中草藥抑制細胞興奮性毒殺模式以治療第十七型脊髓小腦共濟失調症

全文

(1)國立臺灣師範大學生命科學系碩士論文. 中草藥抑制細胞興奮性毒殺模式以治療第十七型脊髓小腦共濟失調症 Chinese herbal medicines for the treatment of spinocerebellar ataxia type 17 via inhibition of excitotoxicity. 研究生：徐銘亨 Ming-Heng Hsu. 指導教授：吳忠信博士 Chung-Hsin Wu, Ph.D. 林榮耀博士 Jung-Yaw Lin, Ph.D.. 中華民國 102 年 7 月.

(2) 誌謝很快的，研究生兩年的生活轉眼即逝，在師大以及台大實驗室的生活，每天都過著很充實、豐富的生活，而此碩士論文的完成，也要很謝謝所有幫助過我的人，不管是實驗上的指導，實驗室規範的提醒，以及心靈上的加油打氣。. 本研究的完成，首先要感謝指導教授吳忠信老師以及林榮耀老師，在學術論文上耐心的指導，給予我鼓勵以及諄諄教誨，總是不斷地在旁給予提點，使得整篇論文的研究更臻於完整，也帶領著學生不斷地努力向前邁進，使我在這兩年期間有很大的收穫，並且得以完成此篇論文。感謝李桂楨老師所提供的小腦脊髓萎縮症第十七型的細胞，以及謝秀梅老師所提供的小腦脊髓萎縮症第十七型的小鼠，讓我的實驗針對小腦脊髓萎縮症此疾病的研究可以更加的完善，也感謝鄭劍廷老師提供自由基偵測儀以及在我實驗上所有給予的指導。. 在研究生兩年的過程中，也要感謝很多實驗室貴人的幫忙。尤其感謝玄原學長以及頂翔學長的幫助，在實驗技術上耐心的指導、實驗想法的提供、論文的訂正等等，真的很感謝兩位學長的提攜。感謝在. 2.

(3) 台大實驗室給我幫助的琮祐學長，同屆研究生戰友綱緒、賀培彼此互相鼓勵以及打氣，實驗室學弟妹柏劭、瑀萱你們的陪伴與幫助；感謝在師大實驗室的雪娥學姊幫我做的組織石蠟塊、佳音學姊對於動物實驗的指導、淳任學長、清隆學長、珍妏學姊、世彬學長、毓國學姊、柏均學長、志翔學長的幫忙，同屆研究生天宇以及鈺埔一起互相勉勵，學弟妹宜珈以及蕍甄的陪伴；感謝鄰近實驗室品豪、俊超以及玥希在論文完成期間給予的加油與打氣，也感謝韻如不斷地給我支持與鼓勵，讓我可以努力的堅持不懈到最後一刻把此篇論文完成。. 最後，由衷的感謝我最愛的父母以及家人們，謝謝您們在這一路上對我無私的付出以及包容，讓我可以順利地在兩年準時完成我的碩士論文，感謝您們！. 3.

(4) 目錄. 目錄................................................................................................................................. I 摘要............................................................................................................................... III Abstract .........................................................................................................................V Figures .........................................................................................................................VII 1. Introduction .......................................................................................................... 1 2. Research aim ........................................................................................................ 4 3. Materials and Methods ........................................................................................ 5 3.1. Materials ................................................................................................. 5 3.2. Cell culture .............................................................................................. 6 3.3. Preparation of Chinese herb medicines................................................... 6 3.4. MTT assay .............................................................................................. 7 3.5. LDH assay............................................................................................... 8 3.6. Western blotting analysis ........................................................................ 8 3.7. Cell apoptotic analysis .......................................................................... 14 3.8. Reactive oxygen species (ROS) analysis .............................................. 15 3.9. SCA17 mice rotarod test ....................................................................... 15 3.10. SCA17 mice footprinting .................................................................... 16 3.11. Acetylcholinesterase (AChE) activity assay ....................................... 17 4.. Results ................................................................................................................. 18 4.1. CHM NH018 rescues SH-SY5Y cell viability after MSG treatment ...... 18 4.2.. Active compound, NH018-1 of NH018 increased SH-SY5Y cell viability after MSG treatment ................................................................................ 18. 4.3.. Effects of NH018-1 on the release of lactate dehydrogenase (LDH) from SH-SY5Y cells treated with MSG ........................................................... 19. 4.4.. Effects of NH018-1 on Bcl-2, Bax and cytochrome C release for SH-SY5Y cells treated with MSG ........................................................... 19. 4.5.. NH018-1 attenuated MSG-induced the activation of Caspase-9, Caspase-3, and PARP expression in SH-SY5Y cells ............................... 20. 4.6.. NH018-1 reduced MSG-induced the activation of Calpain-2 and Calpain specific-SBDP expression in SH-SY5Y cells .......................................... 21. 4.7.. Effects of NH018-1 on MSG-induced phosphatidylserine (PS) externalization and apoptotic induction in SH-SY5Y cells ..................... 22 I.

(5) 4.8.. NH018-1 inhibited the ROS production induced by MSG treatment in SH-SY5Y cells ......................................................................................... 22. 4.9.. Effects of CHMs and NH018-1 on cell viability of DOX induced SCA17 (79Q) cell model ...................................................................................... 23. 4.10.. NH018-1 attenuated nTBP-EGFP(79Q)-induced activated Caspase-9, Caspase-3, and PARP expression in SCA17 cells ................................... 23. 4.11.. Effects of NH018-1 on body weight changes and rotarod performance in SCA17 mice model .................................................................................. 24 Effects of NH018-1 on SCA17 mice footprinting ................................... 25. 4.12. 4.13.. NH018-1 attenuated SCA17 mice-induced activated TBP (N12), Calapin-2, and cleaved-Caspase-3 expression ......................................... 25. 4.14.. Effects of NH018-1 on acetylcholinesterase (AChE) activity of cerebrum and cerebellum in SCA17 mice ............................................................... 26. 5.. Discussion............................................................................................................ 28. 6. 7.. References ........................................................................................................... 33 Figures ................................................................................................................. 46. II.

(6) 摘要神經退化性疾病 (neurodegeneration diseases) 當中存在著由麩醯胺酸活化的興奮性毒殺 (excitotoxicity) ，如多麩醯胺酸疾病 (polyglutamine diseases) 、阿茲海默氏症疾病. (Alzheimer’s. diseases) 和柏金森氏症疾病 (Parkinson’s disease) ，會導致過多的鈣離子流入、粒線體內細胞色素 C 的釋放、導致神經細胞凋亡的蛋白質活化，最後使細胞存活率的下降。許多研究報導指出，中草藥的使用，可能是一個治療退化性疾病新方法。因此，我們研究在人類神經母細胞 (neuroblastoma) SH-SY5Y 細胞以麩醯胺酸 (glutamate) 誘導的興奮性毒殺以及使用多西環素 (doxycycline) 誘導出小腦脊髓萎縮症第十七型 (Spinocerebellar. ataxia,. SCA17). nTBP/Q79-EGFP. 細胞中. TBP/79Q-EGFP 融合蛋白的表現後，探討中草藥是否有保護神經細胞的作用。加入麩醯胺酸後導致神經細胞生存率下降，另一項實驗結果顯示 NH018 和其純化物 NH018-1 可能為有效的藥物可以增加神經細胞的存活率、 NH018-1 可以抑制神經細胞的凋亡 (apoptosis) 、降低乳酸脫氫酶 (LDH) 的釋放以及自由基的生成，並且降低鈣離子所造成的 Calpain-2 及 α-spectrin breakdown products (SBDPs) 的表現，降低從粒線體釋放出的細胞色素 C、增加細胞存活相關蛋白質： Bcl-2、 Bax 及降低凋亡相關：蛋白質. cleaved-caspase-9 、. cleaved-caspase-3 和 cleaved-PARP 等的表現。在誘導 TBP/79Q-EGFP 融合蛋白表現的細胞上，NH018-1 為有效的藥物可以增加細胞的存活率以及降低 III. cleaved-caspase-9 、.

(7) cleaved-caspase-3 和 cleaved-PARP 等的表現。而施打 NH018-1 藥物在 SCA17 的基因轉殖鼠上，能夠有效地延長其在滾輪 (rotarod) 上面跑的時間、步行實驗 (footprint) 上的好轉，在其運動失調的症狀上發揮療效，以及降低 TATA box-binding protein (TBP) 、 Calpain-2 和 cleaved-caspase-3 的表現。總合以上，實驗結果顯示出 NH018 和 NH018-1 藥物在加入麩氨酸以及誘導出小腦脊髓萎縮症第十七型細胞 TBP/79Q-EGFP 融合蛋白的表現後，藉由抑制細胞的凋亡可以增加 SH-SY5Y 細胞以及小腦脊髓萎縮症第十七型細胞的存活率，因此 NH018 和 NH018-1 可能為有潛力治療小腦脊髓萎縮症第十七型疾病的藥物。. 關鍵字：神經退化性疾病，興奮性毒殺，麩氨酸，小腦脊髓萎縮症第十七型，細胞凋亡。. IV.

(8) Abstract Excitotoxicity induced neurodegeneration, and polyglutamine (polyQ) diseases, such as Alzheimer’s disease, and Parkinson’s disease, via glutamatergic activation, resulting in excessive calcium influx, cytochrome C release from mytochondria, pro-apoptotic protein activation, and finally leads to decrease the cell viability. It was believed that Chinese herbal medicines (CHMs) might be one of new approaches to treat neurodegenerative diseases. Therefore, we investigated whether CHMs could protect neurons from monosodium glutamate (MSG)-induced excitotoxicity in human neuroblastoma SH-SY5Y cells and doxycycline (DOX) induced-nTBP-EGFP expression in spinocerebellar ataxia type 17 (SCA17) nTBP/Q79-EGFP cells. Our study showed that NH018 and its active compound NH018-1 were effective against MSG induced neurotoxicity by increasing the cell viability measured by MTT assay, and decreased the LDH release and the production of reactive oxygen species, and deduced cell apoptosis after MSG treatment in SH-SY5Y cells, DOX induced SCA17 nTBP/Q79-EGFP cells. NH018-1 also showed the remarkably protective activity against the neuronal cell death as demonstrated by : (1) reducing the Annexin V-FITC and Propidium Iodide (PI) staining, (2) the decrease of cytochrome C released from mitochondria, (3) the reduction of apoptosis-related proteins such as m-calpain (calpain-2), cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP expression, (4) the decrease of survival-related proteins such as Bcl-2, and (5) the V.

