以SCA17基因轉殖小鼠初級細胞培養評估中草藥之藥效

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(1)國立臺灣師範大學生命科學系碩士論文. 以SCA17基因轉殖小鼠初級細胞培養評估中草藥之藥效 Evaluation of the effects of Chinese herbs on SCA17 through transgenic mouse cerebellar primary culture. 研究生：吳紫綾 Tzu-Ling Wu 指導教授：謝秀梅博士 Hsiu-Mei Hsieh,Ph.D. 中華民國一Ｏ一年六月.

(2) Index. Abbreviation list ........................................................................ Abstract. 1. ...................................................................................... 3. Introduction. ............................................................................... 7. Material and method. ................................................................. 17. Results .............................................................. ......................... 21 Discussion. .................................................................................. 31. Reference. .................................................................................. 37. Appendix. .................................................................................. 52. Figure .......................................................................................... 53.

(3) Abbreviation list. Abbreviation. Full name. AraC. arabinofuranosyl cytidine. ADCA. autosomal dominant cerebellar ataxias. BDNF. brain-derived neurotrophic factor. COX 2. cyclooxygenase 2. DRPLA. dentatorubral pallidoluysian atrophy. GCSF. Granulocyte colony-stimulating facto. HSP27. Heat shock protein 27. HSP70. Heat shock protein 70. HAT. histone acetyltransferases. HDAC. histone deacetylase. HD. Huntington’s disease. IC50. inhibitory concentration. LD50. lethal dose. NGF. nerve growth factor. NT-3. neurotrophin-3. NT-4. neurotrophin-4. pI3K. phosphatidyl inositol 3-kinase. pERK. phospho-ERK. polyQ. Polyglutamine. PKC. protein kinase C. SBMA. spinaland bulbar muscular atrophy. SCA17. Spinocerebellar ataxias type 17 -1-.

(4) SCA. Spinocerebellarataxias. SAHA. Suberoylanilide hydroxamic acid. TBP. TATA-binding protein. TK2. thymidine kinase 2. TG. Transgenic. WT. wild type. -2-.

(5) Abstract. Spinocerebellar ataxia (SCA) is a complex group of heterogeneous autosomal dominant neurodegenerative disease. The main clinical symptoms in patients consist of ataxia, dystonia, parkinsonism, dementia and seizures, and with a significant cerebellum atrophy in pathology. Many SCAs are caused by the trinucleotide expansion of disease-causing genes. Spinocerebellar ataxia type 17 (SCA17) is resulted from CAG repeat expansion of TBP gene, which encodes polyglutamine (polyQ) stretch in mutant TBP protein. Pathological studies have observed neuron loss in cerebella of patients and hTBP transgenic mice. To study the early pathogenesis and screen potential Chinese herbs for SCA17, we have established a primary culture system from the transgenic mouse cerebella. In the cerebellar primary culture, we observed approximately 70% NeuN positive cells after a long-term culture. That IP3R1 and calbindin positive cells were sustained, revealing this culture system could maintain Purkinje cells survived for more than 4 weeks in vitro. Furthermore, we found that the neurites undergo processing during the culture period, and the reduced processing of SCA17 primary culture can be identified compared to wild-type culture in 6 parameters. In line with expectations, no degenerative cells were observed after 5 days in vitro, however, significant degeneration could be identified in transgenic culture after 14 and 21 days. In addition, severe aggregation was formed in SCA17 primary culture at day 21, which was later than the neurite degeneration. We suggest that aggregation may not an initial event but a late stage -3-.

(6) symptom of degeneration. We further applied G-CSF to this primary culture to confirm that this system was an appropriate platform for drug screening. Finally, with this culture system, we have evaluated several Chinese herbs and found NH-003 and NH-015 could be potential in neuronal protection. In summary, we have established the SCA17 cerebellar primary culture and which could be a system to study the pathogenesis and screen potential treatments for SCA17.. Keywords: primary culture, SCA17. -4-.

(7) 摘要. 脊髓小腦運動失調症(Spinocerebellar ataxias, SCAs)為一群體染色體顯性遺傳的神經退化性疾病，大部分亞型肇因於基因中的重複片段異常擴增所致。病人主要的臨床症狀有步伐不穩、肢體障礙、癡呆、癲癇等，在病理上則有明顯的小腦萎縮及神經細胞退化等現象。第十七型脊髓小腦運動失調症(SCA17)主要致病原因為 TATA-box binding protein (TBP)基因 5’端之 CAG/CAA 三核苷重複過度擴增所引起，產生 N 端帶有過度擴增多麩胺酸(polyglutamine, polyQ)的 TBP 蛋白，這種帶有 polyQ 過度擴增的蛋白會傾向在細胞內形成不溶解的聚集物 (insoluble aggregate)，進而慢慢地影響神經細胞的活性，最後導致神經細胞死亡。目前臨床上多半使用藥物減緩該疾病症狀，但並無合適的藥物可以治療。因此本論文研究我們建立小腦組織的初級細胞培養，並利用此系統進行疾病的早期病理分析與潛力藥物的篩選。我們參照前人文獻並加以修改，成功的建立小腦的初級細胞培養系統，根據實驗結果顯示，此方法可以維持大量神經細胞長時間存活並且減低神經膠細胞的過度增生。利用此系統我們分析了 Purkinje cell 在型態上的差異，發現疾病細胞確實會隨著時間而出現明顯的退化。除此之外，我們也發現在此系統中，同樣亦可看到 Purkinje cell 隨著培養時間增 -5-.

(8) 長而表現累積的錯誤折疊蛋白，但出現大量錯誤折疊蛋白的時間晚於型態的退化，因此我們認為此病徵可能是疾病晚期的現象，但可能不是造成疾病開始出現退化的原因。由上述結果顯示此平台確實可以模擬活體動物所看到的退化病徵。同時我們利用白血球生長激素 (granulocyte colony-stimulating factor)評估本細胞平台是否可應用於 SCA17 潛力藥物篩選並確認此細胞平台確實可以有效評估藥物效果。因此我們利用此平台測試了許多中草藥並篩選出 NH-003 及 NH-015 兩種潛力藥物。總結本研究結果，我們成功的建立了 SCA17 小腦初級細胞培養並且確認此平台可以作為 SCA17 病理機制研究與潛力藥物篩選。. 關鍵字：初級細胞培養、脊髓小腦運動失調症. -6-.

