建立和分析過量表現PPP2R2B 基因Bβ1之細胞與基因轉殖小鼠

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(10) . i. ABSTRACT. i. INTRODUCTION Autosomal dominant cerebellar ataxias (ADCA) Spinocerebellar ataxias (SCA). . . 1 2. Spinocerebellar ataxias12 (SCA12). 3. Protein phosphatase 2A (PP2A). 5. Relationship between SCA12 and PP2A. 7. Oxidative stress. 9. MATERIALS AND METHODS SCA12 mouse model. 12. SCA12 cell model. 16. RESULTS SCA12 mouse model. 21. SCA12 cell model. 30. DISCUSSION SCA12 mouse model. 34. SCA12 cell model. 41. REFERENCE. 43. APPENDIX SCA12 mouse model. 51. SCA12 cell model. 88. .

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(17) Abstract Spinocerebellar ataxia type 12 (SCA12) is an autosomal dominant neurodegenerative disorder which has been described in pedigrees of German American and Indian descent. The phenotype typically begins with tremor in the fourth decade, progressing to include ataxia and other cerebellar and cortical signs. SCA12 is associated with an expansion of a CAG repeat in the 5’region of the gene PPP2R2B isoform 1 (Bβ1), which encodes a brain-specific regulatory subunit of the protein phosphatase PP2A. PP2A is a heterotrimeric enzyme and is a major Ser/Thr phosphatase contains three subunits, structural subunit (A), regulatory subunit (B) and catalytic subunit (C). B subunit that define PP2A isoform and their physiological roles are implicated in multiple cellular functions, including cell cycle regulation, tau phosphorylation, and apoptosis. The repeat size ranges from 55 to 78 triplets in the mutant allele of affected individuals, and from 9 to 28 triplets in normal alleles. It is speculated that the repeat expansion in Bβ1 may influence Bβ1 expression and in turn alter the activity of PP2A which may influence the cellular signaling pathways and induce the neurodegeneration. We have established Bβ1 inducible cell lines and transgenic mice to test this hypothesis. Of the mouse model, we already established heterozygous and homozygous transgenic mice. We found their behavioral abnormality through rotarod, locomotor and HomeCageScan analysis. In conclusion, our results suggest that the overexpression of PPP2R2B cause pathogenesis in the mouse cerebellum, including PCs and neuronal cell loss gliosis and also some emotional change according to their behavior abnormality. Of the.

(18) inducible cell lines, by western blot analysis, MTT assay and morphology observation, we observed that our clonal cell lines have decreased survival rate compared to pEGSH control group after treatment with oxidative drugs, indicate that these cells are more vulnerable under the oxidative stress. In summary, the overexpression of Bβ1 did have neurotoxicity effect in both mouse and cell models and finally cause the neurodegeneration..

(19) Introduction Autosomal dominant cerebellar ataxias (ADCA) Autosomal dominant cerebellar ataxias are hereditary neurodegenerative disorders that are known as spinocerebellar ataxias (SCA) in genetic nomenclature. The syndromes of these diseases were characterized by a slowly progressive cerebellar degeneration, and were presenting as ataxia of gait, stance and limbs, dysartrhia and/or oculomotor disorder. The degenerative process also including retina degeneration, or spreading to optic nerve, ponto-medullary systems, basal ganglia, cerebral cortex, spinal tracts or peripheral nerves. Harding (1982) classified ataxias according to their mode of inheritance and clinical features (Schols et al., 2003). Other researches district the molecular classification base on ataxia loci and genes, and the prevalence has been estimated between 0.8 and 3.5:100,000 (Silva et al., 1997). ADCA type I with variable additional signs like optic atrophy, ophthalmoplegia, bulbar signs, spasticity, extrapyramidal features, peripheral neuropathy, sphincter disturbance and dementia. ADCA type II with pigmentary macular degeneration, but also been described in rare cases of SCA2 and SCA3, which were definited in type I. ADCA type III only show cerebellar degeneration but not shortening life-span (Table 1)(Singh et al., 1993). As a rule, phenotype and onset age were highly variable within generically defined even within single families, the age at onset as well as severity of symptoms. Because of these variability, the genotype can’t be predicted by only clinical analyses. 1.

(20) Spinocerebellar ataxias (SCA). Spinocerebellar ataxias (SCAs) are a clinically heterogeneous of inherited neurodegenerative disorders caused by genetic locus mutation. SCA is also been used to mark recessive and occasional diseases. Patients exhibit a slowly progressive cerebellar degeneration and usually accompanied with progress subsequently to extra-cerebellar structures. There is highly difficult to clinical diagnosis of SCA subtypes, because of phenotypes always have slight overlap between genetic subtypes and its’ heterogeneity. Depending on the gene loci mutation, SCAs have been divided into 28 subtypes, SCA1 to SCA28.. Currently, SCAs are classified into three major groups based on the molecular level, a CAG expansion encoding a polyglutamine (polyQ) repeat (SCA1, 2, 3, 7, 17)(Zoghbi, 2000), nucleotide repeats expansion in the untranslated region (UTR) thus alter gene/protein expression (SCA8, 10, 12)(Koob et al., 1999), and the channelopathy (SCA6)(Margolis, 2002). Most types of SCAs cause by CAG repeat expansion in the coding region of the diseases-causing genes, such as spinal and bulbar muscular atrophy (SBMA), dentatorubral and pallio-luysian atrohy (DRPLA), SCA types 1, 2, 3, 6, 7, and 17, but the functions or the disease-caused machinery of the affected proteins are still unknown. Indeed, the pathogenesis is directly linked to the expanded polyQ stretch (Zoghbi, 2000), when polyQ expansion repeat length is over around 39 glutamine residues, which will develop a neurological phenotype and neuropathology (Ordway et al., 1997). Furthermore, longer polyQ repeat 2.

(21) will cause earlier disease onset and more severe phenotypes (Schols et al., 2004). Expanded polyQ repeats can form insoluble aggregates, when repeats length at normal range remain soluble (Scherzinger et al., 1997). Protein aggregates are a hallmark of all polyglutamine diseases. These protein aggregates called nuclear inclusions (NI) was thought to be neurotoxic property (Manto, 2005). Otherwise, the formation of inclusions could be a protective mechanism against the toxicity of expanded polyQ proteins (Saudou et al., 1998). Some SCA subtypes were due to repeat expansions in untranslated regions or by point mutations, for example, SCA12 is associated with an expansion of a CAG repeats in the 5’region of the gene PPP2R2B (Holmes et al., 2001).. An interesting feature of SCA is anticipation, that is, the CAG expansions have instability of repeats, thus resulting in longer length of CAG repeat tracts. The relationship between CAG repeat length and phenotypic presentation has been reported. Another feature is penetrance, which is age-dependent but almost 100% if gene carriers get old enough. Genetically counseling might be difficult, because family history in SCAs may be negative in cases of false paternity or subtypes with late onset of symptoms. Offspring of affected individuals are about 50% risk of carrying the disease. Consequently, development of the molecular mechanisms acknowledge of SCAs will be helpful for future therapies.. Spinocerebellar ataxias12 (SCA12). SCA12 is one of the SCA groups, which has been associated with 3.