(9) improving of SCA17 mice performance in rotarod and footprint experiments. In conclusion, present studies showed that NH018 and NH018-1 protect cell viability after MSG treatment in SH-SY5Y cells, and DOX treatment in SCA17 nTBP/Q79-EGFP cells by inhibition of cell apoptosis. Furthermore, NH018-1 of NH018 could improve the SCA17 mice performance in rotarod test and footprint analysis as well as decrease the expression of TATA box-binding protein (TBP), calpain-2, and cleaved-caspase-3. Thus, NH018 and NH018-1 could be potential CHMs for the treatment neurodegenerative diseases.. KEYWORD: excitotoxicity, neurodegeneration, glutamate, spinocerebellar ataxia type 17, apoptosis.. VI.

(10) Figures Figure 1. Effects of CHMs on MSG-induced cell death in SH-SY5Y cells .................................................................................................... 46 Figure 2. NH018-1 decreased the glutamate-induced cell death in SH-SY5Y cells. .................................................................................. 47 Figure 3. NH018-1 inhibited the LDH release from SH-SY5Y cells induced by MSG. ............................................................................... 48 Figure 4. Effects of NH018-1 on Bcl-2 and Bax expression induced by MSG in SH-SY5Y cells...................................................................... 49 Figure 5. NH018-1 suppressed cytochrome C release from mitochondria to cytosol mediated MSG in SH-SY5Y cells. ............... 50 Figure 6. NH018-1 decreased the cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP expression induced by MSG in SH-SY5Y cells. .................................................................................. 51 Figure 7. NH018-1 suppressed Calpain-2 and SBDP expression induced by MSG in SH-SY5Y cells. .................................................. 52 Figure 8. NH018-1 reduced MSG-induced apoptosis in SH-SY5Y cells. ........................................................................................................... 53 Figure 9. NH018-1 suppressed MSG-induced ROS production. ........ 54 Figure 10. Effects of three CHMs and NH018-1 on DOX-induced SCA17 (79Q) cell model .................................................................... 55 Figure 11. Effects of NH018-1 on cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP expression after MSG exposure in SCA17 cells ................................................................................... 56 VII.

(11) Figure 12. Effects of NH018-1 on body weight and motor performance in SCA17 transgenic (TG) mice ......................................................... 57 Figure 13. NH018-1 improved gait abnormalities .............................. 59 Figure 14. Effects of NH018-1 on TBP (N12), Calpain-2, and cleaved-Caspase-3 expression in SCA17 transgenic mice .................. 60 Figure 15. Effects of NH018-1 on cerebrum and cerebellum AChE activity in SCA17 mice ...................................................................... 61 Table 1. IC50- of nine CHMs studied. ................................................. 62 Table 2. IC50-cytotoxicity of nine compounds studied. ....................... 62 Supplementary figure 1. Effects of nine compounds on MSG-induced cell death in SH-SY5Y cells. ..................... 63. VIII.

(12) 1. Introduction. Glutamate, primarily an excitatory neurotransmitter, could act as an excitotoxin when highly accumulating in brain regions. Cysteine uptake inhibits by glutamate, then subsequently leads to consumption of glutathione levels 19 which may increase production of reactive oxygen species (ROS) and elevate Ca2+ levels 44. The numerous ROS levels increased may cause a variety of pathological processes, including neurodegenerative diseases 45. The high level of glutamate is related to several neurodegenerative diseases, such as Spinocerebellar ataxias type (SCAs) 10, amyotrophic lateral sclerosis 41, 54, 57, Parkinson’s disease (PD) 5. , Huntington’s disease (HD) 82, and Alzheimer’s disease (AD) 22, 30. The. mechanisms overlying imply that excess activation of glutamate on ionotropic receptors such as N-Methyl-D-aspartate (NMDA), 2-amino-3-(3-hydroxy-5-methyl-wasoxazol-4-yl) propanoic acid (AMPA), and Kainate receptor, as well as metabotropic receptors like metabotropic glutamate receptor 49 causing intracellular Ca2+ overloading, which in turn results in lossing of ion homeostasis, ATP levels depletion, and excitotoxic apoptosis 2, 8, 63, 64, 71, 74.. In addition, higher intracellular concentration of Ca2+ may induce mitochondrial apoptotic pathway, such as inhibition of anti-apoptotic protein (such as Bcl-2 or Bcl-xL), induction of pro-apoptotic protein (like Bax, Bim and Bid) 16, 21, 53, 67, 79, and release of cytochrome C from 1.

(13) mitochondria 9, 17, 38, 42, 67, 72 , leading to the activation of downstream caspase-9 and caspase-3. Then, the nuclear enzyme poly (ADP-ribose) polymerase (PARP) is cleaved by activated caspase-3, and cleaved PARP is considered as one of the apoptotic marker 1.. It was also shown that overexpression of glutamate receptors cause Ca2+ influx, activation of calpain (a cytosolic Ca2+-dependent protease), and then neuron cells death 7, 38, 42, 56. Calpain cleaves different substrates, including compositions of cytoskeletal proteins (such as α-spectrin), elements of Ca2+ transport machinery (such as glutamate/NMDA, AMPA receptors, and the Ca2+-ATPase of the plasma membrane) 52, 68, 76, 80, 81, enzymes, transcription factors, that induce morphological alterations and apoptosis 18, 75.. In this study, neuroblastoma SH-SY5Y cell line was used to establish excitotoxicity model and SCA17 disease mice models. SCA17 is caused by polyglutamine expansion (>43 CAG repeat) located at the N-terminus of TATA box-binding protein (TBP) in affected neuron cells 37, 50. . In SCA17, the main symptoms are ataxia, dementia, and involuntary. movements, containing dystonia and chorea 61. MRI findings reveal cerebral and cerebellar atrophy 46. It is also expressed as psychiatric symptoms, pyramidal signs, and rigidity with the incidence ranging are from age 3 to 75 year-old 58. Despite courses of SCA17 are variable, the involuntary movement, dementia, parkinsonism, and pyramidal signs ataxia and psychiatric abnormalities are frequently the initial indications. 2.

(14) Many efforts, such as SCA17 knock-in mouse model 29 and transgenic mouse models 12, 24 had been put into SCA17. Recent study showed that the diffusion tensor imaging could provide a biomarker for SCA17 patients from SCA17 animal model 33. Although much attention had been drawn to SCAs, they are still incurable.. Current approaches Chinese herbal medicines (CHMs) for identifying potential neuroprotective drugs, such as Toki-shakuyaku-san for the treatment of AD 23, Tianqi Pingchan Granule for PD 78, and Ginseng for HD 77. Active principles of CHMs, such as Tanshinone IIA 40, Paeoniflorin 65, Gua Lou Gui Zhi decoction exerts 28, and schizandrin 39 exert neuroprotective effects via inhibition of glutamate-induced excitotoxicity. It is believed that the potential neuroprotective effects of CHMs may be the trend in the future. Accordingly, this study aimed to screen CHMs and develop effective principles targeting SCA17-related glutamatergic neurotoxicity.. 3.

(15) 2. Research aim. Present study aims to find potential CHMs to treat and improve the symptoms of SCA17. Three steps were set up: (1) To establish the SH-SY5Y cell model to study whether CHMs or pure compounds which can rescue the neurotoxicity mediated by glutamate, (2) To investigate the effects of CHMs and pure compounds on inducible SCA17 cell model, and (3) to study the effects of CHMs and compounds obtained from the first step on the performance SCA17 transgenic mouse model. Taken together, it is expected that potential drugs for the treatment of SCA17 disease will be developed.. 4.

(16) 3. Materials and Methods. 3.1. Materials Human neuroblastoma SH-SY5Y cells were from ATCC, USA. Dulbecco’s Modified Eagle Medium with nutrient mixture F-12 (DMEM/F12), 0.5% Trypsin-EDTA, penicillin/streptomycin (P/S), and Fluo-4 AM were obtained from Invitrogen Corporation. Fetal bovine serum (FBS) was from Falcon. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from SIGMA-Aldrich. Lactate dehydrogenase (LDH) Cytotoxicity Kit was from Promega, Annexin V - FITC Kit was from Bio Vision Corporation, and Acetylcholinesterase (AChE) Assay Kit was obtained from Abnova. Protease inhibitors cocktail were obtained from Roche Applied Science, Indianapolis, IN. CHMs were supplied by Sun-Ten Pharmaceutical Company, Taipei, Taiwan.. Primary antibodies against cytochrome C, Bcl-2, and TBP (N12) were purchased from Santa Cruz Biotechnology. Calpain-2, Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP were from Cell Signaling Technology. SBDP was obtained from Millipore. Secondary antibodies of Horse radish peroxidese (HRP)-conjugated goat anti-rabbit and goat anti-mouse were obtained from Minipore Corporation, Billerica, MA, USA. 5.

(17) The transgenic SCA17 mice were kindly provided by Dr. Hsiu-Mei Hsieh, National Taiwan Normal University. SCA17 nTBP/Q79-EGFP cell line was kindly supplied by Dr. Guey-Jen Lee-Chen, NTNU.. 3.2. Cell culture Human neuroblastoma SH-SY5Y cells and SCA17 nTBP/Q79-EGFP cells were routinely maintained in Dulbecco’s Modified Eagle Medium with nutrient mixture F-12 (DMEM/F12) supplemented with 10 % fetal bovine serum (FBS) and 10 U/ml penicillin/streptomycin (P/S). The cells were cultured at 37oC in a humidified atmosphere with 5 % CO2.. 3.3. Preparation of Chinese herb medicines The water extracts of CHMs were supplied by Sun-Ten Pharmaceutical Company. Each 100 g of CHMs and 1500 ml H2O were boiled for 30 min at 100oC, and then these water extracts were concentrated to 100 ml with an evaporator at room temperature. The extracts were prevented from light and stored at -40 oC.. 6.