(9) Introduction. Spinocerebellar ataxias. Spinocerebellarataxias (SCAs), also known as autosomal dominant cerebellar ataxias (ADCAs), are a heterogeneous group of inherited neurodegeneration diseases. Patients are diagnosed with several abnormal behaviors, such as gait and limb ataxia or dystonia, etc; furthermore, some patients accompanied with symptoms of dementia, epilepsy, and visual disorders (Harding, 1982). As a result of progressions in biological techniques, more and more subtypes of SCAs have been identified. So far there are 31 subtypes of SCAs identified with specified genes or genetic loci (Sato et al., 2009).. Subtypes of SCAs could be further divided into four groups according to their phenotypes. First, group I with ataxia and degeneration of other neuronal systems includes SCA1-4, 8, 12-13, 17-21, 23-25,27 and 28, and it was the largest group of this classification. Group II with ataxia and specific degeneration in retina and only SCA7 was included. Group III that only display pure cerebellar ataxia syndrome including SCA 5, 6, 8, 10-12, 14-16, 26, 29, and 30. Finally, group IV that combine with myoclonus and ataxia symptoms containing SCA14, 19 and 24 (Teive, 2009). SCA 31 was not classified yet for only few cases studied so far.. -7-.

(10) Several genetic mutations were found in SCAs. At least 16 genes and 29 loci are identified involved in these diseases (Teive, 2009); however, the disease-associated genes or detail molecular effects are still unknown excepting few of common type like SCA3 or SCA10. Most of them are still uncertain, such as type 4, 9, 12, 16, 18-26, and 28-30.Some SCAs are caused by missense mutation, such as type 5, 13, 14 and 27. The type 5 is a rare case which causes by deleted mutation in Beta-III Spectrin, and type 31 is an insertional mutation in introns of thymidine kinase 2 (TK2) and brain expressed, associated with Nedd4 (BEAN) genes (Duenas et al., 2006, Sato et al., 2009, Teive, 2009). Some SCAs are caused by expanded nucleotide repeat including types 1-3, 6-8, 10, 12 and 17 (Gatchel and Zoghbi, 2005).. Polyglutamine diseases. Polyglutamine (PolyQ) diseases which are with expanded glutamines in target proteins in at least 9 diseases including the spinocerebellar ataxias SCA1-3, 6, 7, and 17, Huntington’s disease (HD), spinal and bulbar muscular atrophy (SBMA) and dentatorubral pallidoluysian atrophy (DRPLA) (Zoghbi and Orr, 2000). Those diseases have some similar characteristics due to the mutant polyQ proteins. One is the age of onset is in inverse proportion of the residues of glutamine (Igarashi et al., 1992, Orr, 2001, Gatchel and Zoghbi, 2005).. -8-.

(11) Recent reports indicate that polyQ toxicity gain of function is correlated to the glutamine residue length (Igarashi et al., 1992, La Spada et al., 1994). Moreover, the mutant proteins have common roles in the disease pathogenesis. The polyQ-containing proteins play an important role in the toxicity induction, but the detail molecular mechanisms are still not fully characterized. It is considered that expanded polyQ repeat will tend to cluster monomer protein into oligomer and move forward to insoluble aggregate in cytoplasm or nuclear. In the generative process of insoluble proteins, some chaperone proteins or ubiquitin–proteasome system involve in the insoluble aggregation and generate toxicity to neuron (Poirier et al., 2002, Kayed et al., 2004).. On the other hand, other studies consider that monomer protein with specific structure is the source of toxicity (Behrends et al., 2006, Lajoie and Snapp, 2010). However, some studies suggest that cleaved protein no matter polyQ-contained or not make neurons dead or dysfunction (Funderburk et al., 2009). Although there have a lot of hypothesis to infer the mechanism, how mutant protein affected is still unknown. But it could be defined that many events in dysfunction by mutation. First, the ubiquitin-proteasome system is highlighted due to its function. With the most important role in cleaning misfolded proteins, the balance in cell with be destroyed if the insoluble aggregate assembles amounts of ubiquitin in it (Paulson et al., 1997, Chai et al., 1999).. -9-.

(12) Additionally, calcium dyshomeostasis is also an important indicator. Neurons are a group of spontaneously active cells. The intercellular concentration of calcium is critical for neuron function and survival (Orrenius et al., 2003). It has also been found calcium unbalanced in SCA6 and HD (Matsuyama et al., 2004, Tang et al., 2005). Furthermore, mitochondria and axonal transport dysfunction are also pathological features of diseases (Cui et al., 2006, Caviston et al., 2007).. Spinocerebellar ataxias type 17. Spinocerebellar ataxias type 17 (SCA17) is a rare neurodegenerative disease whose phenotypes are similar to Huntington's disease (HD). There are some common pathological symptoms between these two diseases, including ataxia, dementia, seizures and psychiatric features (Nakamura et al., 2001, Maltecca et al., 2003, Rolfs et al., 2003, Zuhlke et al., 2003). SCA17, first reported in 1999, iscaused by over-expansion of trinucleotide CAG/CAA on TATA-binding protein (TBP) gene which located on chromosome 6q27. The trinucleotide repeat in normal population is between 25 to 42 repeats; patient would appear abnormal phenotype if their repeats above the range (Koide et al., 1999).. The CAG/CAA trinucleotides are transcribed and translated into polyglutamine (polyQ) in the N-terminal of TBP protein, an essential transcription factor of the cell. The tract of expanded polyQ would affect - 10 -.

(13) protein folding and conformation, and further affected the interaction among TBP and other proteins (Tarlac and Storey, 2003). Of the pathological features, SCA17 likes most of the SCAs; significant atrophy is also observed in cerebellum, especially the Purkinje cell lost. Intranuclear inclusions were also identified in immunohistochemistry analysis of patient’s brain such as putamen and frontal cortex (Nakamura et al., 2001).. Purkinje cell. Purkinje cells are a group of specified neurons in cerebellum. It is one of pyramidal neurons, and it has the most elaborate dendritic trees among neurons in the CNS. It is the only one interneuron in cerebellum to be responsible for integrate input motor and sensory information and output to form a motor coordination (Apps and Garwicz, 2005). Based on their function, Purkinje cells account very few population in cerebellum, about 0.1% of total cerebellar cells (Altman and Bayer, 1996). However, Purkinje cell is one of late-stage development cell types. They appear at days 11-13 in mouse embryo, but develop and migrate at postnatal stage (Miale and Sidman, 1961, Hendelman and Aggerwal, 1980, Armengol and Sotelo, 1991).. According to in vivo study, Purkinje cell would develop slowly in postnatal days 1 to 9 (stage I). In this stage, cell body would grow rapidly, - 11 -.