(22) expansions of non-coding repeats. SCA12 was identified in a pedigree of German descent (Holmes et al., 1999) and a second pedigree of Indian descent (Fujigasaki et al., 2001). In previous study, magnetic resonance data of affected patients showed central nervous system (CNS) atrophy, especially cerebral cortex and cerebellum (Holmes et al., 2001), microscope image reveals neuron loss and shrink in cerebellar and brain stem Patients present with action tremor of the upper extremities and progress to develop a wide range of signs and symptoms, including mild cerebellar dysfunction, hyperreflexia, subtle parkinsonian features, and, in the oldest subjects, dementia.. Genetically, SCA12 is associated with an expansion of a CAG repeats in the 5’region of the gene PPP2R2B, which encodes a brain-specific regulatory subunit of the protein phosphatase 2A (PP2A). Because of the CAG expansion located within the promoter region, thus might effect the phosphatase activity, furthermore, even cause neuron cell damage. Normal repeat length is 7-32 triplets, India population contain more than 12 triplets than others (Fujigasaki et al., 2001). The threshold of repeats from normal and disease length is unclear, but expanded alleles of length are about from 55–78 triplets. The onset age of SCA12 is ranged from 8 to 55, with an average onset at 30 years old, but the correlation between repeat expansion size and age of onset in SCA12 are still unknow. It is also the only SCA in which the mutation may increase gene expression; in this case, because the gene product is involved in regulation of PP2A, abnormal phosphatase activity may be critical to SCA12 pathogenesis (Holmes et al., 2003) 4.

(23) Protein phosphatase 2A (PP2A). In eukaryotes, the phosphorylation state of key proteins is critical in most cellular process and depends on the balance of protein kinase and phosphatase activity. Compared to kinases, very little was known about protein phosphatase (Strack, 2002). PP2A is one of the four major classes of serine/threonine phosphatase, which is also including PP1, PP2B and PP2C. This family is abundant in cells and with important functions involved in regulation of cell cycle, cellular morphogenesis, ion channel function, neurotransmitter releasing, microtubule assembly and apoptosis (Wang et al., 1998; Price and Mumby, 1999; Calin et al., 2000; Virshup, 2000; Gotz et al., 2004).. PP2A has a heterotrimeric structure.Several holoenzyme complexes have been isolated from a variety of tissues and species. The core dimer consists of a ~36 kDa catalytic (C) and a ~65 kDa structural scaffolding (A) subunits (Janssens and Goris, 2001). The AC core enzyme can bind to a third subunit, which is variable to form PP2A holoenzyme, called regulatory (B) subunit. The regulatory subunits are encoded by four multigene families (B, B’, B’’ and B’’’). The B family (also know as PR55) consists of four genes, Bα, Bβ, Bγ and Bδ. The B’ family (know as B56 or PR61) consists of al least seven isoforms encoded by five genes, B’α, B’β, B’γ, B’δ and B’ε, which will participate in wnt/β-catenin signaling (Seeling et al., 1999). B’ subunits bind to cyclins at G1 and G2 stages, inducate that PP2A holoenzyme containing subunits involved cell cycle regulation (Okamoto et al., 2002). Four members of B’’ family were 5.

(24) encoded by three genes, PR72/130, PR48 and PR59, and control the G1/S cell cycle transition (Voorhoeve et al., 1999; Janssens et al., 2003).. B’’’. (striatin, SG2NA), which are important and necessary for living of mammalian cells (Gotz et al., 1998; Kong et al., 2004; Strack et al., 2004). Thus, the total PP2A holoenzymes are about 75 (Martens et al., 2004; Schild et al., 2006). In addition, the regulation of PP2A activity is due to the complexity of different PP2A holoenzyme and posttranslational modifications, such as phosphorylation and methylation of the C subunit (Janssens and Goris, 2001).. PP2A is particularly abundant in brain during development and maturation, even though, when its AC core enzyme binding with various regulatory subunits will show a subunit specific expression (Janssens and Goris, 2001), but roles of large B-type family are still abstrusely. One in vitro evidence indicated that regulatory subunits affect enzymatic activity and substrate specificity of PP2A (Kamibayashi et al., 1994). In addition to alternative splicing or promoter used, although the amino acid sequences of Bα to Bδ have more than 80% similarly, the very slight difference of amino acid sequence will also modulate PP2A activity (Strack et al., 2002). Several PP2A regulatory subunits show restricted tissue expression. Bα is the large member of B-family, and expressed in different cells to mediate dephosphorylation of tau and vementin (Sontag et al., 1995; Sontag et al., 1996; Turowski et al., 1999). Bα and Bδ are expressed widely in different tissues, but Bβ and Bγ mediate specifically neuronal function and only expressed in brain, suggesting that different B subunit controll neuron development and neuron-neuron junction in 6.

(25) different phase. Overexpression of Bγ subunit can stimulate PC12 cell differentiation through MAPK cascade (Strack et al., 2002).. An important role of Bβ in neuronal survival was suggested by the discovery that the neurodegenerative disorder SCA12 is caused by a trinucleotide repeat expansion in the promoter region of the human Bβ gene (Holmes et al., 1999). By alternative promoter splicing, Bβ gene transcribed into two mRNA, Bβ1 and Bβ2 (Dagda et al., 2003). The divergent N terminus of Bβ2 does not affect phosphatase activity but encodes a subcellular targeting signal. Bβ2 is expressed predominantly in forebrain areas, and PP2A holoenzymes containing Bβ2 are about 10-fold less abundant than those containing the Bβ1 isoform(Dagda et al., 2003). Bβ2 was suggested to colocalizes with mitochondria because of the unique N terminal is target to mitochondria, where Bβ2 accelerates neuronal cell death after survival factor deprivation (Dagda et al., 2003).. Relationship between SCA12 and PP2A. Through previous study, SCA12 is due to repeat expansion alters the level of expression of PPP2R2B, how does the repeat length on 5’UTR region alter the gene expression still unknow. The pathogenic mechanisms are proposed. First, CAG repeat expansion could alter splicing pattern of nearby exon, which as a role to be enhancer or inhibitor (Holmes et al., 2003). The variant splicing translated at least six different N-terminal proteins, might affect subcellular localization of the PP2A enzyme (Holmes et al., 2003). Second, mutation may be toxicity on 7.

(26) RNA level, such as myotonic dystrophy (DM)(Mankodi et al., 2000; Liquori et al., 2001).This mechanism is because of presence of the SCA12 repeat within the PPP2R2B transcript 5’ UTR. The repeat length of DM is larger than SCA12, which is within transcript level, thus the pathogenic mechanism is similar to SCA12 (Holmes et al., 2003). Third, mutation might inhibit the transcription, in case of Friedreich’s ataxia (Patel and Isaya, 2001). Forth, PPP2R2B was associated with PP2A protein, when PPP2R2B expression level altered, could lead toxicity to PP2A through different pathways. Although these are yet been identified, but A subunit will interact with at least one B subunit, such as protein phosphatase 5 (PP5), demonstrate that B sununit not only play a role to regulate PP2A but also control other signal transduction pathway.. The most straight forward model of SCA12 pathogenesis remains an alteration of PP2A function as follows: 1) the CAG repeat expansion in PPP2R2B leads to abnormal levels of expression of PPP2R2B;2) abnormal levels of PPP2R2B, result in abnormal PP2A function; 3) changing PP2A function results in altered protein phosphorylation;4) altered phosphorylation of essential neuronal proteins leads to neurotoxicity.. To understand the pathogenesis of SCA12, and elucidation of the pathways between PP2A to neuronal death should provide of considerable important informations in understanding SCA12 and other neurodegenerative disorders.. 8.