(18) 3.4. MTT assay Cell viability was measured by using MTT assay. SH-SY5Y cells were plated in 96-well culture plates at a density of 3.0 x 104 cells/well for complete attachment at 37oC with 5 % CO2 for 24 hr. After the removal of culture medium, and cells were treated with 1/2X IC50, 1/5X IC50 CHMs or pure compounds co-treated with 80 mmole/L monosodium glutamate (MSG). NMDA antagonist, MK801 (10 μmole/L), was used as a positive control.. To investigate the effects of CHMs or compounds on SCA17 inducible cell line, SCA17 nTBP/Q79-EGFP cells were plated in 96-well culture plates at a density of 2.0 x 104 cells/well for 24 hr. Cells were treated with 5, 10, or 20 μg/mL DOX for 24, 48, and 72 hr to induce nTBP-EGFP (79Q) expression. Then, the cells were treated with 10 μg/mL DOX, the 1/2X IC50, 1/5X IC50 CHMs, active pure compound, or 10 μmole/L MK801 for 48 hr to investigate whether CHMs or pure compound can rescue the cell death.. After the removal of culture medium, 100 μl of MTT solution (0.5 g/L) was added and incubated at 37 oC for 3 hr. Then, 100 μl of 10% SDS-0.01N HCl solution was added into each well and incubated at 37 oC overnight to dissolve the formazan. The absorbance was measured at 570 nm with an ELISA reader (uQuant, bio-tek Inc., Vermont, USA). Results were expressed as the relative cell viability of treated-cells against that of 7.

(19) controls.. 3.5. LDH assay Lactate dehydrogenase (LDH) activity was measured with LDH Cytotoxicity Kit. SH-SY5Y cells were seeded in 24-well culture plates at a density of 1.0x105 cells/well at 37oC for 24 hr. After treating with 80 mmole/L MSG with either NH018-1 (2.5, 5, 10, 20, or 40 μmole/L) or 10 μmole/L MK801 for 24 hr, the 24-well culture plates were centrifugated at 250 X g at room temperature for 4 min, and the 50 μl of supernatants were transferred to fresh 96-well culture plates. Then, 50 μl of Reconstitute Substrate Mix was added and incubated at room temperature for 30 min. Finally, 50 μl of Stop Solution was added in each well and used an ELISA reader to measure the absorbance at 490 nm. The absorbance of control was normalized to 100 %, and the results were expressed as the relative absorbance of treated-cells against that of controls.. 3.6. Western blotting analysis 3.6.1. Preparation of cell culture and lysate SH-SY5Y cells were seeded in 60 mm culture plates at a density of 8.

(20) 1.2 x 106 cells for 24 hr. Each well was treated with 80 mmole/L MSG and 6 mg/ml NH018, 10 μmole/L NH018-1 or 10 μmole/L MK801 for 24 hr. Cells were washed with cold PBS and scraped into 40 μl RIPA buffer (50 mM Tris (pH8.0), 150 mM NaCl, 1 % NP-40, 1 % DOS, 0.1 % SDS, 2 mM EDTA, and protease inhibitors cocktail) and incubated on ice for 10 min. After sonication 10 times and centrifugation at 13,000 rpm at 4 o. C for 20 min, the supernatants were transferred to fresh eppendorf tubes. and stored at -20oC.. 3.6.2. Fractionation of mitochondria and cytosol to analyze cytochrome C SH-SY5Y cells were seeded in 100 mm dishes at a density of 2 x 106 cells for 24 hr. Each dish was treated with 80 mmole/L MSG and 6 mg/ml NH018, 10 μmole/L NH018-1 or 10 μmole/L MK801 for 24 hr. Cells were washed twice with ice-cold PBS, and 0.5 ml cytochrome C buffer (20 mM HEPES-KOH (pH7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 250 mM sucrose and protease inhibitors cocktail) was added to resuspend the cells. All of the samples were homogenized with 30 strokes and centrifuged at 500 X g at 4 oC for 10 min to remove nuclei and unbroken cells. The supernatants were transferred to fresh eppendorf tubes and centrifuged at 8000 X g at 4 oC for 10 min to separate the mitochondria and cytosol. The resulting pellet 9.

(21) containing mitochondrial fraction was resuspended in RIPA buffer, sonicated 10 times, and centrifuged at 13,000 rpm at 4 oC for 20 min. The supernatants were collected as cytosolic fraction.. 3.6.3. Quantification of protein concentrations All of the protein concentrations of cell lysates were measured with the Bicinchoninic acid assay (Pierce, Rockford, IL), which is based on the following two chemical reactions. In an alkaline solution, Cu2+ was reduced to Cu+ with protein and the level of Cu+ increased is proportional to the level of protein present. Then Cu+ could combine with bicinchoninic acid to form a purple-colored product, which strongly absorbs light at a 562 nm wavelength.. One μl of sample was diluted with 19 μl ddH2O in duplicate. A series of dilutions of known bovine serum albumin (BSA) concentration to get a standard curve and determine the protein concentration. The BCA working reagent was prepared by mixing reagent A and reagent B with 50:1 ratio (100 μl/well) and incubated at 37oC for 30 min. The absorbance prtoein samples and BSA standards were measured with ELISA reader at 562 nm absorbance.. 3.6.4. Sodium dodecyl sulfate-polyacrylamide gel 10.

(22) electrophoresis (SDS-PAGE). 3.6.4.1. Preparation of the protein samples Protein samples (40 μg) were mixed with 4X SDS-loading buffer, and were boiled at 95oC for 5 min to denature the protein, and then incubated on the ice for 5 min to prevent protein refolding.. 3.6.4.2. Preparation of the SDS-polyacrylamide gels The BIO-RAD system was used to perform SDS-PAGE. The desired concentration of acrylamide of the separating solution was poured into the gap between the glasses and overlaid with 0.1% SDS to prevent the air and flat the gel for 30 min. 0.1% SDS was removed after polymerization was complete, and then the stacking solution was poured onto the separating gel and put the appropriate comb into stacking gel for 30 min to agglomerate. The gel could be kept in a wet paper and saran wrap at 4 oC.. Components Concentration Solution A Solution B. 10 % 2.0 ml 1.5 ml. Separating gel 12.5 % 2.5 ml 1.5 ml. 11. Staking gel 15 % 3.0 ml 1.5 ml. 0.45 ml 一一.

(23) Solution C ddH2O 10% APS TEMED Solution A Solution B Solution C. 0.75 ml. 一一一一一一 2.5 ml. 2.0 ml 60 μl 9.0 μl. 1.5 ml. 1.80 ml 26 μl 4.5 μl. 30 % Acrylamide and 0.8 % Bisacrylamide 1.5 M Tris-HCl, pH 8.0 and 0.4% SDS 1.5 M Tris-HCl, pH 6.8 and 0.4% SDS. 3.6.4.3. Procedure of polyacrylamide gel electrophoresis The comb was removed from the stacking gel after it agglomerated, the SDS-polyacrylamide gel was placed into the BIO-RAD electrophoresis tank and poured 1X running buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine and 0.1 % SDS) into the tank. The prestained protein ladder as the marker and the denatured protein and 4X loading buffer mixed samples were loaded into the wells. The BIO-RAD system was linked to a power supply (80 V) until the marker dye reached the bottom of the separating gel. After the dye reached the end, the power supply was shutted down and the protein could be started to transfer.. 3.6.4.4. Procedure of Semi-Dry transfer system The 3 MM papers were cut to fit the size of SDS-polyacrylamide gel (7 cm X 9 cm), and polyvinylidence fluoride (PVDF) membrane was cut to 6.5 cm X 9.6 cm. One piece of paper was wet in Anode I buffer, 12.

(24) two pieces of paper were wet in Anode II buffer, three pieces of paper were wet in Cathode buffer, and PVDF membrane was wet in 100 % methanol. The SDS-polyacrylamide gel was placed between in the 3 MM papers and PVDF and removed all of the air bubbles to ensure transfer successful. Two kg object was put on the semi-dry transfer system and linked to a power supply (30 V, 70 mA) for 80 min.. Anode I Anode II Cathode. 0.3 M Tris, pH 10.4 and 10 % methanol 25 mM Tris, pH 10.4 and 10 % methanol 25 mM Tris, pH 9.4, 40 mM glycine and 10 % methanol. 3.6.5. Immunoblotting Protein on the PVDF was stained with Ponceau S solution to check the molecular weight and the efficiency of transfer, and then washed thrice with dH2O to destain the membranes. Non-specific protein binding was blocked by saturating the PVDF membranes with 5 % milk solution in Tris-buffered saline solution containing 0.1 % Tween 20 (TBST) buffer (20 mM Tris-HCl, pH7.4, 137 mM NaCl, and 0.1 % Tween 20) overnight at 4 oC. The membranes were washed thrice for 5min each time, and then incubated overnight at 4 oC with primary antibodies against TBP (N12) cytochrome C, actin, Bcl-2, Bax, Calpain-2, α-spectrin breakdown product (SBDP), cleaved caspase-9, cleaved caspase-3, or cleaved PARP. After washing the membranes with TBST buffer thrice to get rid of unbound antibody, the membranes were incubated with TBST buffer 13.

