BV2 co-treated with LPS/IFN-γ and rADI 25 3.7.5 Prolonged treatment of rADI for 3 days in the co-culture system 26 3.7.6 Immunocytochemistry (ICC) of cells co-treated with LPS/IFN-γ and rADI in the co-culture system 26 3.7.7 Dopamine uptake assay of cells co-treated with LPS/IFN-γ and rADI in the co-culture 27 3.7.8 Neuroprotective effects of rADI treatment at different start time in the co-culture system 27 3.8 Mechanism studies of the neuroprotective effect of rADI in the co-culture system 28 3.8.1 Replenishment of L-arg in the co-culture co-treated with LPS/IFN-γ and rADI 28 3.8.2 Treatment of 1400W in the co-culture system 28 3.8.3 Treatment of vinyl L-NIO in the co-culture system 29 3.9 Basic protocols 30 3.9.1 Cell counting 30 3.9.2 MTT assay 30 3.9.3 Agarose gel electrophoresis 31 3.9.4 BCA assay 32 3.9.5 Griess method 32 3.9.6 Cells lysis with RIPA buffer 33 3.9.7 Western blotting 33 3.9.7.1 SDS-PAGE 34 3.9.7.2 Coomassie Brilliant Blue staining (not for Western blotting) 35 3.9.7.3 Transfer 36 3.9.7.4 Immunodetection 36 3.9.8 Immunocytochemistry (ICC) 38 3.9.8.1 Fixation of cells 38 3.9.8.2 Immunostaining 38 3.9.9 Calculation of neuronal viability by Image Pro-Plus 4.5.1.22 39 3.9.9.1 Image taking 40 3.9.9.2 Image analysis with Image Pro Plus 4.5.1.22 40 3.9.10 Dopamine uptake assay 41 3.10 Recipes of reagents and solutions 42 3.11 Statistical analysis 45 4 RESULTS 46 4.1 Characters of the expressed and purified rADI 46 4.1.1 DNA sequence of the transformed rADI cDNA 46 4.1.2 Enzymatic activity of the purified rADI 46 4.2 Effect of rADI in SHSY5Y and BV2 cultures 47 4.2.1 Effect of rADI in SHSY5Y and BV2 cultures 47 4.2.2 Absence of AS expression in SHSY5Y and BV2 48 4.2.3 Recovery cell proliferation of SHSY5Y and BV2 from the rADI-mediated cell viability inhibition 49 4.3 Neurotoxicity mediated by NO● over-production in the co-culture system 49 4.3.1 Expression of iNOS induced by combination of LPS and IFN-γ in BV2 50 4.3.1.1 Determination of optimal dose of LPS 50 4.3.1.2 Combination of LPS and IFN-γ 50 4.3.1.3 Determination of optimal dose of IFN-γ 50 4.3.2 Prolonged effect of iNOS in BV2 treated with LPS/IFN-γ 51 4.3.3 Absence of NO● production in SHSY5Y cultured alone treated with LPS/IFN-γ 52 4.3.4 Neuronal death induced by over-expression of iNOS in the co-culture system 52 4.4 Neuroprotective effect of rADI in the co-culture system 53 4.4.1 Determination of the timing of rADI treatment in suppression of NO● production induced by LPS/IFN-γ in BV2 54 4.4.2 Neuroprotection of rADI in the co-culture system 54 4.4.3 More neuroprotection with the longer treatment of rADI 56 4.4.3.1 MTT assay and Griess assay 56 4.4.3.2 Immunostaining assay 57 4.4.3.3 Dopamine uptake assay 57 4.4.4 Protective effect of rADI with different start time in the co-culture system 58 4.5 Mechanism studies of the neuroprotection of rADI in the co-culture system 59 4.5.1 Abolishment of neuroprotection of rADI by L-arg replenishment 59 4.5.2 Neuroprotection of 1400W in the co-culture system 60 4.5.3 Neuroprotection of vinyl-L-NIO in the co-culture system 61 5. DISCUSSION 62 5.1 Purification of rADI 62 5.2 Cytotoxicity of rADI in SHSY5Y and BV2 62 5.3 iNOS over-expression mediated neurotoxicity induced by the combination of LPS/IFN-γ in the co-culture 64 5.4 Neuroprotective ability of rADI in the co-culture system 68 5.5 Therapeutic application of rADI in iNOS-mediated neuronal diseases 76 6 CONCLUSIONS 78 7 PERSPECTIVES 80 8 REFERENCES 119 List of Figures Figure 1. NOS catalyzing synthesis of NO● 82 Figure 2. ADI catalyzing hydrolysis of L-arg 82 Figure 3. Chemistry of the Griess method 83 Figure 4. Composition of transfer sandwich. 