(27) Oxidative stress. Reactive oxygen species (ROS) are well recognized as critical signaling molecules that take part in a variety of normal cellular functions (Irani, 2000). When exceed physiological levels, ROS will trigger cellular dysfunction by attacking a variety of biomolecules. ROS levels in cells and tissues are related to antioxidant molecules and detoxifying enzyme level (Reiter et al., 2005). Oxidants have widely been shown to initiate the cellular apoptotic cascade by perturbing the balance between the cellular signals for survival and suicide (Chandra et al., 2000). ROS in trauma of cells can damage cellular components such as lipids, proteins and DNA, which leads to cell death. Oxidative modification of proteins of ROS has been implicated in a variety of physiopathologies of both normal aging and neurodegenerative diseases, such as cancer, cardiovascular diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD) and Spinocerebellar ataxias (SCAs).. tert-butylhydroperoxide (TBH) and hydrogen peroxide (H2O2) are thought to be the major precursor of highly reactive free radicals, and have been reported to induce apoptosis in cells of the central nervous system. Oxidative stress can cause oxidative damage to polyunsaturated fatty acids in biological peroxidation. Lipid peroxidation will generate many reactive by-products including lipid hydroperoxides. TBH is an organic lipid hydroperoxide analogue, which is usually used as a pro-oxidant to evaluate oxidative stress in cells and tissues (Rush et al., 1985). Several studies show that TBH can induce cell death and apoptosis 9.

(28) by the mitochondrial pathway in many cell types. In living cells, H2O2 is mainly generated from mitochondria, microsomes, and peroxidomes (Chance et al., 1979). Peroxisomes contain H2O2–producing oxidases, while mitochondria and microsomes produce the superoxide anion as a by-product in the O2 reduction, and the anion quickly dismutates to H2O2 and O2 spontaneously or by the action of superoxide dismutase (Makino et al., 1994). H2O2 is not very reactive with cellular constituents, but in the presence of transition metal ions and appropriate reductants and converted to the hydroxyl radical which is highly reactive with organic compounds and is hazardous to living cells (Makino et al., 1994). H2O2 has been known to activate the mitochondrial permeability transition pore and the release of the mitochondrial protein cytochrome c during apoptosis (Stridh et al., 1998).. Staurosporine (STS) is a natural product originally isolated from bacterium Streptomyces staurosporeus. STS was discovered to have biological activities ranging from anti-fungal to anti-hypertensive (Ruegg and Burgess, 1989). The main biological activity of STS is the inhibition of protein kinases through the prevention of ATP binding to the kinase. This is achieved through the stronger affinity of staurosporine to the ATP-binding site on the kinase (Hashimoto and Hagino, 1989). STS induced apoptosis can activate Bax and the mitochondrial caspase-dependent apoptotic pathway. It also induced apoptosis of melanoma by caspase independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to STS and was associated with release of cytochrome c and Smac/DIABLO 10.

(29) (second mitochondrial-derived activator of caspase /direct IAP binding protein with low pI)from mitochondria and cleavage of poly (ADP-ribose) polymerase and inhibitor of caspase-activated DNase (Zhang et al., 2004).. To study the Bβ1 during neuron differentiation, we established an in vitro model, with Bβ1 overexpression in PC12 (rat pheochromocytoma) cell. PC12 cells have been used widely as an experimental system to study various aspects of neurons, Upon nerve growth factor (NGF) treatment, PC12 cells undergo growth arrest and develop into sympathetic neuron-like cells with elaborating neurites capable of generating action potentials (Toledo-Aral et al., 1995; Schulz et al., 1997). TBH and H2O2 were applied to Bβ1 overexpression PC12 cells to evaluate their susceptibility to oxidative stress.. 11.

(30) Materials and methods I. Mouse model. SCA12 transgenic mice SCA12 transgenic mice overexpressed PP2P2B isoform 1 (B1) was established in our lab (Lin, 2006). (Pronuclear microinjection was helped by the Transgenic Core Facility of Institute of Molecular Biology, Academia Sinica). The transgene B1 was driven by NSE promoter, which is rat neuron-specific enolase, mainly effective expression in the brain in a variety of neuronal cells.. Genotyping analysis We genotyped the SCA12 transgenic mice by isolating genomic DNA from 0.5cm length of mouse tails by lysis tissue in 10 mg/mL proteinase K (in 2% SDS, 5 M NaCl, 0.5 M EDTA (pH 8.0), 1 M Tris (pH 7.4)) at 65 overnight followed by salt extraction and ethanol precipitation. Transgenic mice were identified using polymerase chain reaction (PCR) and a set of primers located in NSE promoter (forward: 5’-TTG GCT GGA CAA GGT TAT GAG C-3’), and B1 cDNA (reverse: 5’-AGA TGG CAG GGA CAG GAT TAC G-3’), respectively. A 492bp fragment was amplified under a PCR condition of 95 30 seconds for denaturing, 56 1 min for primer annealing, 72 30 seconds for elongation, and 30 cycles repeated. PCR reaction mixture contained 1 µl cDNA, 2.0 µl PCR 12.

(31) buffer (500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl, pH 9.0), 1.5 µl 10 mM dNTPs, 0.3 µl Taq DNA polymerase, 0.5 µl of primer, and H2O added to a total volume of 20 µl. Reaction products obtained were applied to electrophoresis in 2.0% agarose gels containing ethidium bromide. In addition, to distinguish between homozygous and heterozygous genotypes of transgenic mice, we use semi-quantitative PCR to analysis mouse genotypes (Fig. 20). We use 750-bp prion-fragment as an internal control.. Rotarod testing This task is used to assess motor coordination, balance, and motor learning. We assessed the ability of SCA12 mice (both heterozygous and homozygous compared with wild type mine) to remain on a rotating rod as the speed of rotation increasing by using of ROTOROD Series8, Model 755 for heterozygous mice, and UGO for homozygous mice. The rotating rod speed was accelerated from 4 to 30 rpm in the first 300 sec, and then maintained in 30 rpm till the maximum duration, 600 sec. An entire testing process required four days (the first three days for training and the last day for testing), three trails per day for each mouse, and series of testing in coming months were completed in two days per month (first day for training and the second day for testing). Series of rotarod analysis were processed on mice at different ages, 7 to 18-month-old for heterozygous mice, 2 to 5-month-old for homozygous mice line TG13.. Locomotor activity monitoring 8-week-old mice were placed in an open field (30 cm * 30 cm * 30 cm 13.