(25) containing HRP-conjugated secondary antibody for 1 hr at room temperature. All of the protein expression signals were visualized by using enhanced chemiluminescence (ECL) detection reagent (Millipore), and detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The amounts of protein were quantified by Image J (National Institute of Health, USA).. 3.7. Cell apoptotic analysis Cell apoptosis was measured with Annexin V – FITC Kit. Cells were plated in 60 mm culture plates at a density of 1.2 x106 cells for 24 hr. Each well was treated with 80 mmole/L MSG in combination of 10 μmole/L NH018-1 or 10 μmole/L MK801 for 24 hr. The cells with the medium were collected and transferred into fresh 15 ml tube at density of 106 cell per tube.. The cells were centrifugation at 2500 rpm at room temperature for 3 min and resuspended with 500 μl 1X binding buffer. The cells were stained with, 5 μl Annexin V – FITC and 5 μl propidium iodide (PI, 50 g/ml). After incubating at room temperature for 5 min, the percentage of apoptotic cells was determined with a ﬂow cytometer (FACScan, USA) for FL1 and FL2 density and analyzed with CellQuest software.. 14.

(26) 3.8. Reactive oxygen species (ROS) analysis Luminol chemiluminescence was measured as indicators for free radical formation. The SH-SY5Y cells were seeded in 60 mm culture plates at the density of 1.2 x 106 cells for 24 hr. Each well was treated with 80 mmole/L MSG, 10 μmole/L NH018-1 or 10 μmole/L MK801 for 24 hr. All of the cells were collected and washed with cold PBS. After removing PBS, 40 μl RIPA was added and sonication was performed on the ice. The protein concentration was measured with BCA protein assay kit and then 25 μg protein was used to detect ROS amount. The 200 μl sample was put on the measurement plate with stir as background for 1 min and then 500 μl luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione, Sigma) was added into plate, and measured with a CLD-110 chemiluminescence detector (Tohoku Electronic Industrial CO., Ltd.) to continually detect signals with a integration time of 10 s for 4 min. Results were expressed as the relative chemiluminescence of treated-cells against that of controls.. 3.9. SCA17 mice rotarod test The sixteen wild type mice and sixteen SCA17 mice were divided to four groups: The first group was intraperitoneal-injected with saline in eight wild type mice, the second group was intraperitoneal-injected with NH018-1 (1.5 mg/kg) in eight SCA17 mice, the third group was 15.

(27) intraperitoneal-injected with saline in eight wild type mice, and the fourth group was intraperitoneal-injected with NH018-1 (1.5 mg/kg) in eight SCA17 mice. Ten weeks after the birth, the wild type mice and the SCA17 mice of first and third groups were intraperitoneal-injected with saline (0.1 ml), and the wild type mice and the SCA17 mice of second and fourth groups were intraperitoneal-injected with NH018-1 (1.5 mg/kg). All of the mice were weighed at eight weeks old, and the mice were subjected to rotarod test on acceleration from 4 rpm to 30 rpm in 5 min, and then 30 rpm for 10 min to measure keeping on the rotarod time at nine weeks after the birth. The rotarod test was carried out once every two weeks after treating with NH018-1. The saline and NH018-1 were intraperitoneal-injected at ten-week after the birth by three times a week. The care and use of animals respected the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan.. 3.10. SCA17 mice footprinting The forepaws and hindpaws were painted with red and blue dye, respectively. The mice were put into the paper-covered straight-line maze that was 80 cm long, 5 cm wide, with 5 cm high walls to record and analyze the footprint at 8th week and then 13th week after treating with NH018-1. The footprint patterns were evaluated quantitatively by the measurements of front/hind footprint overlap, stride length, and parallel length as described previously 13, 60. 16.

(28) 3.11. Acetylcholinesterase (AChE) activity assay AChE activity was measured by using AChE Assay Kit. For experimental AChE activity animal model, wild type mice and SCA17 mice were given an intraperitoneal-injection with saline or 1.5 mg/kg NH018-1 from 10th to 21st week after birth. Then the mice were sacrificed, and the cerebrum and the cerebellum were subjected to brief homogenization with 0.1 M phosphate buffer (pH = 7.5). The homogenates were centrifugated at 14000 rpm for 5min, and the supernatants were used for AChE activity assay. The 200 µL water and 200 µL calibrator were transferred separately into clear 96-well plate, 10 µL samples were added per well, and then 190 µL fresh Working Reagent was transferred to all sample wells. The absorbance was measured at 412 nm with an ELISA reader at 2 min and at 10 min.. Under the assay conditions, the number “200” in AChE activity formula is the equivalent activity of the calibrator and the “n” is the dilution factor (n = 40 for whole blood).. 17.

(29) 4. Results. 4.1. CHM NH018 rescues SH-SY5Y cell viability after MSG treatment MSG was used to induce excitotoxicity in neuroblastoma SH-SY5Y cell model to screen the effective CHMs. At first, cells were treated with 40, 80, 120, or 160 mM MSG for 24 hr to determine the IC50. As shown in figure 1 (A), the IC50 of MSG is 80 mmole/L after 24 hr treatment. Then, the IC50 of various CHM water extracts on SH-SY5Y cells was measured, and half or one-fifth of CHM IC50 (1/2 X or 1/5 X IC50) (IC50 listed in Table 1) was used to screen effective CHMs against MSG by using NMDA receptor antagonist, MK801, as a positive control. As shown in figure 1 (B), Compared to MSG (reduced relative cell viability to about 60%), NH018, NH021, and NH027 exhibited 75%, 70%, and 80% increased of cell viability, respectively. Therefore, NH018 was chosen for further studies.. 4.2. Active compound, NH018-1 of NH018 increased SH-SY5Y cell viability after MSG treatment The IC50 of various compounds isolated from CHMs against SH-SY5Y cells were determined, and the results were summarized in Table 2. Then 1/2 and 1/5 IC50 of the compounds were used to study their effects on the cell excitotoxicity induced by MSG, and the results showed 18.

(30) that NH018-1 was found to be the most effective compound. As shown in figure 2 (A), 10 μmole/L NH018-1 was sufficient to significantly rescue cell death induced by MSG (increased relative cell viability from 65%±1.2 (SEM) to 75%±0.5, p < 0.05). Because NH018-1 is the major active component of NH018, the above mentioned results imply that NH018-1 could be the effective compound that exerts protective effect against glutamatergic cytotoxicity.. 4.3. Effects of NH018-1 on the release of lactate dehydrogenase (LDH) from SH-SY5Y cells treated with MSG To verify the membrane integrity, the passage of substances that are normally detained inside cells to the outside was assessed by monitoring the release of LDH into the culture medium 20. As shown in figure 3, NH018-1 at 2.5-40 μmole/L effectively inhibited the release of LDH induced by MSG (suppressed relative LDH release from ~215% to ~125%). It suggests that NH018-1 exhibits protective effects against MSG-induced cell membrane damage.. 4.4. Effects of NH018-1 on Bcl-2, Bax and cytochrome C release for SH-SY5Y cells treated with MSG To investigate whether mitochondrial apoptotic pathway is involved 19.

(31) in NH018-1-mediated protection against glutamatergic excitotoxicity, the cell-survival protein Bcl-2 and pro-apoptotic protein Bax, which mediate the release of pro-apoptotic cytochrome C from mitochondria 3 were investigated. Treatment of 80 mmole/L MSG resulted in the 12% reduction of Bcl-2 and the 60% increment of Bax. When cells were co-treated with MSG and NH018-1, the levels of Bcl-2 and Bax were increased 30% and suppressed 50%, respectively (Figure 4 (A), (B) and(C)). It indicates that NH018-1 is able to reverse the decline of Bcl-2/Bax ratio.. To further study the effect of NH018-1 on the cytochrome C release from mitochondria to the cytosol, cytoplasm and mitochondria were fractionated and analyzed. As shown in figure 5 (A), by Western analyze, NH018-1 significantly decreased the release of cytochrome C from mitochondria to cytosol induced by MSG (relative fold change 2.8 versus 1.2 , p < 0.05). 4.5. NH018-1 attenuated MSG-induced the activation of Caspase-9, Caspase-3, and PARP expression in SH-SY5Y cells Previous study showed that the overactivation of glutamate receptors by NMDA receptor triggered neuronal cell death, associated with caspase-family downstream of Bcl-2/Bax and cytochrome C such as 20.

(32) caspase-9, caspase-3 as well as nuclear enzyme PARP activated 2, 8. As shown in figure 6 (A), (B), (C), and (D). MSG remarkably increased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by a relative fold changes, 1.3, 4.3, and 8.1, respectively. Co-treatment of NH018-1 with MSG for 24 hr, it decreased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by a relative fold changes, 1.0, 3.2, and 5.0, respectively. Based on the above-mentioned results, it suggests that NH018-1 inhibits the excitotoxicity via the suppression of the mitochondria apoptotic pathway.. 4.6. NH018-1 reduced MSG-induced the activation of Calpain-2 and Calpain specific-SBDP expression in SH-SY5Y cells It was reported that excessive glutamate leads to intracellular calcium inflow, and then to activation of calpain. The calpain-specific α-spectrin cleaved by the activation of calpain produces the fragments of 150 kD and 145 kD [also known α-spectrin breakdown product (SBDP)]. Accordingly, SBDP could be used for the indicator of calpain activity. As shown in figure 7 (A), 7 (B), and figure 7 (C), compared to MSG (increased relative fold changes of Calpain-2 expression to about 1.2 and SBDP expression to about 1.3), NH018-1 significantly reduced the levels of Calpain-2 (0.9 fold) and SBDP (1.0 fold). It suggests that NH018-1 indeed alleviates glutamatergic excitotoxicty.. 21.

(33) 4.7. Effects of NH018-1 on MSG-induced phosphatidylserine (PS) externalization and apoptotic induction in SH-SY5Y cells In order to study effects of NH018-1 on cell apoptosis, Annexin-V was used to stain externalization of PS, which is an apoptotic marker and measured by flow cytometer method. As shown in figure 8 (A) and (B), MSG significantly increased cell apoptosis, and the co-treatment of NH018-1 with MSG decreased the percentage of cell apoptosis (11% versus 17%).. 4.8. NH018-1 inhibited the ROS production induced by MSG treatment in SH-SY5Y cells To investigate whether NH018-1 could block the ROS production induced by MSG treatment in SH-SY5Y cells, luminol-dependent chemiluminescence was used. Luminol is activated with an oxidant to exhibit its luminescence, the emission of energy as a photon, indicating the change of electrons from excited state to ground state. The emission produces is blue glow and detected with a chemiluminescence detector. As shown in figure 9 (A) and (B), the degree of luminescence intensity increased to about 2000 after MSG treatment, but decreased to about 1500 after co-treating with NH018-1. It demonstrated that NH018-1 reduced MSG-induced ROS production.. 22.

