成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	林學賦 Lin, Shiue-Fu
論文名稱：	Smap1及Ccdc75 在大鼠海馬迴神經元軸突與樹突分支與延長的不同角色 Smap1 and Ccdc75 differentially regulate axonal and dendritic branches and elongation in rat hippocampal neurons
指導教授：	黃阿敏 Huang, A-Min
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	56
中文關鍵詞：	樹突、軸突、海馬迴神經、延長、分支
外文關鍵詞：	Smap1, Ccdc75, hippocampal neuron, axon, dendrites, elongation, branching
相關次數：	點閱：102 下載：1
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

軸突和樹突生長在神經細胞分化及再生扮演非常重要的角色。我們先前證實SMAP1和CCDC75受到 NRF-1調控。SMAP1是Arf6的GTP酶激活蛋白，而利用生物資訊預測CCDC75為核內蛋白並具有DNA結合區域，但這兩個基因在軸突和樹突生長的角色仍未知。本篇論文以初代體外培養海馬迴神經元探討SMAP1和CCDC75是否參與神經元軸突和樹突生長過程。從大鼠胚胎18天培養海馬迴神經元並抽取各發育階段的total RNA進行半定量RT-PCR分析，發現Smap1表現量從初期到晚期階段逐漸上升，但Ccdc75則是下降。過度表現Smap1會縮短軸突和樹突的長度，並且延後分支形成; 相反的，過度表現Ccdc75則增加軸突及樹突分支的數量但不影響長度。另一方面，調降Smap1的表現可促進軸突和樹突的長度但不影響分支;相反的，調降Ccdc75的表現減少軸突和樹突分支的數量但不影響長度。利用Smap1-EGFP或 Ccdc75-EGFP融合蛋白觀察其蛋白在細胞的定位，發現Smap1位在細胞質而Ccdc75則在細胞核。我們進一步證實Smap1與網格蛋白共同位於大鼠海馬迴神經元的細胞質中，過度表達Smap1則可抑制轉鐵蛋白受體藉由網格蛋白內吞的現象。這些結果顯示，在大鼠海馬迴神經元，Smap1可透過網格蛋白內吞的機制抑制軸突和樹突的延長，而Ccdc75 則透過未知的機制促進軸突和樹突的分支形成。本研究結果不僅增加神經發育過程中軸突和樹突生長的新知，並且提供神經再生的可能治療標的。

Axonal and dendritic growth is important for neuronal differentiation and nerve regeneration. We have identified that SMAP1 and CCDC75 are two genes downstream of nuclear respiratory factor 1 and differentially regulate neurtie outgrowth in human neuroblastoma cells. SMAP1 encodes the GTPase activating protein of Arf6 GTPase and CCDC75 is a novel gene predicted to encode a nuclear protein with the potential of DNA binding. It remains unknown whether these two genes play different roles in axonal and dendritic growth in neurons. Here, we used the development of cultured rat hippocampal neurons as a model to investigate whether these two genes have differential function in axonal and dendritic growth. Rat hippocampal neurons were cultured from embryonic day 18. Semi-quantitative RT-PCR analysis suggested that the mRNA level of Smap1 increased but Ccdc75 decreased from early to later stages. Overexpression of Smap1 decreased the length of axons and dendrites and delayed the initiation of axonal collaterals and dendritic branches. In contrast, overexpression of Ccdc75 increased the number of axonal collaterals and dendritic branches, but has no effects on the length of axons or dendrites. On the other hand, knockdown of Smap1 increased the length of axons and dendrites, but has no effects on the number of axonal collaterals and dendritic branches. In contrast, knockdown of Ccdc75 decreased the number of axonal collaterals and dendritic branches, but has no effects on axonal and dendritic length. Smap1-EGFP fusion protein localized in the cytosol and Ccdc75-EGFP fusion protein localized in the nucleus. Smap1 co-localized with clathrin in cultured rat hippocampal neurons. Overexpression of Smap1 blocked clathrin-dependent endocytosis which was demonstrated by the inhibition of transferrin receptor internalization. These results suggest that Smap1 may inhibit axonal and dendritic elongation through clathin-dependent endocytosis and Ccdc75 may enhance axonal and dendritic branching through an unknown mechanism. These results not only increase our knowledge on the molecular network underlying axonal and dendritic growth, but also provide potential therapeutic targets for neuronal regeneration after injury.