(14) but without dendrite growth. Postnatal days9 to 18 (stage II) is a rapid dendrite growth period. Dendrite grows rapidly and cells have more difference at this stage compare to the previous stage. Between stages I and II (about postnatal day 9), some cells begin to grow many short peri-somatic dendrites and the others grow a single primary stem dendrite. In spite of two different cell types at P9, they look similar after P12. From P18 to P90 (stage III), dendritic trees become more mature in morphology and function (McKay and Turner, 2005). A similar result could also be observed in the in vitro culture condition, with a faster process than in vivo (Tanaka et al., 2006).. Due to the amount of dendrites and larger soma, Purkinje cells have been well studied in neurite developmental regulation. The Purkinje cell morphology is regulated by several factors such as thyroid hormones, progesterone and estrogen. Recent studies show that reduced thyroid hormones in Purkinje cell will reduce the dendritic development and branch growth (Nicholson and Altman, 1972, Lauder, 1978, Vincent et al., 1982). Progesterone synthesis is increased during the period of Purkinje cell development (Tsutsui et al., 2000). As expected, progesterone and estrogen also show similar effect like thyroid hormones in Purkinje cell development and upgrade cell survival rate in vitro (Sakamoto et al., 2002, Sakamoto et al., 2003).. However, it is unexpected that growth factors like nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) have promote - 12 -.

(15) cell survive in vitro but without promoting dendritic growth (Mount et al., 1993, Larkfors et al., 1996). Brain-derived neurotrophic factor (BDNF) is not only promote cell survive but also affect spine density(Haraguchi et al., 2011).The most recent researches focus on extrinsic factors. However, the intrinsic factor is made a few discussions. The more detailed studies are the role of protein kinase C (PKC) in Purkinje cell development. Showing in recent report, it is a negative regulation in development whiles the PKC is active (Metzger and Kapfhammer, 2000, 2003).. Primary culture. Primary culture technique was established in the early 20th century, and has been developed for more than 100 years. It is a powerful technique for neuroscience research. There are a lot of cell types in nerve system, such as neurons, astrocytes, oligodendrocyte, microglia, endothelial cells, and other perivascular cells. However, the most interesting cell type for neuroscientists, neuron, is a small population in nerve system. A large number of non-neuron cells and the complex interaction between neuron and glia cells would interfere of the neurophysiology observation and experimental evaluation. The above difficult problem could be solved by primary culture. The most advantage of primary culture is to control experiment easily and rules out unnecessary interference. Researchers could easily base on their research topics to isolate specific cell type in specific culture condition. - 13 -.

(16) Primary culture of Purkinje cell was developed in 1980s (Fischer and Schachner, 1982, Gruol and Franklin, 1987) . Several methods had been tried during the recent years. Due to the small cell population, it is very difficult to maintain high density Purkinje cells after long term culture(Furuya et al., 1998, Tomomura et al., 2001, Heuer and Mason, 2003, Yoshimi et al., 2003, Tanaka et al., 2006). Until 2006, Gimenez-Cassina developed a serum-free culture condition, which substantially improved the efficiency. Based on the condition, it could maintain approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells (Gimenez-Cassina et al., 2007). Thus, we could use this method in our study even if significant neuron death of the SCA17 mouse cerebellum is expected.. Histone deacetylase inhibitor. Genetic modification is an important regulation of cell function. One of the important modifications is histone acetylation. Upon histone was acetylated by histone acetyltransferases (HAT), chromosome would be more friable and increase genetic transcription. It balances by histone deacetylase (HDAC). When HDAC remove the acetyl-group from histone, DNA and histone will compact and reduce transcription. In previous studies, lose of HDAC and HAT homeostasis is an important characteristic in many neurodegenerative diseases (Jiang et al., 2003, - 14 -.

(17) Rouaux et al., 2003, Sugars and Rubinsztein, 2003). Thus, maintaining acetylation of histone is a potential therapy in these diseases.. HDAC inhibitor seems a well candidate of this strategy and was reported in many positive cases before (McCampbell et al., 2001, Ryu et al., 2003). Suberoylanilide hydroxamic acid (SAHA), with one hydroxamate group, shows protective effect in many neurodegenerative disease, such as Huntington disease and polyQ-mediated diseases (Steffan et al., 2001, Hockly et al., 2003).. Granulocyte colony-stimulating factor. Granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic hormones that could stimulate myeloid cells growth (Cottler-Fox et al., 2003). It is used widely in clinical therapy to cure neutropenia after cytostatic therapy. In healthy brain, G-CSF plays an important role in learning and memory formation and motor skills (Diederich et al., 2009) Furthermore, G-CSF has been considered with protective effect in neurodegeneration diseases or injury such as amyotrophic lateral sclerosis, Parkinson’s disease, Alzheimer’s disease or acute ischemic (Cao et al., 2006, Meuer et al., 2006, Shyu et al., 2006, Tsai et al., 2007, Pitzer et al., 2008, Pollari et al., 2011). Moreover, G-CSF is considered to protect toxicity-induced dopaminergic neuron death via ERK pathway (Huang et al., 2007). Our previous study has - 15 -.

(18) shown that G-CSF could increase number of Purkinje cells, promote Purkinje cells’ dendritic complexity, and suppress gliosis in hTBP transgenic mice (Chang et al., 2011).. - 16 -.

(19) Materials and Methods. SCA17 transgenic mice. SCA17 transgenic mice which over-expressed mutant human TBP (hTBP) gene with 109 CAG repeats had been established (Chang et al., 2011). The FVB background transgenic mice were maintained by crossing transgenic male mice with wild type FVB female mice (FVB/NJNarl, NALC, Taipei, Taiwan). All of the mice were held in IVC system, and under 12 hour/12 hour day and night cycle, and food and water were made available ad libitum. All experiments were performed during the light phase between 7AM and 7 PM. All experimental procedures involving animals were performed according to the guidelines established by the Institutional Animal Care and Use Committee of National Taiwan Normal University, Taipei, Taiwan.. Genotyping analysis. We cut out some tissue from each mouse during the process of sacrificing. Tissues were then incubated in 0.35 mg/ ml proteinase K (Biovovas, Canada) - containing Direct PCR reagent (Viagen Biotech, Los Angeles, CA, USA) for 5.5 hr under 55℃. The proteinase K activity was stopped by incubating the reaction at 85℃ for 45 min. Extracted DNA samples were used for genotyping according to the protocol - 17 -.