(34) 4.9. Effects of CHMs and NH018-1 on cell viability of DOX induced SCA17 (79Q) cell model The abovementioned results imply that NH018-1 possesses neuroprotective effects on glutamatergic excitotxicity. To further study effects of NH018-1 on SCA17, SCA17 inducible cell model (SCA17 nTBP/Q79-EGFP cells) was used. At first, cells were treated with 5, 10, or 20 μg/mL DOX to induce the expression of nTBP-EGFP(79Q) for 24, 48, and 72 hr to determine the IC50 of DOX. As shown in figure 10 (A), the IC50 of DOX is about 10 μg/mL after 48 hr treatment. From Result 4.1., NH018, NH021, or NH027 significantly increased SH-SY5Y cell viability after MSG treatment. 1/5X IC50 dose of NH018, NH021, or NH027 increased cell viability significantly after nTBP-EGFP(79Q) induction for 48 hr. As shown in figure 10 (B), comparing to DOX (reduced relative cell viability to about 75%), NH018, NH021, and NH027 increased the relative cell viability to 85%, 90%, and 100%, respectively which indicates the CHMs exhibited protective effects against nTBP-EGFP(79Q)-induced cell death. Furthermore, NH018-1 of NH018 also suppressed nTBP-EGFP(79Q)-induced cytotoxicity [increased relative cell viability to about 70%, figure 10 (C)].. 4.10. NH018-1 attenuated nTBP-EGFP(79Q)-induced activated Caspase-9, Caspase-3, and PARP expression 23.

(35) in SCA17 cells To study the effects of NH018-1 on nTBP-EGFP(79Q) induction of activated Caspase-9, Caspase-3, and PARP expression in SCA17 cells, the cells were treated with 10 μg/mL DOX for 5 days. As shown in figure 11 (A), (B), (C), and (D). nTBP-EGFP(79Q) induction significantly increased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by relative fold changes, 1.8, 3.2, and 7.2, respectively. Co-treatment of NH018-1 with DOX for 5 day, it decreased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by relative fold changes, 1.6, 2.6, and 4.0, respectively. Take together the results, nTBP-EGFP(79Q)-induced activation of caspase-family, and PARP were suppressed significantly by the NH018-1 in SCA17 inducible cell model.. 4.11. Effects of NH018-1 on body weight changes and rotarod performance in SCA17 mice model To study the effects of NH018-1 on behavioral aspects in SCA17 mice, the rotarod performance was used to evaluate its efficacy 11. After 1.5 mg/kg NH018-1 administration for 12 weeks, there was no significant difference in body weight change between wild-type and transgenic mice (figure 12 (A)), implying that the NH018-1 dosage used here was not toxic to mice. As shown in figure 12 (B), the SCA17 transgenic mice that received saline vehicle performed poorly on an accelerating rotarod (from 24.

(36) 4 to 30 rpm in 5 min) and the latency to fall kept declining from 15- to 21-week. On the contrary, the motor coordination SCA17 transgenic mice were significantly improved after receiving 1.5 mg/kg NH018-1 from 17to 21-week.. 4.12. Effects of NH018-1 on SCA17 mice footprinting The efficacy of NH018-1 on SCA17 transgenic mice was further investigated by assessing gait abnormalities at 8- and 21-week (figure 13 (A)). The front/hind footprint overlap was significantly increased in the SCA17 transgenic mice that received saline (figure 13 (B) and (C)), but the stride length and parallel length were not altered (figure 13 (D) to (I)) even at 8 and 21 week. NH018-1 significantly suppressed gait abnormalities of SCA17 transgenic mice by reducing front/hind footprint overlap (figure 13 (B) and (C)), and intensifying the stride and parallel lengths (figure 13 (D) and (I)).. 4.13. NH018-1 attenuated SCA17 mice-induced activated TBP (N12), Calapin-2, and cleaved-Caspase-3 expression To study the effects of NH018-1 on aggregation (TBP (N12)), and the expression of Calpain-2, and cleaved-Caspase-3 was measured in cerebellum of SCA17 mice, mice were sacrificed after 21 weeks. As 25.

(37) shown in figure 14 (A), (B), (C), and (D). The expression of TBP (N12), Calpain-2, and cleaved-Caspase-3 were increased by a relative fold changes, 1.8, 1.3, and 1.7, respectively in SCA17 mice. The treatment of NH018-1 from 10- to 21-week in SCA17 mice, it decreased the expression of TBP (N12), Calpain-2, and cleaved-Caspase-3 by a relative fold changes, 1.3, 0.9, and 0.6, respectively. Based on the above-mentioned results, it suggests that NH018-1 inhibits the aggregation and excitotoxicity-induced cell death.. 4.14. Effects of NH018-1 on acetylcholinesterase (AChE) activity of cerebrum and cerebellum in SCA17 mice The significant reduction of AChE activity in the cerebral and cerebellar cortex with inherited olivopontocerebellar atrophy (OPCA) was observed in the postmortem 35. It was also reported that AChE activity was decreased in SCA3 and SCA6 47, but the AChE activity in the cerebellum in SCA17 disorder is remained to be studied. To investigate whether NH018-1 improves the SCA17 disorder via inhibition of AChE activity in the cerebellar, mice were sacrificed, and the AChE activity in cerebellar was measured. As shown in figure 15 (A), cerebral AChE activities were similar within control and NH018-1 treated groups. The cerebellar AchE activity, however, was remarkably increased in SCA17 transgenic mice (figure 15 (B)). Treatment of NH018-1 did not inhibit cerebellar AChE activity in SCA17 transgenic mice. It suggests that amelioration of SCA17 by 26.

(38) NH018-1 is not via inhibiting of AChE activity.. 27.

(39) 5. Discussion. The present study demonstrates that NH018-1 exhibits beneficial effects on MSG-induced excitotoxicity, SCA17 expanded-polyQ-mediated cell death, and SCA17 transgenic mice. The results show several pieces of evidences that NH018-1 inhibited glutamatergic cytotoxicity by deducing downstream mitochondrial apoptotic pathway. Moreover, NH018-1 ameliorated glutamatergic excitotoxicity by down-regulating Bax and up-regulating Bcl-2 expressions, resulting in increasing Bcl-2/Bax ratio. NH018-1 inhibited MSG-mediated cytochrome C release and activation of downstream Caspase-9, Caspase-3, and PARP. In addition, NH018-1 decreased the levels of Calpain-2 and its substrate SBDP induced by MSG. Besides, MSG-induced cell apoptosis and production of ROS were decreased by NH018-1 treatment. Furthermore, application of NH018-1 to SCA17 cell model revealed that NH018-1 alleviated expanded polyQ-mediated cytotoxicity by decreasing the expression of cleaved-Caspase-9, -Caspase-3, and -PARP fragments. Importantly, NH018-1 treatment improved the performance of motor coordination in SCA17 transgenic mice.. In this study, by screening nine CHMs and nine compounds isolated from CHMs, and NH018 and compound NH018-1 were found to have high capability to increase cell viability of MSG-induced SH-SY5Y cell 28.

(40) death (figure 1 and 2). In addition, NH018-1 inhibited the release of LDH into the culture medium after SH-SY5Y cell damage caused by MSG (figure 3). It was reported previously that the binding of glutamate to NMDA receptor leads to the influx of Ca2+ into the cells, and the elevation of intracellular Ca2+ levels in CNS neurons 43, 48. The excess of Ca2+ inside cells is related to variety pathological phenomena such as neuronal toxicity 15. Thus, Ca2+ is an important indicator in several neurondegenerative disorders. Higher intracellular Ca2+ may disrupt the mitochondrial membrane potential in hippocampal neurons, cortical neurons 32, and cerebellar granule neurons 73. Bcl-2 could block loss of mitochondrial membrane potential to reduce the extent of apoptosis 55. The mitochondrial apoptosis pathway is mediated by the Bcl-2 family of proteins, and apoptosis will be inhibited at high levels of Bcl-2. In contrast, the initiation of apoptosis is associated with an increased expression of Bax 36. Our results showed that NH018-1 increased the expression of Bcl-2 but decreased that of Bax after glutamatergic treatment (figure 4) in SH-SY5Y cells. However, the changes of intracellular Ca2+ concentration and mitochondrial membrane potential need to be further examined.. The regulation of the mitochondrial permeability transition plays an important role in the mitochondria-mediated apoptosis pathway 27, 34, 59. In this process, cytochrome C releases from the intermembrane into cytoplasm 6 and binds Apaf-1, leading to the activation of protease caspase-9, and then caspase-3. Our data indicated that NH018-1 inhibited 29.

(41) cytochrome C release (figure 5) and decreased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved PARP (figure 6) in SH-SY5Y cells, suggesting that NH018-1 could prevent mitochondria-mediated apoptosis.. The calcium-dependent protein, calpain, had been known to directly participate in tau proteolysis and degradation 31, and inhibition of calpains was found to ameliorate synaptic transmission and memory in AD mouse 62. . The Cyclin-dependent kinase (cdk5) activity was regulated by p25,. which is generated upon the cleavage of p35 by activated calpain increased cdk5 kinase activity 51 enhances tau phosphorylation 4, and finally causes cell death. On the other hand, Ca2+ may trigger the calpain-dependent formation of polyglutamine containing fragments in neuroblastoma cells, and ALLN or calpeptin (both are calpain inhibitors) treatment can lead to complete disappearance of aggregates. Additionally, overexpression of calpastatin (endogenous calpain inhibitor) could inhibit cleavage and aggregation of mutant ataxin-3 26. Our findings showed that NH018-1 decreased the expression of Calpain-2 and Calpain specific-SBDP (figure 7) in SH-SY5Y cells, which indicates that NH018-1 might affect calpain/p35/p25/cdk5 pathway and decrease neuronal cell death.. High concentrations of extracellular glutamate can block cystine uptake into cells, leading to depletion of intracellular cysteine and antioxidant glutathione 25, then inducing ROS accumulation, oxidative 30.