(32) white box) and allowed to explore the environment for 10 min Using Etho-Vision video tracking system, we evaluated distance of horizontal movement, velocity of movement and rearing frequency of each mouse in every five min and collected parameters for one-way ANOVA analysis. This task provides us the information of the gross behavioral effect of mice.. Immunohistochemistry SCA12 mice were detected by calbindin, NeuN, GFAP, CART, GH and flag. Mice were anesthetized with 205% avertin (17L/g mouse weights) and then perfused with 4% paraformaldehyde. First, we fixed the anesthetized animal in plastic tray, opened abdominal skin by a longitudinal incision; removed skin and gut to expose abdominal aorta and vena cava; inserted the butterfly 23 gauge 3/4 needle in the aorta below the renal arteries; started perfusion with 0.9% normal saline for replacing blood and then without interrupting of the flow perfused 4% paraformaldehyde instead of 0.9% normal saline until the limbs of mice were totally rigid. Mouse brain was dissected out from the mouse. Cerebellum was fixed in 4% paraformaldehyde in 1X PBS for 24 hours, transferred into 10% sucrose in 1X PBS for 48 hours, and then dissected (30 m thick) for sagittal saw.. RNA extraction and microarray analysis Total RNA was extracted from half of mouse brain and cerebellum tissue using 1ml TRIzol reagent, and centrifuge at 12,000 rpm for 5 min 14.

(33) at 4°C, take supernatant. Add 0.2 ml chloroform/ml TRIzol, mix gently, and centrifuge at 12,000 rpm for 10 min at 4°C, following take upper, clear part into new tubes. Add 0.5ml isopropanol/ml TRIzol, mix, put on ice 10 minutes, Centrifuge at 12,000 rpm for 10 min at 4°C. Keep pellets, and using 1ml 70% ethanol for wash 2 times, after that, air dry 2 minutes. Dissolve the pellets in proper amount DEPC (diethyl pyrocarbonate) treated H2O (water was treated with 0.1% DEPC at room temperature for overnight, and then autoclave for 45 minutes at 121°C to remove the DEPC). RNA yield and quality were determined spectrophotometrically (Nano-drop ND-100,NanoDrop. The microarray analysis was conducted by Chang Gung Memorial Hospital co-facility.. Electronic microscope analysis Mice were first perfused with 0.9% normal saline for replacing blood and then perfused with 4% paraformaldehyde in 1 X PBS with 2.5% glytaraldehyde and post-fixed for 24 hours. We dissected cerebellum in 1mm3, and replace in wash buffer (0.1 M phosphate buffer (0.2 M NaH2PO4H2O and 0.2 M Na2HPO4, pH 7.2) in 5% sucrose), following wash by wash buffer 15 minutes three times. The cerebella were fixed by Taipei Medical University Hospital, images were acquired for Electron Microscope Center of National Taiwan University.. Home cage scanning Mice were single-housed and analyzed in their home cage. Four digital video cameras were mounted perpendicular to the cages. The cameras 15.

(34) input into a video processor connected to computer and video data were analyzed by HomeCageScan software. During recording, mice were housed in standard cages, which were recorded for 3 hours at night. II. Cell model. Establishment of of B1 expression construct The pEGMT- B1 vector was transformed into E. coli to amplify the B1 fragment. XbaI and XhoI restriction enzymes were used to diget and isolate B1 cDNA from pEGMT vector. B1 cDNA was then sub-cloned into xbaI/xhoI cutting site of pEGSH vecto. The expression construct pEGSH- B1 was then transformed into E. coli, screened by PCR analysis and confirmed by sequencing.. Transfection of pEGSH- B1 intoPC12/pERV3 cell line The PC12 (rat adrenal gland pheochromocytoma)/ pERV3 cell line was originally prepared in our lab (Wang. 2007). The cell was maintained in 85% RPMI1640 media (GIBCO) and supplemented with 10% horse serum, 5% fetal bovine serum(Greene and Tischler, 1976), 1% penicillin/streptomycin, 2mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5g/L D-glucose, 1M HEPES, and 1mM sodium pyruvate. Cells were seeded on poly-L-lysine coated plate 24 hrs prior to transient transfection. Clonal PC12/pERV3 cells were transfected with 3 µl of pEGSH- Bβ1 cDNA at 60% confluency by 6 µl of Lipofectamine 2000 (Invitrogen). We 16.

(35) utilized 200 ng/µl G418 (genetomycin) for clonal pERV3 cell selection and 50 ng/µl hygromycin B for clonal pEGSH/Bβ1 cell selection for two weeks. The medium was changed every threedays.. Nerve Growth Factor (NGF) treatment of PC12 cells We seeded PC12 cells with regular medium for 24 hours, and then transfer the medium to low serum medium (0.5% horse serum (HS) and fetal bovine serum (FBS) ) with 50 ng/mL NGF to induce neuritis sprouting for 48 hours.. RT-PCR RNA yield and quality were determined by spectrophotometer (ND-100,NanoDrop), and 2 µg of total RNA was reverse transcribed using the Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. In order to control the yield of cDNA synthesis as well as detecting any genomic DNA contamination, a PCR with primers locate on pEGSH vector and one of Bβ1 exon (forward: 5’-ACA GCT ATG GA CCG AAG C-3’ and reverse: 5’-TCA CCC CTA CGA TGA ACC TG-3’) for amplification of the B1 mRNA, was performed. PCR reaction mixture contained 1 µl cDNA, 2.0 µl PCR buffer (500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl, pH 9.0), 1.5 µl 10 mM dNTPs, 0.3 µl Taq DNA polymerase, 0.5 µl of primer, and H2O to a total volume of 20 µl. Reaction products obtained were applied to electrophoresis in 2.0% agarose gels containing ethidium bromide.. 17.

(36) MTT assay MTT assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and form a dark blue formazan crystals which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. We added each well 0.5 mg/mL MTT into three groups of cells cultured in 24-well plate: pEGSH/Bβ1 expressed clone 3 and clone 8, and pEGSH-vector only clone. After two hrs incubation in 37, we dissolved formazan crystals with dimethyl sulfoxide (DMSO), measured OD570 values with a multiwell scanning spectrophotometer (Bio-Tek Microplate autoreader EL311). Through MTT assay, we first confirmed the survival rate of differentiated and non-differentiated PC12 cells with induced overexpression Bβ1 protein respectively. Furthermore, we used oxidative stress drugs to test the cells’ survival rate.. Immunocytochemistry Differentiated PC12 cells grown on 6-cm culture dish were fixed in cold 4% paraformadehyde solution for 10 minutes. After washes with 1X PBST (0.2% Tween-20 included) four times, the non-specific protein binding sites will be blocked with 5% fetal bovine serum (FBS) for 2-4 hours, then the cells were incubated overnight in 4 or one hour in room temperature with primaty anti-flag antibody (1:2000, Sigma), which was diluted with 5% FBS solution. After four times wash with 1X PBS for 10 minutes each, cells were incubated with anti-mouse secondary antibody (green fluorescence, Alex Fluor® 488 Donkey anti-mouse IgG) for 1 18.