(20) provided by the Direct PCR kit (Viagen Biotech).. PCR was conducted with two pairs of primers, SRY forward (5’ -AGA GAT CAG CAA GCA GCT GG-3’) and backward (5’-TCT TGC CTG TAT GTG ATG GC-3’) primers were used for gender identification; while PL-7 forward (5’-TAT GGT GAG AGC AGA GAT GG-3’) and TBP3R backward (5’-CTG CTG GGA CGT TGA CTG CTG-3’) primers were used for SCA17 genotyping. Fragment sizes of ~290 bps and 765 bps would be amplified respectively, after 25 cycles of reaction with temperature condition of 1 min at 94℃ for denaturing, one min at 68℃ with -0.1℃ touch-down of each cycle for annealing, and 1 min at 72℃for elongation.. Tissue isolation and primary culture. Our protocol was modified from several previous reports (Gruol and Franklin, 1987, Furuya et al., 1998, Gimenez-Cassina et al., 2007, Tanaka et al., 2009). Culture medium bases on NEUROBASAL® medium (GIBCO-Invitrogen, Carlsbad, CA, USA) supplement with 2% B-27 (GIBCO-Invitrogen), 1 mM adenine (Sigma Aldrich, St Louis, MO, USA), 2 mM GlutaMax-I (GIBCO-Invitrogen), 3 mM potassium chloride (Sigma), 10 μg/ml Gentamicin (GIBCO-Invitrogen), 100U/ml Penicillin and Streptomycin (GIBCO-Invitrogen). P0-P1 neonatal mice were sacrificed and cerebella were separated from whole central nervous - 18 -.

(21) system. After meninges were removed, the cerebellum were cut into small pieces and incubated in papain-DNase-containing culture medium [8 U/ml papain and 20 U/ml DNase (Sigma)] for 30 min at 37℃. To stop the proteolytic reaction, medium was replaced with 10% horse serum (GIBCO) and 20 U/ml DNase (Sigma) and tissues were then triturated with 1 ml and 0.2 ml tips sequentially. After centrifugation, cells were resuspended in 1% horse serum containing medium. Finally, cells were seeded into 24-well culture dish which was coated with 100 μg/ml poly-L-lysine. (Sigma).. Drug treatment. All Chinese herbs were boiled water-extracted, and the determination of treatment dose was based upon half of maximal lethal dose (LD50) and half of maximal HDAC inhibitory concentration (IC50). G-CSF (Kirin, Tokyo, Japan) was diluted in 0.1M PBS. SAHA (Nature Wise Biotech and Medicals Corporation; NBM, Taipei, Taiwan) was diluted in DMSO. All treatments are added to fresh medium and exchanged all medium at DIV 7.. Immunocytochemistry. After culture, cells were washed by 0.1M PBS to remove the residual medium, and then fixed in cold 4% paraformadehyde solution for - 19 -.

(22) 30 min. After 3 times washes with 0.1% PBST [0.1M PBS (pH7.4), 0.05% Tween 20] , non-specific protein epitopes were blocked by 5 % horse serum containing PBS-Triton [0.1M PBS (pH7.4), 0.1 % Triton-X-100, 5 % serum] over night at 4℃. Primary antibody (IP3R1, 1:1000, Santa Cruz; GFAP, 1:2000, Chemicon; NeuN, 1:1000, Santa Cruz; pERK, Cell signaling, 1:500; COX2, Chemicon, 1:500; HSP27, Santa Cruz, 1:500; HSP70, Cell signaling, 1:500) was diluted with 5 % horse serum with PBS-Triton [0.1M PBS (pH7.4), 0.1 % Triton-X-100, 5 % serum] and applied to the cells for reaction over night at 4℃. Fluorescence-conjugated secondary antibody (1: 500) and 4',6-diamidino-2-phenylindole (DAPI, 1:50000) were diluted with PBST and incubated with cells for 2 hr at room temperature. All samples were observed by Live image microscope (Leica DMI4000, Germany) and High throughput scanning system (Molecular Devices, Sunnyvale, CA, USA).. Analysis and statistic. All data was analyzed by Metaxpress application software – Neurite Outgrowth (Molecular Devices, Sunnyvale, CA, USA) and compared by independent t test (SPSS Software, Endicott, NY, USA). Critical value of all statistics was 0.05. If p-value < 0.05 it was considered having significant difference between each other.. - 20 -.

(23) Results. Establishment of cerebellum primary culture system. In order to characterize early disease pathology and screen potential drugs for SCA17, we attempted to establish a cerebellum primary culture. At first, we used wild type neonatal mice to identify suitable protocol for culture. Based on previous reports, we tried a lot of methods including parameters involved in different culture medium formulas, mouse age and cell isolation procedure, etc. Some of the results are shown in Figure 1, we tested and examined the growth difference during culture. All of the test condition was modified from previous reports (Gimenez-Cassina et al., 2007). It included serum free, 1% serum, 4mM and 2 mM arabinofuranosyl cytidine (AraC) treatment 1 day after plated. The cell number is significantly reduced in groups with 2 or 4 mM AraC treatment at DIV6, and most cells died after cultured for 14 days. Furthermore, the cells in serum containing group showed better attachment on the plates than the cells in serum-free groups after culture for 6 days. There was also significant difference between groups with or without 1% serum (Figure 1). After cultured for 14 days, we utilized immunocytochemical staining to identify Purkinje neuron and glia cell survival and population. As expected, glia cells were expaned in serum-containing condition. However, there was nerely no cell survival after 4 mM AraC treatment (Figure 1). Finally, we established the SCA17 cerebellar primary culture used the media and culture condition modified from the report of - 21 -.