(42) stress, and subsequent neuronal cell death. Thus, ROS plays an important role in neurodegenerative diseases, and our results showed that NH018-1 reduced MSG-induced ROS production (figure 9) in SH-SY5Y cells.. The neuroprotective effects of NH018-1 on AD are well defined. NH018-1 could suppress hydrogen peroxide-, β-amyloid protein-, hypoxic-ischemic-, and glutamate-induced cytotoxicity 69. Nevertheless, the effects of NH018-1 on neurodegenerative diseases in addition to AD are rarely defined. Therefore, this study was designed to investigate the effects of NH018-1 on SCA17 cell and animal model. Present study showed that NH018-1 increased cell viability by inhibiting mitochondrial-mediated cell death pathway, it indicates that NH018-1 might rescue the cell death of SCA17 (79Q) by the same way. Our results showed that NH018-1 increased cell viability (figure 10) and decreased the expressions of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP (figure 11) after induction of SCA17-79Q expression in SCA17 cells. The SCA17 transgenic mice used here recapitulate SCA17 phenotypes by exhibiting Purkinje cell loss, cerebellar atrophy, and worse performance on the rotarod 82. We found NH018-1 improved the motor performance on an accelerating rotarod (figure 12) and ameliorated gait abnormalities such as reducing the front/hind footprint overlaps as well as increasing the front/hind stride and parallel length (figure 13). NH018-1 also decreased the expression of TBP (N12), Calpain-2, and cleaved-Caspase-3 in cerebellum of SCA17 mice (figure 14).. 31.

(43) Besides, previous studies indicated that NH018-1 is a reversible and selective inhibitor of AChE 14 and the potency of AChE inhibition is similar or better to physostigmine, galanthamine, donepezil and tacrine 66, 70. . NH018-1 could inhibit the AChE activity to improve memory and. cognitive function in AD cerebrum. As shown in figure 15, our findings showed that cerebral AChE acitivities are similar between wild type and SCA17 transgenic mice, but the AChE activity is significantly increased in the cerebellum of SCA17 transgenic mice comparing to wild type mice. However, no significant changes were observed after treating SCA17 transgenic mice with NH018-1. According the above-mentioned results, it suggests that NH018-1 improves the SCA17 disorder through the mitochondrial apoptosis pathway but not through the inhibition of AChE activity.. Taken together, NH018-1 has protective effects on MSG-induced cell death of SH-SY5Y cells and SCA17-79Q -induced cell death, and improved the motor coordination of SCA17 animal model via inhibition of glutamate excitotoxicity. It is the first time to demonstrate the effects of NH018-1 on SCA17 cell and animal models, therefore, NH018-1 could be a potential drug to improve SCA17 disorder.. 32.

(44) 6. References 1. A. Agarwal, R. Z. Mahfouz, R. K. Sharma, O. Sarkar, D. Mangrola, and P. P. Mathur, 'Potential Biological Role of Poly (Adp-Ribose) Polymerase (Parp) in Male Gametes', Reprod Biol Endocrinol, 7 (2009), 143.. 2. M. Ankarcrona, J. M. Dypbukt, E. Bonfoco, B. Zhivotovsky, S. Orrenius, S. A. Lipton, and P. Nicotera, 'Glutamate-Induced Neuronal Death: A Succession of Necrosis or Apoptosis Depending on Mitochondrial Function', Neuron, 15 (1995), 961-73.. 3. B. Antonsson, 'Mitochondria and the Bcl-2 Family Proteins in Apoptosis Signaling Pathways', Mol Cell Biochem, 256-257 (2004), 141-55.. 4. K. Baumann, E. M. Mandelkow, J. Biernat, H. Piwnica-Worms, and E. Mandelkow, 'Abnormal Alzheimer-Like Phosphorylation of Tau-Protein by Cyclin-Dependent Kinases Cdk2 and Cdk5', FEBS Lett, 336 (1993), 417-24.. 5. F. Blandini, J. T. Greenamyre, and G. Nappi, 'The Role of Glutamate in the Pathophysiology of Parkinson's Disease', Funct Neurol, 11 (1996), 3-15.. 6. E. Bossy-Wetzel, D. D. Newmeyer, and D. R. Green, 'Mitochondrial Cytochrome C Release in Apoptosis Occurs Upstream of Devd-Specific Caspase Activation and Independently of Mitochondrial Transmembrane Depolarization', EMBO J, 17 (1998), 37-49. 33.

(45) 7. J. R. Brorson, C. J. Marcuccilli, and R. J. Miller, 'Delayed Antagonism of Calpain Reduces Excitotoxicity in Cultured Neurons', Stroke, 26 (1995), 1259-66; discussion 67.. 8. S. L. Budd, and D. G. Nicholls, 'Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells', J Neurochem, 67 (1996), 2282-91.. 9. S. L. Budd, L. Tenneti, T. Lishnak, and S. A. Lipton, 'Mitochondrial and Extramitochondrial Apoptotic Signaling Pathways in Cerebrocortical Neurons', Proc Natl Acad Sci U S A, 97 (2000), 6161-6.. 10. K. M. Carlson, J. M. Andresen, and H. T. Orr, 'Emerging Pathogenic Pathways in the Spinocerebellar Ataxias', Curr Opin Genet Dev, 19 (2009), 247-53.. 11. R. J. Carter, L. A. Lione, T. Humby, L. Mangiarini, A. Mahal, G. P. Bates, S. B. Dunnett, and A. J. Morton, 'Characterization of Progressive Motor Deficits in Mice Transgenic for the Human Huntington's Disease Mutation', J Neurosci, 19 (1999), 3248-57.. 12. Y. C. Chang, C. Y. Lin, C. M. Hsu, H. C. Lin, Y. H. Chen, G. J. Lee-Chen, M. T. Su, L. S. Ro, C. M. Chen, and H. M. Hsieh-Li, 'Neuroprotective Effects of Granulocyte-Colony Stimulating Factor in a Novel Transgenic Mouse Model of Sca17', J Neurochem, 118 (2011), 288-303.. 13. X. Chen, T. S. Tang, H. Tu, O. Nelson, M. Pook, R. Hammer, N. Nukina, and I. Bezprozvanny, 'Deranged Calcium Signaling and Neurodegeneration in Spinocerebellar Ataxia Type 3', J Neurosci, 34.

(46) 28 (2008), 12713-24. 14. D. H. Cheng, H. Ren, and X. C. Tang, 'Huperzine A, a Novel Promising Acetylcholinesterase Inhibitor', Neuroreport, 8 (1996), 97-101.. 15. D. W. Choi, 'Glutamate Neurotoxicity and Diseases of the Nervous System', Neuron, 1 (1988), 623-34.. 16. C. G. Concannon, L. P. Tuffy, P. Weisova, H. P. Bonner, D. Davila, C. Bonner, M. C. Devocelle, A. Strasser, M. W. Ward, and J. H. Prehn, 'Amp Kinase-Mediated Activation of the Bh3-Only Protein Bim Couples Energy Depletion to Stress-Induced Apoptosis', J Cell Biol, 189 (2010), 83-94.. 17. S. P. Cregan, A. Fortin, J. G. MacLaurin, S. M. Callaghan, F. Cecconi, S. W. Yu, T. M. Dawson, V. L. Dawson, D. S. Park, G. Kroemer, and R. S. Slack, 'Apoptosis-Inducing Factor Is Involved in the Regulation of Caspase-Independent Neuronal Cell Death', J Cell Biol, 158 (2002), 507-17.. 18. D. E. Croall, and G. N. DeMartino, 'Calcium-Activated Neutral Protease (Calpain) System: Structure, Function, and Regulation', Physiol Rev, 71 (1991), 813-47.. 19. J. B. Davis, and P. Maher, 'Protein Kinase C Activation Inhibits Glutamate-Induced Cytotoxicity in a Neuronal Cell Line', Brain Res, 652 (1994), 169-73.. 20. T. Decker, and M. L. Lohmann-Matthes, 'A Quick and Simple Method for the Quantitation of Lactate Dehydrogenase Release in Measurements of Cellular Cytotoxicity and Tumor Necrosis Factor 35.

(47) (Tnf) Activity', J Immunol Methods, 115 (1988), 61-9. 21. G. P. Dietz, B. Dietz, and M. Bahr, 'Bcl-Xl Protects Cerebellar Granule Neurons against the Late Phase, but Not against the Early Phase of Glutamate-Induced Cell Death', Brain Res, 1164 (2007), 136-41.. 22. A. Doble, 'The Role of Excitotoxicity in Neurodegenerative Disease: Implications for Therapy', Pharmacol Ther, 81 (1999), 163-221.. 23. N. Egashira, K. Iwasaki, Y. Akiyoshi, Y. Takagaki, I. Hatip-Al-Khatib, K. Mishima, K. Kurauchi, T. Ikeda, and M. Fujiwara, 'Protective Effect of Toki-Shakuyaku-San on Amyloid Beta25-35-Induced Neuronal Damage in Cultured Rat Cortical Neurons', Phytother Res, 19 (2005), 450-3.. 24. M. J. Friedman, A. G. Shah, Z. H. Fang, E. G. Ward, S. T. Warren, S. Li, and X. J. Li, 'Polyglutamine Domain Modulates the Tbp-Tfiib Interaction: Implications for Its Normal Function and Neurodegeneration', Nat Neurosci, 10 (2007), 1519-28.. 25. M. Fukui, H. J. Choi, and B. T. Zhu, 'Mechanism for the Protective Effect of Resveratrol against Oxidative Stress-Induced Neuronal Death', Free Radic Biol Med, 49 (2010), 800-13.. 26. A. Haacke, F. U. Hartl, and P. Breuer, 'Calpain Inhibition Is Sufficient to Suppress Aggregation of Polyglutamine-Expanded Ataxin-3', J Biol Chem, 282 (2007), 18851-6.. 27. A. P. Halestrap, G. P. McStay, and S. J. Clarke, 'The Permeability Transition Pore Complex: Another View', Biochimie, 84 (2002), 36.

