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研究生:陳聖音
研究生(外文):Shen-Yin Chen
論文名稱:探討CDK12在海馬迴神經突觸可塑性中的功用
論文名稱(外文):Characterization of CDK12 Functions in Hippocampal Synaptic Plasticity
指導教授:范明基
指導教授(外文):Ming-Ji Fann
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:神經科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:44
中文關鍵詞:海馬迴神經突觸可塑性
外文關鍵詞:hippocampussynapticplasticity
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CDK12 是一個需要依賴細胞週期蛋白而活化的磷酸激酶,目前已知它參與在RNA之選擇性剪接而並非細胞週期的調控。經過細胞實驗證明, CDK12會和它的細胞週期蛋白伴侶Cyclin L1及Cyclin L2互相聯結,進而改變E1a 微小報導基因之選擇性剪接。 CDK12大量表現在大鼠胚胎時期之神經系統裡,並隨著發育的進行而表現量逐漸下降,但在成熟的腦區域中 CDK12 仍然於海馬迴有表現。我們因此推測CDK12 在成熟的海馬迴中還保有功能並可維持神經細胞之間的連結。
CDK12 的基因轉殖小鼠已完成製作,其目的為研究生物體內CDK12之功能。我們將帶有精胺酸/絲胺酸區塊及類細胞分裂週期激酶區塊之短式CDK12 的cDNA 嵌入thy-1 啟動匣,用以促進轉殖之短式CDK12特定表現在神經系統內。如此我們獲得三種CDK12 基因轉殖小鼠,分別為Tg-1,Tg-11和Tg-17,基因型分析顯示牠們各自帶有不同量的轉殖基因。利用西方墨點法看到在基因轉殖小鼠腦內的短式CDK12 加強表現並不與牠們所攜帶轉殖基因的多寡成正比,推測是此三種小鼠體內轉殖基因嵌入位置之差異所致。有趣的是,轉殖的短式CDK12 表現在某些腦區域會抑制內生性的長式CDK12 表現;然而整體而言,CDK12 的表現量在CDK12 基因轉殖小鼠的神經系統內是被加強的。
我們利用電生理實驗方法研究CDK12 基因轉殖小鼠,觀察海馬迴神經突觸的可塑性是否受到改變。我們發現利用高頻刺激 (100赫茲)引發的長期增益效應並不因為CDK12 增量而改變,但利用原本不改變突觸連結的低頻刺激 (10赫茲)卻可以在CDK12 基因轉殖小鼠中引發長期增益效應;CDK12 的增量表現降低了海馬迴被引發長期增益效應的刺激門檻。我們需要更多延伸的實驗來探討CDK12 如何影響神經突觸之可塑性,進而了解CDK12 在生理上的意義。
Cyclin-dependent kinase 12 (CDK12) is a recently identified CDK that regulates alternative splicing rather than cell cycle progression. Together with its cyclin partners, cyclin L1 and cyclin L2, CDK12 changes the splicing patterns of E1a reporter minigene. The endogenous expression of CDK12 was found abundantly in the nervous system at embryonic stage and gradually decreased as development proceeds. In the mature brain, CDK12 remained its expression in hippocampus. We thus hypothesized that CDK12 activity can maintain neuronal connectivity in adult hippocampus. CDK12 transgenic mouse lines were generated in order to study CDK12 function in vivo. Thy-1 expression cassette was used to carry cDNA of the short isoform of CDK12 (CDK12S) which contains arginine/serine-rich (RS) domain and CDC-like kinase domain. Three transgenic mouse lines, Tg-1, Tg-11 and Tg-17, were obtained and found to carry different copy numbers of the transgene. Analysis of CDK12 by Western blotting showed that the protein expression level is not proportional to the transgene copy number, possibly resulted from different integration sites in genomes of these transgenic lines. Interestingly, expression of transgenic CDK12 protein suppressed amounts of endogenous CDK12 protein in several brain regions, but the total expression of CDK12 was increased. Electrophysiological approaches were then performed to examine changes of synaptic plasticity in CDK12 transgenic mice. Overexpression of CDK12 did not alter LTP induction by a 100 Hz high-frequency stimulus (HFS), but it did lead to LTP induction by a 10 Hz low-frequency stimulus (LFS) which normally has no effect on synaptic plasticity. CDK12 seemed to lower the induction threshold of hippocampal LTP. More experiments are required to determine whether CDK12 regulates synaptic plasticity and what physiological function CDK12 exerts.