(24) Gimenez-Cassina (Gimenez-Cassina et al., 2007). To simplify the condition and maintain high cell survival rate so that we could avoid variance from the culture, we removed both the 1% serum and AraC from the media. It has been reported that serum-free condition has been used to reduce the glia (Wallace and Johnson, 1989).. During the in vitro culture, we immunostained the primary culture at four stages after culture: 7 days in vitro (DIV 7), 14 days in vitro (DIV 14), 21 days in vitro (DIV 21), and 28 days in vitro (DIV 28) (Figure 2). Those cultures were stained with the antibody of Inositol 1,4,5-Triphosphate Receptor type 1 (IP3R1),a specific Purkinje cell marker, to observe Purkinje Neuron development during culture. We found that Purkinje neurons could survival well even cultured until DIV 28. Moreover, Purkinje cells showed significant developments in neurite length and complexity along with culture time (Figure 2). These results were consistent with previous in vitro and in vivo studies (McKay and Turner, 2005, Tanaka et al., 2006). In the culture of DIV 14, we could see two different types of Purkinje cells (Figure 2 panel of DIV 14). One of them has several processes extended from soma and form a sea star-like shape. The other one has a single and evident process and without many branches. Both of these two types of Purkinje cells were also identified in postnatal day 12 mouse cerebella simultaneously (McKay and Turner, 2005).. - 22 -.

(25) We also used another antibody, Neuronal Nuclei (NeuN), to recognize mature neuron in the primary culture (Figure 3A). In all stages of observation, we could see that mature neurons account for a majority of cells under the observation view of microscope. Although less NeuN-positive signals were identified at DIV 28 compared to that of DIV 14 and DIV 21, there was still about 60% of the cells are NeuN-positive cells.. In addition, we also utilized Glial fibrillary acidic protein (GFAP) as a glia cell marker to analyze whether abundant glia cell proliferate during culture. Compared to original condition, which used AraC in medium to reduce glia cell growth, with our modified culture condition, serum-free condition used to replace the function of AraC, we could see less glia cells in immunocytochemistry results (Figure 3B).. These results reveal that cerebellar primary culture could be used to characterize the early phenotypes of SCA17 transgenic mice.. Examination of SCA17 transgenic mouse early pathology with cerebellar primary culture. We used the primary culture system to analyze early neuronal degeneration of SCA17. We utilized IP3R1 as a marker of Purkinje cell to analyze morphological difference between wild-type and transgenic - 23 -.

(26) mouse culture. We could not identify any difference between each other at DIV 7. However, significant difference was observed at DIV 14. Shorter neurites and fewer branches were shown in transgenic group and which was continued until DIV 21(Figure 4).. By using the Neurite Outgrowth application program (MetaXpress, Molecular Devices, Sunnyvale, CA, USA) to analyze these neurite outgrowth data and found some of the results were statistic differences between these two groups, including total outgrowth, mean process length, maximum and median process length, number of branch and number of process. We chose four of the differential parameters to represent the morphological status of Purkinje cell (Figure 5), and which will be used for the rescuing index during drug screening. Total outgrowth means summation of all neurite length in each neuron; process number is the number of primary neurite; mean processes length is total outgrowth divided by processes number; and branch number is all branches on process no matter they are primary or secondary branches.. We found that three of the four parameters, including total outgrowth, mean process length and number of branch, show significant difference at both DIV 14 and DIV 21 stages, which is consistent with the significant cerebellar culture degeneration of transgenic mice. However, significant difference in processes number between wild-type and transgenic culture was observed at DIV 7, which reveals the - 24 -.

(27) degeneration of transgenic cerebella might occur earlier than DIV 7. We therefore further characterized the process numbers of cultures before DIV 7 and found there was no difference between wild-type and transgenic cell at DIV5.. In addition, it’s interesting that a declined tendency in processes number was observed between DIV 14 and DIV 21. The similar result was also reported in previous in vivo and in vitro studies (McKay and Turner, 2005, Tanaka et al., 2006).. Evaluating aggregated protein generation in SCA 17 primary culture system. It has been indicated that aggregated proteins were accumulated in patients’ Purkinje neurons with polyQ disease progression (Rolfs et al., 2003). It was also observed in our transgenic mouse studies (Chang et al., 2011). Thus, we analyzed the aggregation formation in the cerebellar primary culture. To identify aggregation formations, we utilize low titer 1TBP18 antibody to detect the over-expressed and aggregated TBP protein in cells at DIV7, DIV14 and DIV21. It showed no aggregation was detected at DIV7 (Figure 6A, DIV7 panel). However, most of the Purkinje cells present significant aggregation at DIV21 (Figure 6A, DIV21 panel). About 80% of the cells showed aggregated 1TBP18 signals in their nuclei. However, at DIV14, transgenic group cells have - 25 -.

(28) significant degeneration compared to wild-type cells (Figure 5), only limited cells showed aggregated protein in their nuclei (Figure 6B).. Although we could observe aggregated proteins at DIV14, their aggregate morphology is very different from DIV21. Based upon the 1TBP18’s staining signal, we found most of aggregates are more uniform and small (Figure 6C, upper panel). Only a small portion of the aggregates have significant signals at DIV14 (Figure 6, lower panel). Our results suggest that aggregation may not an initial event but a late stage symptom of degeneration.. Evaluating the therapeutic effects of potential compounds with SCA17 primary culture. To evaluate whether the SCA17 cerebellar primary culture is a reliable platform used for drug screen, we need to have a positive control compound to test this system. Suberoylanilide hydroxamic acid (SAHA) an inhibitor of histone deacetylases, has been proved to have neuroprotective effect in many neurodegenerative diseases, such as Huntington's disease and frontotemporal dementia (Steffan et al., 2001, Cenik et al., 2011, Chen et al., 2012). We intended to use SAHA as a positive control in our primary culture system. However, after 100 nM of SAHA treatment, we observed significant decrease of neurite length, process and branch numbers of Purkinje cells (Figure 7). - 26 -.

(29) Therefore, we tried to use another compound, granulocyte colony-stimulating factor (G-CSF) as positive control. G-CSF is one of the hematopoietic hormones that could stimulate myeloid cells growth (Cottler-Fox et al., 2003). It is used widely in clinical therapy to cure neutropenia after cytostatic therapy. Our previous study has shown that GCSF could increase number of Purkinje cells, promote Purkinje cells’ dendritic complexity, and suppress gliosis in SCA17 transgenic mice (Chang et al., 2011). Furthermore, G-CSF has also been reported to show protective effect in other neurodegeneration diseases such as amyotrophic lateral sclerosis (Pollari et al., 2011), and Alzheimer’s disease animal models (Tsai et al., 2007).. We were interested in the protective effects of G-CSF on our cerebellar primary culture. First, we used 25 ng/ml concentration in the primary cell culture followed previous studies (Schabitz et al., 2003, Pan et al., 2010). After treatment for 7 days, there was no significant difference identified between the two groups (Figure 8A). Therefore, we applied higher concentrations, 50 ng/ml and 100 ng/ml, respectively on the culture in the further treatments. There was still no effect under the treatment of concentration of 50 ng/ml, but effect on promoting neurite outgrowth was shown under 100 ng/ml of concentration (Figure 8B).. - 27 -.

