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Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the TATA-binding protein (TBP), an essential transcription factor. Despite its widespread expression, mutant TBP causes age-dependent and late-onset neurological symptoms and neurodegeneration, a phenomenon that also occurs in Huntington’s disease and other 7 inherited neurological disorders caused by polyQ expansion in the associated disease proteins. It remains unclear how late-onset neuropathology is caused by polyQ expansion. We hypothesize that aging process is important for the development of neurological symptoms in SCA17. Conditional SCA17 mice via Cre/loxP recombination system will allow us to examine the relationship between age-dependent neuropathology and the expression of mutant TBP. Using conditional SCA17 mice, this project will determine the expression of mutant TBP in mice at different ages after Cre/loxP recombination. The expression of mutant TBP will be examined using real-time PCR, western blotting and immunohistochemistry. Determining the expression of mutant TBP in conditional SCA17 mice will allow us to discover age-dependent neurological phenotypes mediated by polyQ expansion and shed light on the polyQ disease pathogenesis.
The goal of the project is to successfully induce expression of mutant TBP in KI mice of different ages. I will extract mRNA and protein and perform PCR and western blotting to confirm such expression. The expression data will be analyzed in association with neurological phenotypes of SCA17 mice.
The hypothesis is that older mice expressing mutant TBP show more severe phenotypes than the younger mice, which may explain late-onset neurological phenotypes in polyQ diseases and also implicates the importance of aging in polyQ diseases.
Animals and breeding strategy:
We have obtained B6.Cg-Tg (cre/Esr1)5Amc/J from The Jackson Lab. These transgenic mice have a tamoxifen-inducible, Cre-mediated recombination system driven by the chicken beta actin promoter/enhancer coupled with the cytomegalovirus (CMV) immediate-early enhancer4. These transgenic mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. When crossed with a strain containing a loxP site-flanked sequence of interest, the offspring mice are useful for generating tamoxifen-inducible and Cre-mediated gene targeted deletions. The cross has yielded four genotypes: Cre/-, loxP/-, Cre/loxP, and wild type. In the case of conditional SCA17 mice, Cre/loxP knocked-in mice with tamoxifen treatment can express Cre and turn on the expression of mutant TBP.
Diagram 1. Cre/ loxP system. Tamoxifen, an estrogen-receptor antagonist, activates the Cre promoter. Cre protein will splice out the stop sequence flanked by loxP, and allow the recombined gene to be transcribed.
Inducing Knocked-in Mice
Two mice were genotyped to confirm the presence of loxP/Cre and polyQ-expanded TBP. Mice expressing Cre, but without loxP-Stop-mutant-TBP, were used as controls in this experiment. Mice were treated with tamoxifen (TM) to activate the knock-in TBP by excising the lox-stop region. Mice were injected intraperitoneally with TM dissolved in corn oil at 20 mg/ml. Dosages are be calculated by 100mg/kg body weight for each treatment. Mice were treated daily for five consecutive days. The mice were monitored for three days after treatment to allow full expression of Cre and polyQ-expanded
Detecting Expression
After monitoring the mice for three days, brain tissues were collected to perform Western blot. Lysis buffer was added to the cerebellum at 1ul for every 50 ug. The lysate was sonicated for 10 seconds to break up non-protein structures, and spun down to separate out the protein. 5x dye was added to the protein solution 4:1, boiled at 100 degree Celsius for 6 minutes, spun down, and sonicated for 5 seconds, and loaded into a 4-20% Western gel. The protein on the gel was transferred to a membrane and tagged with antibodies to detect expression level. The membrane was treated with developing solution and exposed to a film for development
The condition of breeding and expressing the Cre/loxP system has been established for additional studies. Expression of polyglutamine expanded TBP is detectable by Western blot. Animals are bred, genotyped, and group by age to allow further studies.
Future studies will involve immunostaining of brain tissues to detect expression polyglutamine expanded TBP. Phenotype studies will be performed using rotarod and other tests. When enough mice has been genotyped for each age-group, studies of age-dependent polyglutamine expanded TBP expression can be done
X.J.L is supported by grants awarded by the NIH
Friedman, MJ; Shah, AG; Fang, ZH; Ward, EG; Warren, ST; Li, S; Li, XJ. Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration. Nat Neurosci. 2007 10: 1519–1528
Guy, J; Gan, J; Selfridge, J; Cobb, S; Bird, A. Reversal of neurological defects in a mouse model of Rett syndrome. Science 2007 315:1143–1147
Zhou, H; Cao, F; Wang, Z; Yu, ZX; Nguyen, HP; Evans, J; Li, SH; Li, XJ. Huntingtin forms toxic NH2-terminal fragment complexes that are promoted by the age-dependent decrease in proteasome activity. J Cell Biol. 2003 163:109–118.
S. Hayashi and A.P. McMahon, Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse, Dev. Biol. 244 (2002), pp. 305–318.
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