(37) hour at room temperature and light protection. The cells were then washed with 1X PBST and counterstained with 4’, 6-diamidino-2-phenylindole (DAPI, Sigma) for 3 minutes.. Flow cytometric detection of apoptotic cells Cells were collected by centrifugation 1,200 rpm for 5 minutes, and thoroughly rinsed with 1 X PBS. The pellets were resuspended in 80% EtOH (dilute with 1 X PBS) and fixed at 20 overnight. The cells were then centrifuged (1,200 rpm for 5 minutes), and ethanol was removed by rinsing thoroughly with PBS. The cell pellets were resuspended in 1 ml DNA staining reagent containing 50 µg/ml RNase, 0.1% triton X-100, 0.1 mM EDTA (pH 7.4), and 50µg/ml PI. The sample was then kept in dark for 30 minute at 4. Red fluorescence (DNA) was detected through a 563 ~ 607 nm band-pass filter by using a FACS 440 flow cytometer (Calibor, BD).. Western analysis Protein lysates prepared from mouse brain, cerebellar and culture cell pallet in 1 M Tris pH 7.4, 0.5 M EDTA pH 8.0, 5 µg/mL PMSF, and 10% Triton-x-100. Proteins were separated by 10% SDS-polyacrylamine gels and transferred onto nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane, which were then blocked in 5% skin milk in 1 X PBS. Membrane was probed with the flag, calbindin, GFAP, NeuN, CART, or PP2A-C antibody individually. All the antibodies were dilute 1:2000 in 1 X PBST. Protein loading levels were normalized to actin levels (50 19.

(38) g/well).. Statistical analyses The results of experiments were expressed as means ± S.D. and were analyzed by ONE WAY ANOVA. The scientific statistic software SPSS 10.0 was used to evaluate the significance of differences between groups. The criterion of statistical significance was represented as *P0.5.. 20.

(39) Result I. Mouse model. A. Behavior analysis of SCA12 heterozygous transgenic mice. The overexpression of Bβ1 transgene were analyzed by western blot using brain and cerebellar protein from offspring of ten transgenic founders. Two lines which with high level Bβ1 protein were used for further characterization, lines TG-13 and TG-20 (Lin, 2006). To investigate whether overexpression of Bβ1 affect the learning and motor ability, we used rotarod analysis to test this mice. This behavior test conducted on SCA12 heterozygous transgenic mice in different stages, 28 to 72-week-old of line TG-13 and 26 to 68-week-old of line TG-20. In the beginning, we tested the mice with speed of 4 to 30 rpm and found even the wild type mice latencies were fewer than 200 sec, when the speed was still in acceleration. There was no significant difference between transgenic and wild type mice when they were before 60 weeks old (Fig. 2). These findings indicate that this condition was too difficult to stay on the rod for these mice, thus, we changed to an easier condition, 2 to 20 rpm. Upon the easier condition, we found that heterozygous transgenic mice starting show shorter latency time compared to wild type mice (line TG-13 began at 68-week-old and line TG-20 began at 64-week-old) (Fig. 3).. 21.

(40) B. Microarray analysis of the differential gene expression between wild-type and heterozygous transgenic mice. Although changing in the activity of a single gene can be directly correlated to a cellular functional, but the molecular definition is still complex, all are depends on the interaction of multiple genes and cellular pathways. Thus, microarray technology offers an opportunity to delineate the complex gene expression profiles of specialized cell types (Alizadeh et al., 2000; Cho et al., 2001), and have revolutionized the way in which the research community addresses biological problems. Many different biological questions can be addressed through gene-expression experiments, so as SCAs. Our cDNA microarray data (Fig. 4) of 64-week-old mice reflect a change in many genes expression and/or mRNA stability (Tables. 2 and 3). According to this result, we choose two neuronal-related genes whose levels have been altered in both lines of transgenic mice compared to the wild type mice. Growth hormone (GH) is upregulated and cocaine and amphetamine regulated transcript (CART) is downregulated in transgenic mouse cerebella. GH has been associated with nervous functions as previous report shown that GH deficient mice may increase in sympathetic firing rate (Sverrisdottir et al., 1998). CART is a recently discovered brain neuropeptide with potent anorexigenic peptide properties and is widely expressed in the central and peripheral, including the enteric, and central nerve systems (Douglass et al., 1995). CART peptides have been reported to inhibit apoptosis and promote survival and differentiation of various neurons in vitro (Louis 1996). We also use a microarray database online analysis software (MetaCore) to 22.

(41) extract the relationship platforms for the differential gene expression profile of the microarray data.. C. Immunohistochemical and western blot analyses of heterozygous transgenic mice in different stages. Since we observed significant difference in the rotarod performance between heterozygous transgenic mice and control group, we doubted of adverse effects of overexpression Bβ1 and further identified the neuropathologies with six antibodies: 1) flag, transgene expression. 2) calbindin, Purkinje cell morphology; 3) NeuN, neuronal nuclei; 4) GFAP, glial cells such as astrocytes and a marker of gliosis; 5) CART, cocaine and amphetamine regulated transcript; 6) GH, growth hormone and. We first use flag antibody to confirm the transgene expression, in heterozygous transgenic mice, flag signal can be detected in cerebellar nucleui of different stages of mice (Fig. 6B, 9B and 12B).. In agreement with the severity of the respective neurological phenotypes, histological analyses reveal more marked abnormalities in the Purkinje cells (PC). Our immunohistochemistry (IHC) data of 3-month-old mice with calbindin antibody showed that there was slight PC degeneration in 3-month-old heterozygous transgenic mice (Fig. 5A). Although no significant Purkinje cell loss or disorganization was noticed in 3-week-old heterozygous transgenic mice, some other neuronal cell loss in cerebellar nuclei were detected by NeuN (Fig. 5B). Cerebral 23.

(42) ischemia produces complex molecular and functional changes in the brain. Among them are strong astroglial and microglial activations. Astrocytes are crucial to the normal function of the central nervous system. They provide neurons with energy substrates and produce precursors of neurotransmitters and important in neuronal antioxidant defense (Forsyth, 1996), also a hallmark of gliosis (Schultz et al., 2004). At young stage, wild type mice have less astrocytes in cerebellar nuclei than heterozygous transgenic mice as shown by the GFAP staining (Fig. 6A). According to our microarray data, we also conducted IHC data to confirm the differential expression of these two genes, CART and GH. The expression level of CART is higher in cerebellar nuclei of wild type mice and the signal of GH expression is higher in cerebellar nuclei of heterozygous transgenic mice at this stage (Fig. 7).. PC degeneration is increased and presented by a reduction in the cell and dendrite numbers at 7-month-old (Fig. 8A). Similar to PC loss, our data indicate that neuronal loss in cerebellar nuclei were more severe in aged animals (Fig. 8B). During progressive degeneration astrocytes were more activated in heterozygous transgenic mice and astrogliosis were observed (Fig. 9A). The differential expression levels of CART and GH showed the same results as early stages (Fig. 10).. A more severe degeneration of PC was identified when these heterozygous transgenic mice were even odder (19-month-old) (Fig. 11A). PCs in wild type mice at the same age were maintained in stable level, which indicates that PCs degeneration is caused by Bβ1 transgene 24.