(30) Molecular effect of G-CSF in SCA17 primary culture cells. G-CSF was reported to have many functions, it could promote cell survival, reduce inflammatory response and anti-apoptosis. Here we tested several directions to elucidate G-CSF effects on SCA17 primary culture system. First, we analyzed chaperone protein 27 (HSP27) and chaperone protein 70 (HSP70) levels after G-CSF treat. HPS27 was reported to be an apoptosis inhibitor via activating phosphatidyl inositol 3-kinase-dependent (pI3K-dependent) pathway (Havasi et al., 2008). HSP70 plays an important role in the cell's machinery for protein folding, and help to protect cells from stress (Tavaria et al., 1996, Morano, 2007). In primary cells, it is interesting that HSP70 shows conspicuous expression in Purkinje cell (Figure 9A). HSP27 was up-regulated and concentrates in most of nuclei after G-CSF treatment (Figure 9B).. Owing to our serum-free culture system, we could not use gliosis to assess the inflammatory response. Thus, we chose an inflammatory-induced enzyme, cyclooxygenase 2 (COX 2), as a marker to evaluate inflammatory effect after G-CSF treatment. We observed that COX 2 was significantly up-regulated after G-CSF treatment (Figure 10). Finally, according to previous studies and HSP27 up-regulated expression, we analyzed phospho-ERK (pERK) to check cell survival effect of G-CSF treatment. We found that pERK signal was up-regulated after G-CSF treatment (Figure 11A-D). In addition, we could find that pERK signal was slightly increased in Purkinje cell (Figure 11G-J), which - 28 -.

(31) indicates that G-CSF might affect Purkinje cell directly. Furthermore, when we utilized antibodies against NeuN and GFAP to detect whether pERK expression in neuron or gila cell, we observed abundant signals shown in neuron cells but not in gila cells (Figure 12-13).. These data reveal that G-CSF treatment protects neuronal survival through the pERK pathway. Screening of Chinese herbs on transgenic mouse cerebellar primary culture. In order to identify potential drugs for SCA17, we have screened several herbs which have ability in inhibiting histone deacetylase (HDAC) or neuron protection. According to the half of maximal lethal dose (LD50) and half of maximal HDAC inhibitory concentration (IC50) of these herbs, we applied the possible effective concentration of herbs to our primary culture system. We assessed total seven herbs. Consideration in a timeand cost-effective strategy, we treated the transgenic primary culture at DIV 7, a mild degeneration stage, and observed the treatment results at DIV 14. All treatments were repeated on the cultures from three different transgenic mice.. First, we evaluate effect of NH-001 in different doses (Figure 14). It showed no difference with dose of 100 μg /ml. However, cell was - 29 -.

(32) degenerated with the concentration increased. It seems NH-001 has no protective effect in SCA17 primary cells. Next, we treated 100, 200 and 500 μg /ml of NH-003 on the transgenic group. The result showed significant increase in total outgrowth, mean processes length and branch number at different doses (Figure 15). Furthermore, when we treatment with 100, 200 and 500 μg /ml of NH-007, it show no difference in any parameters (Figure 16). We also tested NH-013 with the same concentration. However, we found that all of the four parameters were significant decreased in the treated group (Figure 17), no matter how low concentration we used. Similar with NH-013, NH-008 was applied to the culture with concentration of 0.1, 1 and 5μg /ml. We found significant decrease in neurite length after treatment (Figure 18). A similar result was also obtained with the treatment of NH-035 in concentrations of 1, 10 and 100 μg /ml, all of the four parameters was reduced after treatment (Figure 19).. Finally, we evaluated the effect of NH-015. Due to the low LD50, we applied 10 μg /ml of NH-015 in the primary culture treatment. The results show that NH-015 has the potential in supporting neurons’ neurite outgrowth, with the all 4 parameters showing significant increased after treatment (Figure 20).. - 30 -.

(33) Discussion. According to our results, we have established a cerebellar primary culture with a more simplified medium and culture condition than previous reports (Furuya et al., 1998, Gimenez-Cassina et al., 2007). With our culture condition, cerebellar cells isolated from postnatal day 0-1 mice could be maintained to have 70% neuron and much less glia cells in the primary cell population during DIV 7-28. Moreover, this system which bypasses a complex medium formula could reduce the risk of variation in medium preparation. In addition, using serum-free condition to replace AraC in reducing glial cell population also reduced the serum interference. This primary system could provide a suitable platform for analyzing early degeneration and screening potential drug for SCA17.. In our system, we found the Purkinje neuron growth pattern was similar to previous studies in vitro and in vivo (Armengol and Sotelo, 1991, Tanaka et al., 2006), including different Purkinje cell morphologies presented at DIV 14 (Fig.2 panel) and reduced process numbers at DIV 21. It might be caused by special neuronal development behavior: in the beginning of neuronal development, neuron cells would extend lots primary neurites to explore the environment. If it could make a correct connect, the correct-connected process could maintain and survive. If not, the processes would lose the trophic factors and progress to “death”. The characteristic is particularly evident in the Purkinje neuron due to its function and cell shape (Bear et al., 2007). However, that the process - 31 -.