84 Figure 5. Agarose gel electrophoresis of the plasmid extracted from the E. coli. 85 Figure 6. SDS-PAGE of the purified rADI. 86 Figure 7. Effect of rADI in SHSY5Y culture. 87 Figure 8. Effect of rADI in BV2 culture. 88 Figure 9. AS protein expression in SHSY5Y and BV2, treated or untreated with rADI. 89 Figure 10. Recovery of cell proliferation of SHSY5Y and BV2 after rADI removed. 90 Figure 11. Titration of LPS at low dose in the induction of cell death and NO● production in BV2 culture. 91 Figure 12. Titration of LPS doses with or without IFN-γ in the induction of cell death and NO● production in BV2. 92 Figure 13. Titration of IFN-γ doses with or without LPS in the induction of cell death and NO● production in BV2. 93 Figure 14. Titration of IFN-γ doses in the induction of cell death and NO● production in BV2 culture. 94 Figure 15. Cytotoxicity and NO● production after withdrawal of LPS/IFN-γ in BV2 95 Figure 16. Insusceptibility of SHSY5Y to LPS/IFN-γ. 96 Figure 17. Neuronal death induced by LPS/IFN-γ in the co-culture. 97 Figure 18. Images of the SHSY5Y and co-culture treated with LPS/IFN-γ. 98 Figure 19. Inhibition of NO● production by rADI treatment at different start time after LPS/IFN-γ treatment. 99 Figure 20. Preservation of cell viability by rADI in the co-culture system. 100 Figure 21. Recovery of LPS/IFN-γ induced NO● production of after rADI-withdrawal on day 3 in BV2. 101 Figure 22. The effect of rADI treatment in iNOS protein expression in the co-culture system 102 Figure 23. Neuroprotection of 3 days and 2 days of treatment of rADI in the system. 103 Figure 24. ICC of SHSY5Y and BV2 with anti-β-tubulin III 104 Figure 25. ICC of SHSY5Y in the co-culture system treated with rADI. 105 Figure 26. Quantification of the ICC images. 106 Figure 27. Dopamine uptake assay of the co-culture system treated with rADI. 107 Figure 28. Different protection and NO● inhibition of rADI treatment with different start time. 108 Figure 29. Effect of restoration of L-arg on the neuroprotection of rADI. 109 Figure 30. Effect of 1400W in the co-culture system 110 Figure 31. Effect of Vinyl-L-NIO in the co-culture system 111 List of Tables Table 1. Comparison of three families of NOS 112 Table 2. The time courses of the expression of NOS isoforms in cerebral ischemia. 113 Table 3. Comparison of NOS inhibitors by in vitro IC50 (μM) 114 Table 4. Preparation of L-cit standard 114 List of Schemes Scheme 1. The mechanism of NO● induced cytotoxicity. 115 Scheme 2. The cascade of events occurs in cerebral ischemia and the roles of each NOS isoform. 116 Scheme 3. Accessibility of iNOS an eNOS to different L-arg-pools 117 Scheme 4. The protection of rADI in the neurons-microglia co-cultures system. 11