(43) overexpression. These results also correlated well to our rotarod data (Fig. 3). Our IHC data also indicate that neuronal loss in cerebellar nuclei were more severe in aged animals (Fig. 11B). Active astroliosis was observed in both wild type and transgenic mice when they were 19-month-old (Fig. 12A). The expression of CART and GH remained similar levels in wild-type and transgenic mice as the other 2 younger stages (Fig. 13).. We also used western blot analyses to confirm these IHC data (Fig. 14-17). However, some of the quantitation results do not show significant changes. We suggest that western blot analysis is to detect each protein from the total proteins of whole cerebellum, but the IHC can detect more specifically the restricted region where protein is expressed.. D. Tau phosphorylation and PP2A C subunit of transgenic mice identified by western blot analysis. PP2A accounts for ~70% of the total tau phosphatase activity in human brain (Liu et al., 2005). PP2A also indirectly promotes the activities of several tau kinases in brain (Li et al., 2004). To investigate whether overexpression Bβ1 alter tau phosphorylation through changed of PP2A activity. We found that both phosphorylated and unphosphorylated tau are not significantly changed in wild type and heterozygous transgenic mice in different stages, and so as PP2A C subunit expression (Fig. 18 and 19).. 25.

(44) E. Behavior analysis of SCA12 homozygous transgenic mice. The behavior performance of homozygous transgenic mice was identified by using rotarod task with rotating speed from 4 to 30 rpm. We found that homozygous TG-13line showed significantly worse performance than control mice (Fig. 20B). In contrast, homozygous TG-20line showed at better performance than control group (Fig. 21A). Why these two homozygouse TG lines show different performance, we will discuss it later. But these behavior results were not affected by mouse body weight because there was no significant difference of the body weight between homozygous transgenic and wild type mice (Fig. 20C and 21B).. Spontaneous locomotor behavior and habituation was assessed using an open field test. Individual mice were placed into a white box and their behavior was recorded on video for a total of 10 min, and was analyzed using the EthoVision 3.1 video-tracking software (Noldus, The Netherlands). In our locomotor analysis, pairwise comparison the distance moved showed that groups of homozygous line TG-13 did not differ from control one. However, the performance of homozygous line TG-20 was significantly better than wild type mice, reflecting their better habituation to the novel environment (Fig. 22A). In the rearing frequency analysis, homozygous line TG-13 displayed markedly lower rearing frequency, but homozygous line TG-20 showed significantly increased rearing frequency in the same amounts of time in the open field (Fig. 22A). Furthermore, compared these two homozygous transgenic lines, 26.

(45) homozygous line TG-20 was more active than line TG-13 in this test. During the behavior test, we found that homozygous line TG-20 were trying to get out of the testing box, which seems they were more nervous to a novel environment.. F. IHC and western blot analysis of homozygous transgenic mice in different stages. First, the transgene signal, flag staining can be detected in cerebellar nuclei of different stages of mice (Fig. 24A, 26A and 28A). To identify the correlation between the behavioral performance and the pathological phenotypes, IHC staining was conducted in sections from the cerebella of homozygous transgenic mice. The reduction of the cerebellar size was not observed. According to our data, there were no difference in calbindin and NeuN stainings between homozygous transgenic and wild type mice at 3 month old , but GFAP signal was slightly increased in homozygous transgenic mice (Fig. 23). The signal of CART expression is lower and GH expression is higher in cerebellar nuclei of homozygous transgenic mice (Fig. 24).. We observed the reduced number of PCs in lobules and neuron cells in cerebellar nuclei when mice were 6-month-old, however, we did not observe the gliosis pathology (Fig. 25). The signal of CART expression is lower and GH expression is higher in cerebellar nuclei of homozygous transgenic mice at different stages (Fig. 26).. 27.

(46) When mice were 14-month-old, we found more severe PC loss in the lobules and neuronal cell loss in the cerebellar nuclei of homozygous transgenic mice. Also observation siginificant GFAP signal in the cerebellar nuclei of homozygous transgenic mice (Fig. 27). The signal of CART expression is lower and GH expression is higher in cerebellar nuclei of homozygous transgenic mice than that in wild-type mice at different stages (Fig. 28). These findings are consistent to our microarray, western blot (Fig. 29 and 30) and heterozygous data.. G. Home cage analysis of homozygous line TG-13 at 5.5-month-old. Automated analysis of mouse behavior will be vital for elucidating the genetic determinants of behavior, for comprehensive analysis of human disease models, and for assessing the efficacy of various therapeutic strategies and their unexpected side effects. In many neurodegenerated transgenic mice model, the methods for detecting phenotype are weight loss, clasping, and declining performance in rotarod and grip-strength test. We use a video-based behavior-recognition technology to analyze home-cage behaviors and might discover previously unrecognized features of our already extensively characterized mouse models of neurodegenerative disease. We recorded the behaviors of nine homozygous transgenic mice and ten wild type mice for three hours at night.. According to our records, the time spent in resting between these 2 groups were not different (Fig. 31A). On the other hand, awakening 28.

(47) events were dramatically increased in transgenic mice (Fig. 31B). Twitching is defined as a movement during rest, which was slightly less in transgenic mice than that in wild type mice (Fig. 31C).. Consistent with previously reported motor abnormalities, we also quantified defects in behaviors requiring significant grip strength and coordination. Hanging was reduced in our homozygous transgenic mice (Fig. 31D). Despite considerable variation in hanging behaviors of diseased and control mice, stretching was similar for two groups (Fig. 31E). Jumping, another complex motor behavior, shows low frequency in two groups (Fig. 31F). Distance traveled provided a robust metric of overall activity and motor performance also was similar in two groups (Fig. 31G).. Alterations in many other behaviors were detected, interestingly, we found homozygous transgenic mice have slightly higher frequency than control group, such as eating, drinking, turning, grooming, sniffing and rearing (Fig. 32). This system is helpful to exceedingly rare behaviors in diseased model mice, and may approach to detect and quantified of phenotypes in the rodent, will greatly aid in testing therapeutics on this mouse models of human disease.. 29.

(48) II. Cell model. A. Establishment and analysis of inducible B1 overexpression PC12 cell system. PC12 cells weretransfected with Bβ1 and under a PonA induction system (Fig. 33). A time course induction was conducted to investigate whether overexpression of Bβ1 affected the morphology and viability of PC12 cells, but we did not find any change in these cells (Fig. 34). Because these transfected cells were a mix population with different insertion sites could cause different gene expression levels, we thus chose thirteen clonal cells for our further studies. The genotyping and RNA expression levels of these clonal cells were characterized (Table 4). Upon nerve growth factor (NGF) treatment, PC12 cells undergo growth arrest and develop into sympathetic neuron-like cells with elaborating neurites capable of generating action potentials (Toledo-Aral et al., 1995; Schulz et al., 1997). Furthermore, two clonal cell lines were chosen because no leaky RNA expression was identified. After NGF treatment for 48 hrs, following PonA induced for 48 hrs, the Bβ1 mRNA expression levels in these 2 clones were significantly increased than non-induction groups (Fig. 35). We thus used these two clones for the following experiments.. B. Identification of Bβ1 expression in inducible clonal cells by western blot and immunocytochemistry (ICC) analyses. 30.