(34) number was significantly reduced at DIV 7 in transgenic primary culture was unexpected, we therefore suspect that process numbers might be the first sign of neuron degeneration.. Based on our morphology and aggregation analysis results, it reveals that aggregated proteins are a later symptom of SCA17 degeneration. In the morphology assessment, Purkinje cells degenerated begin at DIV7 and significantly at DIV14. However, only few Purkinje cells had aggregation at DIV14. The aggregation showed various morphologies at this stage, but with most of them were small and uniform. Although the aggregation formation observed started at around DIV14, the spread of aggregation was in a very high speed. Eighty percent of Purkinje cells showed aggregation in their nuclei at DIV21. It is interesting to further investigate the aggregation formed in such a short period and how the process is regulated. It is suggested that risk of aggregation formation is random in individual, and lifestyle-association was considered (Clarke et al., 2000). But it is sure that risk is correlated with protein concentration in the cell (Soto, 2003, Dobson, 2004). However, TBP protein is an important transcription factor in cell function, and previous study indicated that TBP concentration is rigorous regulation in cell based on its function (Friedman et al., 2007). It also shows that the level of mutant TBP is lower than normal TBP level in our SCA17 transgenic mice (Chang et al., 2011). Therefore, there may be other mechanisms in involved in TBP aggregation formation.. - 32 -.

(35) In addition, it has been reported the insoluble aggregation may cause toxicity in many poly glutamine diseases (DiFiglia et al., 1997, Sanchez et al., 2003, Kaytor et al., 2004). However, we observed that neurite degeneration occurred earlier than the aggregation formation (Fig. 5 and 6B). It revealed that aggregation may not be a necessary event for disease initiation. Some of studies also suggested that aggregation is not initiation of symptoms but help to protect against cellular dysfunction (Klement et al., 1998, Saudou et al., 1998, Lin et al., 1999). According to the several previous reports and our observation, we suggest that toxicity of aggregated protein may not be an initiator but an enhancer of disease. TBP aggregation may recruit other transcription factors, like TFIIB or NF-YA (Friedman et al., 2007, Lee et al., 2012) to cause transcriptional dysregulation, and further leads to cell degeneration.. SAHA is a potential compound for neurodegenerative disease. It can ameliorate spinal muscular atrophy (SMA) phenotype (Riessland et al., 2010, Mutsaers et al., 2011). Therefore, HDAC inhibitor also shows protective effects in poly-glutamine mediated disease such as Huntington's disease (Mielcarek et al., 2011, Soragni et al., 2011). Moreover, HDAC6 could negatively regulate synaptic plasticity and memory formation (Guan et al., 2009). However, the 100 nM of SAHA treatment showed a significant toxicity to the Purkinje cells in our primary culture system.. G-CSF was used as another candidate to evaluate the primary culture - 33 -.

(36) system. The protective effect was shown when we applied 100 ng/ml of dose to the culture (Fig. 8B). The explanation for higher concentration of G-CSF to exert a positive effect in our system could be due to cell type or treatment strategy. In previous report, G-CSF treatment was combined with taurine, an inhibitory neurotransmitter (Schabitz et al., 2003, Pan et al., 2010). G-CSF was used alone in our study. Thus, a higher concentration of G-CSF may be applied next to see if higher effect can be exerted on Purkinje cells.. G-CSF was reported to have anti-apoptosis and anti-inflammation effects. In our study, we observed that pERK level was up-regulated after G-CSF treatment. It is similar to ours in vivo study (Chang et al., 2011). The increased pERK expression was distributed in Purkinje cells and neuronal cell but not in glial cells, which suggests that G-CSF could affect Purkinje cell and other neuron survival directly. We speculate that the survival of other neurons could also benefit to the Purkinje cells.. COX1/2 was used as inflammatory signaling markers to evaluate the inflammation status of the cerebellar primary culture after G-CSF treatment; it is interesting that we observed that COX2 level was significant increased after treatment. It is reported that polyglutamine overexpression will suppress the activation of transcription factor NFκB activity and reduce the down-stream gene COX2 expression (Goswami et al., 2006). It was also suggested that NFκB activity was increased for an early protective response to oxidative stress or mitochondrial dysfunction - 34 -.

(37) (Lezoualc'h et al., 1998). We speculate that the G-CSF treatment may provide an early protective effect to the oxidative stress or mitochondrial dysfunction of neuronal cells resulted from the polyglutamine expansion. The similar result was also observed in cancer cell experiments, with COX2 inhibitor induced apoptosis and inhibited cell growth (Chang and Weng, 2001). Therefore, we consider that the G-CSF induced COX2 may have an anti-apoptotic effect in primary culture system.. In addition, we also observed HSP27 level was up-regulated after G-CSF treatment. HSP27 plays an important role in anti-apoptotic response. It would be up-regulated when cells under stress. Previous reports indicated that HSP27 is working through mitochondria-associated pathway to against heat shock or injury (Samali et al., 2001, Stetler et al., 2008). Most of studies are considered that it acts through inhibiting caspase activity to protect cell apoptosis (Garrido et al., 1999, Samali et al., 2001).. To evaluate the therapeutic potential of Chinese herbs, we tested different doses of several herbs and examined the Purkinje cell morphology after the treatments. Most of the treatments show reduction in Purkinje neurite growth. We conjecture that all herbs were extracted by water and without purification. Several compositions may interfere of cell growth at the same time. Therefore, we could find some herbs showed toxicity in low dose but not in high dose, such as NH-035 (Fig. 13). However, we still found some drugs showed potential protective effects - 35 -.

(38) in SCA17 primary cells. NH-003 is one of them, and showed dose-depended effects and low toxicity. These results indicate that our cerebellar primary culture could be a platform to assess potential treatments for SCA17.. In summary, we established a simplified system to maintain cerebellar primary neuron culture. We indentified early degeneration of Purkinje cells through this system. We confirmed the neuron protective effect of G-CSF on this system. Additionally, we screen Chinese herbs with this system and found herb NH-003 and NH-015 could be potential in SCA17 treatment.. - 36 -.

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(54) Appendix 1. List of primary antibodies used in this study.. Name IP3R1. Goal Purkinje neuron. Species Goat. Source Santa Cruz. Titer 1:1000. marker NeuN. Mature neuron maker. Mouse. GFAP. Gila cell marker. Mouse. Chemicon Chemicon. 1:1000 1:2000. 1TBP18 Aggregation marker. Mouse. QED. 1:30000. pERK. Cell survival signal. Rabbit. Cell signaling. 1:500. COX2. Inflammation signal. Rabbit. Chemicon. 1:500. HSP27. Chaperone marker. Mouse. Santa Cruz. 1:500. HSP70. Chaperone marker. Rabbit. Cell signaling. 1:500. - 52 -.

(55) Figure 1. Fig. 1. Evaluation of the effects of different culture conditions for cerebellar primary culture. The cellular growth images record during culture after 2, 6, 10 and 14 days. Immunocytochemistry staining using IP3R1 and GFAP to identify Purkinje cells and gila cells, respectively. - 53 -.