Abstract (In Chinese)……………………………………………………… 2
Abstract
……………………………………………………………3
Table of contents
……………………………………………………………4
Introduction
……………………………………………………………6
CDK12 regulates alternative splicing
CDK12 and neuronal development
Synaptic plasticity in the hippocampus
Specific aims of the research
Materials and Methods
……………………………………………………11
Animals and reagents
Cell culture and transfection
Generation of CDK12 transgenic mice
Isolation of genomic DNA
Probe preparation for Southern blot analysis
Southern blot analysis
Protein extraction
Western blotting
Immunoflourescence staining
Morphological analysis
Electrophysiology
Passive avoidance
Results
……………………………………………………………19
Genotyping of CDK12 transgenic mice
CDK12 expression in cortex, hippocampus, striatum and cerebellum
Structures of hippocampus is normal in CDK12 transgenic mice
Synaptic plasticity change in CDK12 transgenic mice
CDK12 overexpression had no effect on passive-avoidance
Discussion
……………………………………………………………23
Bidirectional synaptic plasticity correlates memory formation
Metaplasticity is the higher level of synaptic plasticity
CDK12 may participate in hippocampal metaplasticity
Further evidences is needed to identify CDK12 role in memory
Molecular pathway of CDK12 remain to be elucidated
References
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Figures
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Abbott LF, Nelson SB (2000) Synaptic plasticity: taming the beast. Nat Neurosci 3 Sup:1178-1183.
Abraham WC, Bear MF (1996) Metaplasticity: the plasticity of synaptic plasticity. Trends Neurosci 19:126-130.
Aiba A, Chen C, Herrup K, Rosenmund C, Stevens CF, Tonegawa S (1994a) Reduced hippocampal long-term potentiation and context-specific deficit in associative learning in mGluR1 mutant mice. Cell 79:365-375.
Aiba A, Kano M, Chen C, Stanton ME, Fox GD, Herrup K, Zwingman TA, Tonegawa S (1994b) Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79:377-388.
Bakkour A, Morris JC, Dickerson BC (2009) The cortical signature of prodromal AD: regional thinning predicts mild AD dementia. Neurology 72:1048-1055.
Bienenstock EL, Cooper LN, Munro PW (1982) Theory for the development of neuron selectivity: orientation specificity and binocular interaction in visual cortex. J Neurosci 2:32-48.
Boutz PL, Chawla G, Stoilov P, Black DL (2007) MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development. Genes Dev 21:71-84.
Brannan CI, Bedell MA, Resnick JL, Eppig JJ, Handel MA, Williams DE, Lyman SD, Donovan PJ, Jenkins NA, Copeland NG (1992) Developmental abnormalities in Steel17H mice result from a splicing defect in the steel factor cytoplasmic tail. Genes Dev 6:1832-1842.
Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406-417.
Chen HH, Wang YC, Fann MJ (2006) Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation. Mol Cell Biol 26:2736-2745.
Chu YH (2009) Identification of the Subtrates of CDK12 by Yeast Two Hybrid Assay. Bachelor Thesis.
Fisher RP (2005) Secrets of a double agent: CDK7 in cell-cycle control and transcription. J Cell Sci 118:5171-5180.
Giese KP (2007) Novel insights into the beneficial and detrimental actions of cdk5. Mol Interv 7:246-248.
Hawasli AH, Benavides DR, Nguyen C, Kansy JW, Hayashi K, Chambon P, Greengard P, Powell CM, Cooper DC, Bibb JA (2007) Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation. Nat Neurosci 10:880-886.
Hebb DO (1949) The Organization of Behavior: A Neuropsycological Theory.
Hering H, Sheng M (2001) Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci 2:880-888.
Hong YH (2008) Identification of the Substrates of CDK12 by Yeast Two Hybrid Screening. Bachelor Thesis.
Hu D, Mayeda A, Trembley JH, Lahti JM, Kidd VJ (2003) CDK11 complexes promote pre-mRNA splicing. J Biol Chem 278:8623-8629.
Jedlicka P (2002) Synaptic plasticity, metaplasticity and BCM theory. Bratisl Lek Listy 103:137-143.
Klamt B, Koziell A, Poulat F, Wieacker P, Scambler P, Berta P, Gessler M (1998) Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms. Hum Mol Genet 7:709-714.
Ko TK, Kelly E, Pines J (2001) CrkRS: a novel conserved Cdc2-related protein kinase that colocalises with SC35 speckles. J Cell Sci 114:2591-2603.
Ladd AN, Stenberg MG, Swanson MS, Cooper TA (2005) Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development. Dev Dyn 233:783-793.
Lai C, Brow MA, Nave KA, Noronha AB, Quarles RH, Bloom FE, Milner RJ, Sutcliffe JG (1987) Two forms of 1B236/myelin-associated glycoprotein, a cell adhesion molecule for postnatal neural development, are produced by alternative splicing. Proc Natl Acad Sci U S A 84:4337-4341.