(49) To identify the Bβ1 protein expression level, flag antibody was used to recognize the Bβ1-flag fusion protein. The overexpression of Bβ1 protein can be detected till 48 hrs induced by PonA in a low serum condition, however, the expression of Bβ1 was rarely detectable after PonA induction for 72 hrs (Fig. 36A). These results suggest that cells may have a feedback system to degrade over-produced Bβ1 proteins or the effective duration of PonA induction is only last for 48 hrs. The Bβ1 expression induced by PonA at 48 hrs was independent on NGF treatment (Fig. 36B). Our data indicate that, whether cells were under proliferation or differentiation, this inducible system works similarly.. Our ICC analysis showed that clonal cell lines had strong homogeneous cytoplasmic Bβ1 staining after NGF treatment 48 hrs followed by PonA induction for 48 hrs under normal serum condition (Fig. 37). In serum starvation condition, homogeneous cytoplasmic Bβ1 staining were also detected (Fig. 38). There was only slight induction signal detected in normal condition (Fig. 39). According to these data, we suggest that NGF treatment to induce the differentiation of PC12 cells is helpful for Bβ1 protein in the neuron-like cells strongly and homogenously. Under a proliferation state, cells can only slightly expressed Bβ1 in cytoplasm. Serum starvation arrests cells in G1 phase, the expression of Bβ1 protein was also reduced.. C. Cell viability test and cell morphology. To investigate the effect of Bβ1 overexpression in PC12 cells, we 31.

(50) characterized the cell survival and morphological change during the PonA induction period. We tested the cell viability in two different stages, proliferation and differentiation (after NGF treatment for 48 hrs). Our preliminary MTT assay results (N=3 in each group) reveal that there was no significant difference between clonal cell lines with overexpression of Bβ1 under proliferation or differentiation states (Fig. 40). Furthermore, to identify the morphology of these cells, we measured the dendrite length and outgrowth spine numbers of these cells at different time point after PonA induction following NGF treatment. The measurement was conducted in ten individual areas of each experiment. The dendrite length and outgrowth spine numbers of overexpressed Bβ1 was similar to the vector pEGSH control cells (Fig. 41). Interestingly, we found that outgrowth spine number of clone 8 cells was even significantly increased after NGF treatment for 72 hrs.. D. Oxidative stress drugs induced cytotoxicity in clonal cells. Hydrogen peroxide (H2O2), tert-butylhydroperoxide (TBH) and staurosporine (STS) are very common chemical oxidants, and have been shown previously to induce apoptosis in several cell types, including PC12 cells and fibroblasts (Makino et al., 1994; Lee et al., 2000). Neurons are very vulnerable to these oxidative drugs for their relatively low levels of antioxidant enzymes and dependence on mitochondrial respiration (Whittemore et al., 1994). In order to study the apoptotic effect of these oxidative drugs in pathological conditions, we investigated three kinds of oxidative stresses on these clonal cells, respectively. Cell 32.

(51) viability under oxidative stress was measure by MTT method. Our study showed that viability of cells exposed to 20 µM H2O2, 10 µM TBH and 125 nM STS were significantly decreased in B1 overexpresses group compared to control group (Fig. 42). These results indicate that oxidative stress caused apoptosis by cytotoxity in this diseased-cell model.. 33.

(52) Discussion I. Mouse model. Our Bβ1 transgenic mice represent a model of cerebellar degeneration, due to loss of PCs caused by the overexpression of Bβ1.. The rotarod test, in which animals walk on a rotating rod, is widely used to assess motor status in laboratory rodents. In our research, motor learning was assessed in male mice started at 2 months of age by using an accelerating rotarod. This accelerating rotarod can test animals’ tolerance of the accelerated speed. The heterozygous mice had difficulty to stay on the rotating rod upon the more restricted condition (4 to 30 rpm in the first 5 min), even the wild type mice could not last for 600 sec. Thus, we modified the rotating speed to an easier condition (2 to 20 rpm in the first 5 min) which wild type mice can stay on for 600 sec. There was no significant difference between transgenic and wild type mice when they were before 60 weeks old. Upon the easier condition, we found that heterozygous transgenic mice show significant shorter latency time compared to wild type mice at older stage, suggest these mice had a late onset disease.. To gain more severe phenotype and early onset disease mice, we generated homozygous transgenic mice in our study. Behaviorally, compared to heterozygous mice, homozygous line TG-13 showed deficits 34.

(53) in rotarod testing and bad motor coordination since they were 2-month-old. Interestingly, homozygous line TG-20 showed better performance on the rotating rod when they were 2-month-old. During the task of rotarod task, we observed that wild type mice knew how to get out of the rotating rod but not felled down when they were tired or loss balance, suggesting an anxiety-like behavior. The limbic system is involved in anxiety and risk evaluation tasks (Millan, 2003), with several classes of neurotransmitters participating in these processes (McEwen and Sapolsky, 1995). Previous research also revealed the correlation between anxiety and behavior. Spinocerebellar ataxia type 3 is a neurodegenerative disease caused by expansion of a polyglutamine domain in the protein ataxin-3 (ATXN3). Atxn3 knockout (ko) mice displayed no overt abnormalities. On the accelerating rotarod Atxn3 ko mice performed as well as wild type animals, but reduced exploratory behavior in the open field suggested a sense of heightened anxiety (Schmitt et al., 2007). G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels mediate the postsynaptic inhibitory effects of many neurotransmitters and drugs of abuse. Both GIRK1(-/-) mice and GIRK2(-/-) mice, however, showed elevated motor activity and delayed habituation to an open field (Pravetoni and Wickman, 2008). This evidence suggests that, homozygous line TG-20 showed better performance might due to anxiety. Thus, we cannot use motor activity to define how overexpression of Bβ1 affected the behavior of mice. Furthermore, we may test mice using plus-maze test and light-dart exploration test, which are useful to definite the anxiety level of the mice. Also accompany with brain pathology analysis in areas correlated to 35.

(54) emotion control, such as frontal cortex and prefrontal cortex would be necessary to confirm our hypothesis.. On the other hand, PC has a main role participating in the motor function of cerebellum. PC of the cerebellum receives excitatory input from the inferior olivary nucleus via climbing fibers, and sends inhibitory projections to the deep cerebellar nuclei, which in turn project to postural and motor structures such as the vestibular nucleus and motor cortex (Patel and Hillard, 2001). This inhibitory projection was sometimes placed at decisive location within the motor system and was therefore important for programming and controlling movement (Linnemann et al., 2006). According to previous studies, due to loss of PC will suffer from functional cerebellar decortication resulting in ataxia and deterioration of cognitive functions (Cendelin et al., 2008). Since PC axons are the only efferent pathway of the cerebellar cortex, the loss of these cells doesn’t lead to complete functional cerebellar decortication. Brain still involves in this multiple motor and learning activity, such as motor cortex, hippocampus and hypothalamus. Thus, we should also conduct brain pathology analysis in areas involved in multiple motor and learning activity in the future.. To demonstrate that the motor and cognitive deficits observed in Bβ1 transgenic mice were not caused by muscle weakness or some other restriction on general locomotor ability, we compared the open field exploratory activity between homozygous and wild type mice at 2 months of age. The measurements of total distance traveled showed no 36.