(56) Figure 2. Fig. 2. Neurite morphology of the SCA17 transgenic mouse cerebellar primary culture in vitro. IP3R1 antibody was used to immunostain Purkinje neuron at four stages, DIV-7, DIV-14, DIV-21, and DIV-28. Scale bar: 40 μm.. - 54 -.

(57) Figure 3. Fig. 3. Cell population in cerebellar primary culture was characterized at different stages. (A) NeuN antibody was used in - 55 -.

(58) immunocytochemistry to analyze mature neurons in the primary culture. (B) GFAP antibody was utilized in immunocytochemistry to stain glia cell in the primary culture. Scale bar: 40 μm.. - 56 -.

(59) Figure 4. Fig. 4. Morphological difference between wild-type and transgenic cerebellar primary cultures. IP3R1 immunocytochemistry identified the reduced neurite outgrowth of Purkinje cells in transgenic group at DIV-14 and DIV-21. Scale bar: 40 μm.. - 57 -.

(60) Figure 5. Fig.5. Quantitation of neurite outgrowth in the cerebellar primary culture between wild-type and transgenic groups. Statistics were conducted with two-factor ANOVA, and post-hoc analysis is Scheffe test. *, P <0.05 ; **, P < 0.01; ***, P < 0.001, n = 100.. - 58 -.

(61) Figure 6. Fig. 6. Assessment of aggregated protein formation at different stages. (A) Immunstaining with IP3R1 and 1TBP18 antibodies to recognizes aggregated protein in Purkinje cells at DIV7, 14 and 21. (B) Quantification the percentage of aggregation at different stages. (C) Aggregation morphology identified in DIV14 transgenic Purkinje cells. Statistics were conducted with t-test, *, P < 0.05; ***, P < 0.001, n > 30. - 59 -.

(62) Figure 7. Fig. 7. Evaluating SAHA effects in transgenic cerebellar primary culture. Solid bars represent the treatment groups, hollow bars represent the control groups. Statistics were conducted with t-test. **, P-valus< 0.01, n = 100.. - 60 -.

(63) Figure 8. Fig. 8. Evaluating G-CSF effects in transgenic cerebellar primary culture. - 61 -.

(64) Solid bars represent the treatment groups at different concentration, hollow bars represent the control groups. Statistics were conducted with one-way ANOVA and post-hoc analysis is Dunnett’s test. *, P-valus < 0.05, n = 100.. - 62 -.

(65) Figure 9. Fig. 9. Chaperone proteins are up-regulated after G-CSF treatment. (A) Immunstaining with IP3R1 (green) and HSP70 (red) antibodies to recognizes chaperone Hsp70 expression in primary culture. Scale - 63 -.

(66) bar = 20 μm. (B) Immunstaining with IP3R1 (green) and HSP27 (red) antibodies to recognizes chaperone Hsp27 expression in primary culture. Scale bar = 40 μm.. - 64 -.

(67) Figure 10. Fig. 10. Inflammatory signal is up-regulated after G-CSF treatment. Immunstaining with IP3R1 (green) and Cox2 (red) antibodies to recognizes inflammatory response in primary culture. Scale bar = 20 μm .. - 65 -.

(68) Figure 11. Fig. 11. Characterization of the pERK signal expression in Purkinje cells after G-CSF treatment. Immunstaining with IP3R1 (green, B, E, H and K) and pERK (red, A, D, G and J) antibodies to recognizes - 66 -.

(69) the localization of pERK survival signal in Purkinje cells. (A-F) Cell images in lower magnification after GCSF treatment. Scale bar = 40 μm. (G-L) Cell images in higher magnification after GCSF treatment. Scale bar = 5 μm .. - 67 -.

(70) Figure 12. Fig. 12. Characterization of the pERK signal expression in neuron cells after G-CSF treatment. Immunstaining with NeuN (green, B, E, H and K) and p-ERK (red, A, D, G and J) antibodies to the - 68 -.

(71) localization of pERK survival signal in neuron cells. (A-F) Cell images in lower magnification after GCSF treatment. Scale bar = 40 μm. (G-L) Cell images in higher magnification after GCSF treatment. Scale bar = 10 μm .. - 69 -.

(72) Figure 13. Fig. 13. Characterization of the pERK signal expression in glia cells after G-CSF treatment.. Immunstaining with GFAP (green, B, E, H and - 70 -.

(73) K)and phospho-ERK (red, A, D, G and J) antibodies to recognizes survival signal in glia cells. (A-F) Cell images in lower magnification after G-CSF treatment. Scale bar = 40 μm. (G-L) Cell images in higher magnification after G-CSF treatment. Scale bar = 10 μm.. - 71 -.

(74) Figure 14. Fig. 14. Evaluating NH-001 effects in transgenic cerebellar primary culture. Solid bars represent treatment groups, hollow bars represent control groups. Statistics were conducted with one-way ANOVA, and post-hoc analysis is Dunnett’s test. **, P < 0.01, n = 100.. - 72 -.

(75) Figure 15. Fig. 15. Evaluating NH-003 effects in transgenic cerebellar primary culture. Solid bars represent treatment groups, hollow bars represent control groups. Statistics were conducted with one-way ANOVA, and post-hoc analysis is Dunnett’s test. *, P <0.05 ; **, P < 0.01; ***, P < 0.001, n = 100.. - 73 -.

(76) Figure 16. Fig. 16. Evaluating NH-007 effects in transgenic cerebellar primary culture. Solid bars represent treatment groups, hollow bars represent control groups. Statistics were conducted with one-way ANOVA, and post-hoc analysis is Dunnett’s test, n = 100.. - 74 -.

(77) Figure 17. Fig. 17. Evaluating NH-013 effects in transgenic cerebellar primary culture. .Solid bars represent treatment groups, hollow bars represent control groups. Statistics were conducted with one-way ANOVA, and post-hoc analysis is Dunnett’s test. *, P <0.05 ; **, P < 0.01; ***, P < 0.001, n = 100.. - 75 -.



(80) Figure 20. Fig. 20. Evaluating NH-015 effects in transgenic cerebellar primary culture. Solid bars represent treatment groups, hollow bars represent control groups. Statistics were conducted with t-test. *, P-value < 0.05, n = 100.. - 78 -.

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