(48) 153-66. 28. J. Huang, J. Tao, X. Xue, S. Yang, P. Han, Z. Lin, W. Xu, J. Lin, J. Peng, and L. Chen, 'Gua Lou Gui Zhi Decoction Exerts Neuroprotective Effects on Post-Stroke Spasticity Via the Modulation of Glutamate Levels and Ampa Receptor Expression', Int J Mol Med, 31 (2013), 841-8.. 29. S. Huang, J. J. Ling, S. Yang, X. J. Li, and S. Li, 'Neuronal Expression of Tata Box-Binding Protein Containing Expanded Polyglutamine in Knock-in Mice Reduces Chaperone Protein Response by Impairing the Function of Nuclear Factor-Y Transcription Factor', Brain, 134 (2011), 1943-58.. 30. M. R. Hynd, H. L. Scott, and P. R. Dodd, 'Glutamate-Mediated Excitotoxicity and Neurodegeneration in Alzheimer's Disease', Neurochem Int, 45 (2004), 583-95.. 31. G. V. Johnson, R. S. Jope, and L. I. Binder, 'Proteolysis of Tau by Calpain', Biochem Biophys Res Commun, 163 (1989), 1505-11.. 32. Y. Kambe, N. Nakamichi, D. D. Georgiev, N. Nakamura, H. Taniura, and Y. Yoneda, 'Insensitivity to Glutamate Neurotoxicity Mediated by Nmda Receptors in Association with Delayed Mitochondrial Membrane Potential Disruption in Cultured Rat Cortical Neurons', J Neurochem, 105 (2008), 1886-900.. 33. A. Kelp, A. H. Koeppen, E. Petrasch-Parwez, C. Calaminus, C. Bauer, E. Portal, L. Yu-Taeger, B. Pichler, P. Bauer, O. Riess, and H. P. Nguyen, 'A Novel Transgenic Rat Model for Spinocerebellar Ataxia Type 17 Recapitulates Neuropathological Changes and 37.

(49) Supplies in Vivo Imaging Biomarkers', J Neurosci, 33 (2013), 9068-81. 34. J. S. Kim, L. He, and J. J. Lemasters, 'Mitochondrial Permeability Transition: A Common Pathway to Necrosis and Apoptosis', Biochem Biophys Res Commun, 304 (2003), 463-70.. 35. S. J. Kish, L. Schut, J. Simmons, J. Gilbert, L. J. Chang, and M. Rebbetoy, 'Brain Acetylcholinesterase Activity Is Markedly Reduced in Dominantly-Inherited Olivopontocerebellar Atrophy', J Neurol Neurosurg Psychiatry, 51 (1988), 544-8.. 36. L. J. Ko, and C. Prives, 'P53: Puzzle and Paradigm', Genes Dev, 10 (1996), 1054-72.. 37. R. Koide, S. Kobayashi, T. Shimohata, T. Ikeuchi, M. Maruyama, M. Saito, M. Yamada, H. Takahashi, and S. Tsuji, 'A Neurological Disease Caused by an Expanded Cag Trinucleotide Repeat in the Tata-Binding Protein Gene: A New Polyglutamine Disease?', Hum Mol Genet, 8 (1999), 2047-53.. 38. S. Lankiewicz, C. Marc Luetjens, N. Truc Bui, A. J. Krohn, M. Poppe, G. M. Cole, T. C. Saido, and J. H. Prehn, 'Activation of Calpain I Converts Excitotoxic Neuron Death into a Caspase-Independent Cell Death', J Biol Chem, 275 (2000), 17064-71.. 39. M. S. Lee, J. Chao, J. C. Yen, L. W. Lin, F. S. Tsai, M. T. Hsieh, W. H. Peng, and H. Y. Cheng, 'Schizandrin Protects Primary Rat Cortical Cell Cultures from Glutamate-Induced Apoptosis by Inhibiting Activation of the Mapk Family and the Mitochondria 38.

(50) Dependent Pathway', Molecules, 18 (2012), 354-72. 40. T. Y. Lin, C. W. Lu, S. K. Huang, and S. J. Wang, 'Tanshinone Iia, a Constituent of Danshen, Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals', J Ethnopharmacol, 147 (2013), 488-96.. 41. A. C. Ludolph, and C. Munch, 'Neurotoxic Mechanisms of Degeneration in Motor Neuron Diseases', Drug Metab Rev, 31 (1999), 619-34.. 42. C. M. Luetjens, N. T. Bui, B. Sengpiel, G. Munstermann, M. Poppe, A. J. Krohn, E. Bauerbach, J. Krieglstein, and J. H. Prehn, 'Delayed Mitochondrial Dysfunction in Excitotoxic Neuron Death: Cytochrome C Release and a Secondary Increase in Superoxide Production', J Neurosci, 20 (2000), 5715-23.. 43. A. B. MacDermott, M. L. Mayer, G. L. Westbrook, S. J. Smith, and J. L. Barker, 'Nmda-Receptor Activation Increases Cytoplasmic Calcium Concentration in Cultured Spinal Cord Neurones', Nature, 321 (1986), 519-22.. 44. P. Maher, and A. Hanneken, 'Flavonoids Protect Retinal Ganglion Cells from Oxidative Stress-Induced Death', Invest Ophthalmol Vis Sci, 46 (2005), 4796-803.. 45. P. Maher, and D. Schubert, 'Signaling by Reactive Oxygen Species in the Nervous System', Cell Mol Life Sci, 57 (2000), 1287-305.. 46. F. Maltecca, A. Filla, I. Castaldo, G. Coppola, N. A. Fragassi, M. Carella, A. Bruni, S. Cocozza, G. Casari, A. Servadio, and G. De Michele, 'Intergenerational Instability and Marked Anticipation in 39.

(51) Sca-17', Neurology, 61 (2003), 1441-3. 47. H. Maruyama, Y. Izumi, H. Morino, M. Oda, H. Toji, S. Nakamura, and H. Kawakami, 'Difference in Disease-Free Survival Curve and Regional Distribution According to Subtype of Spinocerebellar Ataxia: A Study of 1,286 Japanese Patients', Am J Med Genet, 114 (2002), 578-83.. 48. M. L. Mayer, and G. L. Westbrook, 'The Physiology of Excitatory Amino Acids in the Vertebrate Central Nervous System', Prog Neurobiol, 28 (1987), 197-276.. 49. J. Naarala, P. Nykvist, M. Tuomala, and K. Savolainen, 'Excitatory Amino Acid-Induced Slow Biphasic Responses of Free Intracellular Calcium in Human Neuroblastoma Cells', FEBS Lett, 330 (1993), 222-6.. 50. K. Nakamura, S. Y. Jeong, T. Uchihara, M. Anno, K. Nagashima, T. Nagashima, S. Ikeda, S. Tsuji, and I. Kanazawa, 'Sca17, a Novel Autosomal Dominant Cerebellar Ataxia Caused by an Expanded Polyglutamine in Tata-Binding Protein', Hum Mol Genet, 10 (2001), 1441-8.. 51. G. N. Patrick, L. Zukerberg, M. Nikolic, S. de la Monte, P. Dikkes, and L. H. Tsai, 'Conversion of P35 to P25 Deregulates Cdk5 Activity and Promotes Neurodegeneration', Nature, 402 (1999), 615-22.. 52. W. J. Pottorf, 2nd, T. M. Johanns, S. M. Derrington, E. E. Strehler, A. Enyedi, and S. A. Thayer, 'Glutamate-Induced Protease-Mediated Loss of Plasma Membrane Ca2+ Pump Activity 40.

(52) in Rat Hippocampal Neurons', J Neurochem, 98 (2006), 1646-56. 53. M. M. Semenova, A. M. Maki-Hokkonen, J. Cao, V. Komarovski, K. M. Forsberg, M. Koistinaho, E. T. Coffey, and M. J. Courtney, 'Rho Mediates Calcium-Dependent Activation of P38alpha and Subsequent Excitotoxic Cell Death', Nat Neurosci, 10 (2007), 436-43.. 54. P. J. Shaw, 'Calcium, Glutamate, and Amyotrophic Lateral Sclerosis: More Evidence but No Certainties', Ann Neurol, 46 (1999), 803-5.. 55. S. Shimizu, Y. Eguchi, W. Kamiike, S. Waguri, Y. Uchiyama, H. Matsuda, and Y. Tsujimoto, 'Bcl-2 Blocks Loss of Mitochondrial Membrane Potential While Ice Inhibitors Act at a Different Step During Inhibition of Death Induced by Respiratory Chain Inhibitors', Oncogene, 13 (1996), 21-9.. 56. R. Siman, J. C. Noszek, and C. Kegerise, 'Calpain I Activation Is Specifically Related to Excitatory Amino Acid Induction of Hippocampal Damage', J Neurosci, 9 (1989), 1579-90.. 57. P. S. Spencer, 'Food Toxins, Ampa Receptors, and Motor Neuron Diseases', Drug Metab Rev, 31 (1999), 561-87.. 58. G. Stevanin, and A. Brice, 'Spinocerebellar Ataxia 17 (Sca17) and Huntington's Disease-Like 4 (Hdl4)', Cerebellum, 7 (2008), 170-8.. 59. G. Szalai, R. Krishnamurthy, and G. Hajnoczky, 'Apoptosis Driven by Ip(3)-Linked Mitochondrial Calcium Signals', EMBO J, 18 (1999), 6349-61.. 60. T. S. Tang, X. Chen, J. Liu, and I. Bezprozvanny, 'Dopaminergic 41.