(55) differences between homozygous line TG-13 and wild type mice, but the total distance traveled of homozygous line TG-20 mice display more active in this test. These results suggest homozygous line TG-20 mice might have more anxiety than homozygous line TG-13 mice. However, rearing frequency of homozygous line TG-13 was significantly lower than that in wild type mice, but homozygous line TG-20 was significantly increased than two other groups, this results indicate that homozygous mice line TG20 have more exploring activity in a novel environment than others. Combine the results from rotarod and locomotor tests, neither the rotarod nor the activity in the open field indicate a sign of motor dysfunction in homozygous line TG-20 mice. How does this distinct impaired locomotion results from two homozygous lines with the alternation outcome of behavioral test happened, we may confirm by the anxiety behavior analysis and more brain pathology in the future.. In our IHC data, we can detect the transgene expression in cerebellar nuclei of transgenic mice by using flag antibody. The flag signals were significantly increased through quantitative analysis. Our mouse models also show some features with some extent of cerebellar pathology. Only the PC mutant transgenic mice show an early loss of dendritic PC arborization and abnormal cell morphology with reduction of the perikaryon and clarification of the nucleus (Weber et al., 2001). However, PC degeneration was not significant in both young heterozygous and homozygous mice. Although PC loss is not fully acknowledged, many experimental results point to apoptosis as the underlying process. Experiments that measure apoptosis-related byproducts in PCs and 37.

(56) cerebellum (cytochrome c release, mRNA expression of pro- and antiapoptotic genes, TUNEL assay, cleaved caspase-3) (Light et al., 2002; Heaton et al., 2003) have shown that apoptotic events occur and in a similar pattern as the cellular degradation labeling. A more severe degeneration of PC was identified when these heterozygous transgenic mice were 7-month-old and homozygous transgenic mice were 6-month-old, or even older. The intensity of calbindin immunostaining in PC diminished as the mice aged, but only with slight absence of arborization and pale cytoplasmic staining caused by the reduction of PC perikarya, which indicates that PC degeneration is caused by Bβ1 transgene overexpression.. We also examined the immunostaining intensity of GFAP in the Bβ1 transgenic mouse cerebella. GFAP is widely used as a marker of astrocyte activation or reactive gliosis. Astrocytes are the only cells in the brain that have the important ability to convert glutamate into glutamine via Glutamine synthetase (GS). Glutamine is released to neurons and used for the synthesis of glutamate (and then GABA, in GABAergic neurons). A cortical GS decrease has also been suggested as a characteristic in Alzheimer’s disease (Robinson, 2001). A major finding of this study is that overexpression of Bβ1 is critically involved in degeneration-induced astrocyte changes similar to gliosis. Compared to control group, GFAP levels in cerebellar nucleui region of astrocytes were slightly higher in young transgenic mice, and were significantly higher when transgenic mice were aged. These results indicate that astrocyte pathology turned into more severity during the disease progressing and increased GFAP 38.

(57) immunostaining intensity, which are typical signs of astrogliosis and cerebellum degeneration.. Based on our microarray data, Growth hormone (GH) is upregulated and cocaine and amphetamine regulated transcript (CART) is downregulated in transgenic mouse cerebella. This was also confirmed by our IHC results at different stages of mice. Both GH and insulin-like growth factor-1 (IGF-1) elicit pleiotropic actions in the CNS and may play a role in age-related changes in cognitive function (Kinney et al., 2001; Bartke, 2003). Unregulated GH-signaling in neural progenitor cells derived from SOCS-2 null mice results in impaired neurogenesis (Turnley et al., 2002) GH-signaling in supporting hippocampal neurogenesis can be evaluated irrespective of localized hippocampal GH synthesis (Ransome and Turnley, 2008). These evidences indicate that GH correlated with aging and neurogenesis, which suggest that GH upregulated in Bβ1 transgenic mouse cerebella may display a protective role to delay the degeneration.. The (CART) is an endogenous peptide which is widely expressed in the CNS and PNS (Douglass et al., 1995; Koylu et al., 1997). The cerebellar coordinates motor behaviors and plays a key role in fine motor control, balance, posture and eye movements (Ito, 1984, 2006). CART peptides can localized in fiber like structures within the vestibular cerebellum (Koylu et al., 1998), part of the cerebellum involved in the maintenance of equilibrium and balance (Barmack, 2003). The presence of CART peptides in the inferior olivary complex has lead to the 39.

(58) suggestion that CART peptides are expressed by climbing fibers. Climbing fibers play a central role in cerebellar processing and signal to the output neurons of the cerebellar cortex (PCs) by the production of a large membrane depolarization, termed a complex spike, (Koylu et al., 1998). CART is also protective against focal cerebral ischemia in vivo and against neuronal cell death (Mao et al., 2007). Thus, the downregulation of CART in transgenic mouse cerebella is consistent to our rotarod data with transgenic mice had worse performance. These data further emphasis that maintaining normal PC activity is critical for maintaining the whole coordination system.. In our HomeCageScan analysis, we have established a rigorous system for characterizing behavioral abnormalities in the mouse and have used it to demonstrate a contrasting array of behavioral changes in our transgenic mice model. Many of these behavioral alterations were previously unreported in other disease models, such as awakening events were dramatically increased in Bβ1 transgenic mice. In addition, we detected differences in some exceedingly rare behaviors, such as stretching. Most of the behavioral testing of these mice is focus on their robust motor phenotypes, such as Hanging was reduced in our homozygous transgenic mice. Our analysis also revealed substantial psychological behaviors in homozygous transgenic mice, such as eating, drinking, turning, grooming, sniffing and rearing. One disadvantage of this system is that we cannot analysis two mice at the same time, mice must be singly housed during each testing. However, with this system we can detect multiple behavioral alterations of these mice and may greatly aid in testing and evaluation of 40.

(59) therapeutics of these disease mouse models. According to our study, we found neurodegeneration of PCs, dendritic atrophy, and behavioral deficits in the transgenic mice. Therefore, these phenotypes will provide valuable parameters for future therapeutic evaluation for SCA12 disease.. II. Cell model. In our ICC results, we can detect cytoplasmic Bβ1 staining after NGF treatment 48 hrs followed by PonA induction for 48 hrs under both normal n and serum starvation condition. But there was only slight induction signal detected in normal condition. The western blot analysis show overexpression of Bβ1 was induced after PonA treatment. Although we observed significant Bβ1 expression, the cell survival and morphological change were not identified during the PonA induction period, thus, we investigate whether oxidative stress would cause pathology of these disease-cell models.. The vast majority of eukaryotic organisms need atmospheric oxygen to survive. Oxygen is the terminal electron acceptor in the respiratory chain of mitochondria during the generation of ATP. An unavoidable by-product during respiration by the mitochondria is the generation of reactive oxygen species (ROS), which are implicated in the pathophysiology of a diversity of chronic diseases (Boveris, 1984). Therefore oxidative stress is a ubiquitous phenomenon in all cell types. It has been pointed out that 41.

(60) the crucial balance between ROS generation and antioxidant defense is regarded as a force in a wide variety of chronic diseases (Machlin and Bendich, 1987). ROS may have several effects including alteration in the activity of transcription factors, phopholipases, protein tyrosine kinases, gene expression, apoptosis and cell injury (Finkel, 1998).Our study showed that viability of cells exposed to 20 µM H2O2, 10 µM TBH and 125 nM STS 48 hrs were significantly decreased in Bβ1 overexpressing group compared to control group. These results suggest that Bβ1 overexpressing cells are more vulnerable to these stress.. 42.

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