Lim S, Naisbitt S, Yoon J, Hwang JI, Suh PG, Sheng M, Kim E (1999) Characterization of the Shank family of synaptic proteins. Multiple genes, alternative splicing, and differential expression in brain and development. J Biol Chem 274:29510-29518.
Lin GT, Fann MJ (2007) Characterization of CDK12 and CDK13 functions in neurite outgrowth. Master Thesis, Institute of Neuroscience, National Yang-Ming University, Taiwan.
Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (2004) Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304:1021-1024.
Loyer P, Trembley JH, Grenet JA, Busson A, Corlu A, Zhao W, Kocak M, Kidd VJ, Lahti JM (2008) Characterization of cyclin L1 and L2 interactions with CDK11 and splicing factors: influence of cyclin L isoforms on splice site selection. J Biol Chem 283:7721-7732.
McHugh TJ, Blum KI, Tsien JZ, Tonegawa S, Wilson MA (1996) Impaired hippocampal representation of space in CA1-specific NMDAR1 knockout mice. Cell 87:1339-1349.
Mineur YS, Crusio WE (2002) Behavioral and neuroanatomical characterization of FVB/N inbred mice. Brain Res Bull 57:41-47.
Morris RG, Anderson E, Lynch GS, Baudry M (1986) Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 319:774-776.
Myers SJ, Dingledine R, Borges K (1999) Genetic regulation of glutamate receptor ion channels. Annu Rev Pharmacol Toxicol 39:221-241.
Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH (1996) The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes Dev 10:816-825.
Oh ES, Savonenko AV, King JF, Fangmark Tucker SM, Rudow GL, Xu G, Borchelt DR, Troncoso JC (2008) Amyloid precursor protein increases cortical neuron size in transgenic mice. Neurobiol Aging.
Oprica M, Hjorth E, Spulber S, Popescu BO, Ankarcrona M, Winblad B, Schultzberg M (2007) Studies on brain volume, Alzheimer-related proteins and cytokines in mice with chronic overexpression of IL-1 receptor antagonist. J Cell Mol Med 11:810-825.
Patrick GN, Zukerberg L, Nikolic M, de la Monte S, Dikkes P, Tsai LH (1999) Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402:615-622.
Pines J (1994) Protein kinases and cell cycle control. Semin Cell Biol 5:399-408.
Pugh PL, Ahmed SF, Smith MI, Upton N, Hunter AJ (2004) A behavioural characterisation of the FVB/N mouse strain. Behav Brain Res 155:283-289.
Sandell LJ, Nalin AM, Reife RA (1994) Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non-cartilaginous tissues during early mouse development. Dev Dyn 199:129-140.
Scheper W, Zwart R, Baas F (2004) Alternative splicing in the N-terminus of Alzheimer's presenilin 1. Neurogenetics 5:223-227.
Scoville WB, Milner B (1957) Loss of recent memory after bilateral hippocampal lesions. J Neurol Neurosurg Psychiatry 20:11-21.
Smith PD, Crocker SJ, Jackson-Lewis V, Jordan-Sciutto KL, Hayley S, Mount MP, O'Hare MJ, Callaghan S, Slack RS, Przedborski S, Anisman H, Park DS (2003) Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A 100:13650-13655.
Stent GS (1973) A physiological mechanism for Hebb's postulate of learning. Proc Natl Acad Sci U S A 70:997-1001.
Taketo M, Schroeder AC, Mobraaten LE, Gunning KB, Hanten G, Fox RR, Roderick TH, Stewart CL, Lilly F, Hansen CT, et al. (1991) FVB/N: an inbred mouse strain preferable for transgenic analyses. Proc Natl Acad Sci U S A 88:2065-2069.
Tao HW, Poo M (2001) Retrograde signaling at central synapses. Proc Natl Acad Sci U S A 98:11009-11015.
Watabe AM, O'Dell TJ (2003) Age-related changes in theta frequency stimulation-induced long-term potentiation. Neurobiol Aging 24:267-272.
Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning induces long-term potentiation in the hippocampus. Science 313:1093-1097.
Winocur G, Moscovitch M, Sekeres M (2007) Memory consolidation or transformation: context manipulation and hippocampal representations of memory. Nat Neurosci 10:555-557.
Wright IC, Rabe-Hesketh S, Woodruff PW, David AS, Murray RM, Bullmore ET (2000) Meta-analysis of regional brain volumes in schizophrenia. Am J Psychiatry 157:16-25.
Yu H, Wang Y, Pattwell S, Jing D, Liu T, Zhang Y, Bath KG, Lee FS, Chen ZY (2009) Variant BDNF Val66Met polymorphism affects extinction of conditioned aversive memory. J Neurosci 29:4056-4064.
Zhou J, Yu Q, Zou T (2008) Alternative splicing of exon 10 in the tau gene as a target for treatment of tauopathies. BMC Neurosci 9 Sup 2:S10.
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