(53) Signaling and Striatal Neurodegeneration in Huntington's Disease', J Neurosci, 27 (2007), 7899-910. 61. Y. Toyoshima, O. Onodera, M. Yamada, S. Tsuji, and H. Takahashi, 'Spinocerebellar Ataxia Type 17', in Genereviews, ed. by R. A. Pagon, M. P. Adam, T. D. Bird, C. R. Dolan, C. T. Fong and K. Stephens (Seattle WA: University of Washington, Seattle, 1993).. 62. F. Trinchese, M. Fa, S. Liu, H. Zhang, A. Hidalgo, S. D. Schmidt, H. Yamaguchi, N. Yoshii, P. M. Mathews, R. A. Nixon, and O. Arancio, 'Inhibition of Calpains Improves Memory and Synaptic Transmission in a Mouse Model of Alzheimer Disease', J Clin Invest, 118 (2008), 2796-807.. 63. M. Tymianski, M. P. Charlton, P. L. Carlen, and C. H. Tator, 'Source Specificity of Early Calcium Neurotoxicity in Cultured Embryonic Spinal Neurons', J Neurosci, 13 (1993), 2085-104.. 64. O. Vergun, J. Keelan, B. I. Khodorov, and M. R. Duchen, 'Glutamate-Induced Mitochondrial Depolarisation and Perturbation of Calcium Homeostasis in Cultured Rat Hippocampal Neurones', J Physiol, 519 Pt 2 (1999), 451-66.. 65. D. Wang, Q. R. Tan, and Z. J. Zhang, 'Neuroprotective Effects of Paeoniflorin, but Not the Isomer Albiflorin, Are Associated with the Suppression of Intracellular Calcium and Calcium/Calmodulin Protein Kinase Ii in Pc12 Cells', J Mol Neurosci (2013).. 66. H. Wang, and X. C. Tang, 'Anticholinesterase Effects of Huperzine A, E2020, and Tacrine in Rats', Zhongguo Yao Li Xue Bao, 19 (1998), 27-30. 42.

(54) 67. H. Wang, S. W. Yu, D. W. Koh, J. Lew, C. Coombs, W. Bowers, H. J. Federoff, G. G. Poirier, T. M. Dawson, and V. L. Dawson, 'Apoptosis-Inducing Factor Substitutes for Caspase Executioners in Nmda-Triggered Excitotoxic Neuronal Death', J Neurosci, 24 (2004), 10963-73.. 68. K. K. Wang, 'Calpain and Caspase: Can You Tell the Difference?', Trends Neurosci, 23 (2000), 20-6.. 69. R. Wang, and X. C. Tang, 'Neuroprotective Effects of Huperzine A. A Natural Cholinesterase Inhibitor for the Treatment of Alzheimer's Disease', Neurosignals, 14 (2005), 71-82.. 70. Y. E. Wang, D. X. Yue, and X. C. Tang, '[Anti-Cholinesterase Activity of Huperzine A]', Zhongguo Yao Li Xue Bao, 7 (1986), 110-3.. 71. M. W. Ward, A. C. Rego, B. G. Frenguelli, and D. G. Nicholls, 'Mitochondrial Membrane Potential and Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells', J Neurosci, 20 (2000), 7208-19.. 72. M. W. Ward, M. Rehm, H. Duessmann, S. Kacmar, C. G. Concannon, and J. H. Prehn, 'Real Time Single Cell Analysis of Bid Cleavage and Bid Translocation During Caspase-Dependent and Neuronal Caspase-Independent Apoptosis', J Biol Chem, 281 (2006), 5837-44.. 73. P. Weisova, U. Anilkumar, C. Ryan, C. G. Concannon, J. H. Prehn, and M. W. Ward, ''Mild Mitochondrial Uncoupling' Induced Protection against Neuronal Excitotoxicity Requires Ampk 43.

(55) Activity', Biochim Biophys Acta, 1817 (2012), 744-53. 74. R. J. White, and I. J. Reynolds, 'Mitochondrial Depolarization in Glutamate-Stimulated Neurons: An Early Signal Specific to Excitotoxin Exposure', J Neurosci, 16 (1996), 5688-97.. 75. B. B. Wolf, J. C. Goldstein, H. R. Stennicke, H. Beere, G. P. Amarante-Mendes, G. S. Salvesen, and D. R. Green, 'Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation', Blood, 94 (1999), 1683-92.. 76. H. Y. Wu, E. Y. Yuen, Y. F. Lu, M. Matsushita, H. Matsui, Z. Yan, and K. Tomizawa, 'Regulation of N-Methyl-D-Aspartate Receptors by Calpain in Cortical Neurons', J Biol Chem, 280 (2005), 21588-93.. 77. J. Wu, H. K. Jeong, S. E. Bulin, S. W. Kwon, J. H. Park, and I. Bezprozvanny, 'Ginsenosides Protect Striatal Neurons in a Cellular Model of Huntington's Disease', J Neurosci Res, 87 (2009), 1904-12.. 78. N. Wu, L. Song, X. X. Yang, J. L. Wei, and Z. G. Liu, '[Effects of Chinese Herbal Medicine Tianqi Pingchan Granule on G Protein-Coupled Receptor Kinase 6 Involved in the Prevention of Levodopa-Induced Dyskinesia in Rats with Parkinson Disease]', Zhong Xi Yi Jie He Xue Bao, 10 (2012), 1018-24.. 79. H. Xiang, Y. Kinoshita, C. M. Knudson, S. J. Korsmeyer, P. A. Schwartzkroin, and R. S. Morrison, 'Bax Involvement in P53-Mediated Neuronal Cell Death', J Neurosci, 18 (1998), 44.

(56) 1363-73. 80. W. Xu, T. P. Wong, N. Chery, T. Gaertner, Y. T. Wang, and M. Baudry, 'Calpain-Mediated Mglur1alpha Truncation: A Key Step in Excitotoxicity', Neuron, 53 (2007), 399-412.. 81. E. Y. Yuen, Z. Gu, and Z. Yan, 'Calpain Regulation of Ampa Receptor Channels in Cortical Pyramidal Neurons', J Physiol, 580 (2007), 241-54.. 82. M. M. Zeron, N. Chen, A. Moshaver, A. T. Lee, C. L. Wellington, M. R. Hayden, and L. A. Raymond, 'Mutant Huntingtin Enhances Excitotoxic Cell Death', Mol Cell Neurosci, 17 (2001), 41-53.. 83. Y. C. Chang, C. M. Hsu, C. Y. Lin, C. M. Chen, and H. M. Hsieh, 'Characterization of Pathogenesis of SCA17 Transgenic Mice', BioFormosa, 45 (2010), 1-9.. 45.

(57) 7. Figures. Figure 1. Effects of CHMs on MSG-induced cell death in SH-SY5Y cells (A) 3.0 x 104 SH-SY5Y cells were treated with various concentrations of MSG for 24hr and cell viability was determined by MTT assay. The IC50 of MSG is found to be 80 mmole/L. (B) 3.0 x 104 SH-SY5Y cells were co-treated with nine CHMs at indicated concentrations (IC50 listed in Table 1), or 10 μmole/L MK801 (antagonist of NMDA), or 80 mmole/L MSG. Cell viability was measured by MTT assay at 24hr treatment. The results were expressed as mean±SEM, n=6. *P < 0.05, ** P < 0.01, comparing to that of MSG treatment. 46.

(58) Figure 2. NH018-1 decreased the glutamate-induced cell death in SH-SY5Y cells. 3.0 x 104 SH-SY5Y cells were treated with 80 mmole/L MSG and various concentrations of NH018-1 or 10 μmole/L MK801 for 24 hr. Cell viability was measured by MTT assay. The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 47.

(59) Figure 3. NH018-1 inhibited the LDH release from SH-SY5Y cells induced by MSG. 1.0 x 105 SH-SY5Y cells were co-treated with 80 mmole/L MSG and various concentrations of NH018-1, or 10 μmole/L MK801 for 24 hr. The cell damage was determined by measuring the amount of LDH release. The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 48.

(60) Figure 4. Effects of NH018-1 on Bcl-2 and Bax expression induced by MSG in SH-SY5Y cells (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 6 hr. The Bcl-2 and Bax expression were measured by immunoblotting. (B) and (C) Quantification of the intensities of Bcl-2 and Bax protein bands was determined by Image J and expressed as the ratio versus actin (internal control). The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 49.

(61) Figure 5. NH018-1 suppressed cytochrome C release from mitochondria to cytosol mediated MSG in SH-SY5Y cells. (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. The level of cytochrome C in cytosol fraction was assayed by immunoblotting. (B) Quantification of the intensities of cytochrome C protein bands was measured by Image J and expressed as the ratio versus actin. The results were expressed in mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 50.

(62) Figure 6. NH018-1 decreased the cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP expression induced by MSG in SH-SY5Y cells. (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 12 hr. The expression of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, was measured by immunoblotting. (B), (C), and (D). Quantification of the intensities of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP protein bands was carried out by Image J and expressed as the ratio versus actin. The results were expressed in mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 51.

(63) Figure 7. NH018-1 suppressed Calpain-2 and SBDP expression induced by MSG in SH-SY5Y cells. (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 12 hr. The expression of Calpain-2 and SBDP, as assessed by immunoblotting. (B), (C) Quantification of the intensities of Calpain-2 and SBDP protein bands was performed by Image J and expressed as the ratio versus actin. The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.. 52.

(64) Figure 8. NH018-1 reduced MSG-induced apoptosis in SH-SY5Y cells. (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. PI/Annexin V-FITC double staining was performed to detect cell death/apoptosis after MSG treatment and measured with a flow cytometer. (B) Quantification of cell apoptosis percentage by measuring the lower right region. The results were expressed as mean±SEM, n=3. 53.

(65) Figure 9. NH018-1 suppressed MSG-induced ROS production. (A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. The amounts of ROS was measured by luminol-dependent chemiluminescence patterns detected with a chemiluminescence detector. (B) Quantification of the degree of ROS production. The results were expressed as mean±SEM, n=3.. 54.