Table of contents
Abstract ............................................................................................................ 1
摘要 ................................................................................................................... 3
誌謝 ................................................................................................................... 4
Table of contents .............................................................................................. 5
I. Introduction
1-1. Physiological importance of axonal and dendritic outgrowth ................... 9
1-2. Cultured rat hippocampal neurons as a model for neuronal differentiation9
1-3. Molecules involved in the regulation of axonal and dendritic growth .... 10
1-4. Nuclear respiratory factor 1 (NRF-1) ...................................................... 11
1-5. Genome-wide screening of genes involved in NRF-1-regulated neurite
outgrowth......................................................................................................... 12
1-6. Stromal Membrane-Associated GTPase-Activating Protein 1, SMAP1 . 12
1-7. Coiled-coil domain containing 75, CCDC75........................................... 14
1-8. Hypothesis and specific aims ................................................................... 14
II. Materials and methods
2-1. Cell cultures ............................................................................................. 16
2-2. Plasmid constructs ................................................................................... 16
2-3. Recombinant lentivirus production .......................................................... 17
2-4. Semi-quantitative RT-PCR ...................................................................... 17
2-5. Hippocampal neuronal culture, transfection, and infection ..................... 18
2-6. Immunocytochemistry ............................................................................. 18
2-7. Measurement of axonal and dendritic parameters ................................... 19
2-8. Internalization of transferrin receptors .................................................... 19
2-9. Statistical analysis .................................................................................... 19
III. Results
3-1. Expression profiles of Smap1 and Ccdc75 in cultured rat hippocampal
neurons ............................................................................................................ 20
3-2. Overexpression of Smap1 decreases the length of axon and dendrites,
whereas overexpression of Ccdc75 increases number of axonal collaterals and
dendritic branches .......................................................................................... 20
3-3. Knockdown of Smap1 increases the length of axon and dendrite, whereas
knockdown of Ccdc75 increases the number of axonal collaterals and
dendritic branches ........................................................................................ 22
3-4. Predicted domains and cellular localization of Smap1 and Ccdc75 ...... 24
3-5. Smap1 co-localizes with clathrin in cultured rat hippocampal neurons 25
3-6. Overexpression of Smap1 blocks internalization of transferrin receptors25
IV. Discussion
4-1. Major findings .......................................................................................... 27
4-2. Expression profiles of Smap1 and Ccdc75 correlate with that of NRF-1 in
cultured rat hipppocampal neurons ................................................................. 27
4.3. Smap1, an ArfGAP, inhibits axon and dendrite elongation through
clathrin dependent endocytosis ....................................................................... 28
4.4. Ccdc75, a nuclear protein, promotes axonal and dendritic branching in rat
hippocampal neurons....................................................................................... 29
4.5. Conclusion................................................................................................30
V. Figures
Figure 1. Functional domains of SMAP1 and CCDC75. ................................ 31
Figure 2. Proposed differential roles of Samp1 and Ccdc75 in axonal and
dendritic growth in primary rat hippocampal neurons. ................................... 32
Figure 3. The time lines for assay of neuronal expression profile and
morphology.. ................................................................................................... 33
Figure 4. The mRNA expression profiles of Smap1 and Ccdc75 in cultured rat
hippocampal neurons at different developmental stages. ............................... 35
Figure 5. Effects of Smap1 or Ccdc75 overexpresssion on axonal and
dendritic growth at 4.5 DIV.. .......................................................................... 36
Figure 6. Effects of Smap1 or Ccdc75 overexpression on axonal and dendritic
growth at 7 DIV.. ............................................................................................. 37
Figure 7. Overexpression of Smap1 decreases axonal length, whereas
overexpression of Ccdc75 increases the number of axonal collaterals.. ......... 38
Figure 8. Overexpression of Smap1 decreases the dendritic length, whereas
overexpression of Ccdc75 increases the number of dendritic branches.. ....... 39
Figure 9. Effects of Smap1 or Ccdc75 knockdown on axonal and dendritic
growth at 4.5 DIV.. .......................................................................................... 40
Figure 10. Effects of Smap1 or Ccdc75 knockdown on axonal and dendritic
growth at 7 DIV. .............................................................................................. 42
Figure 11. Knockdown of Smap1 increases axonal length, whereas
knockdown of Ccdc75 decreases the number of axonal collaterals.. ............. 43
Figure 12. Knockdown of Smap1 increases dendritic length, whereas
knockdown of Ccdc75 decreases number dendritic branches... ..................... 44
Figure 13. Cellular localization of Smap1 and Ccdc75. ................................. 45
Figure 14. Smap1 co-localizes with clathrin in primary rat hippocampal
neurons. ........................................................................................................... 46
Figure 15. Effects of Smap1 overexpression on transferrin receptor
internalization in rat hippocampal neurons. .................................................... 47
Figure 16. Smap1 and Ccdc75 in axonal and dendritic elongation and
branching in primary rat hippocampal neurons. ............................................. 49
VI. Appendix
Table 1. shRNA clones. ................................................................................... 50
Table 2. Primer sequences and PCR parameters for semi-quantitative, full
length constructs and fusion protein constructs. ............................................. 51
VII. References. ............................................................................................. 52
                                    

Becker TS, Burgess SM, Amsterdam AH, Allende ML, Hopkins N (1998) not really finished is crucial for development of the zebrafish outer retina and encodes a transcription factor highly homologous to human Nuclear Respiratory Factor-1 and avian Initiation Binding Repressor. Development 125:4369-4378.
Bilimoria PM, Bonni A (2013) Molecular control of axon branching. The Neuroscientist 19:16-24.
Bushlin I, Petralia RS, Wu F, Harel A, Mughal MR, Mattson MP, Yao PJ (2008) Clathrin assembly protein AP180 and CALM differentially control axogenesis and dendrite outgrowth in embryonic hippocampal neurons. The Journal of neuroscience 28:10257-10271.
Cam H, Balciunaite E, Blais A, Spektor A, Scarpulla RC, Young R, Kluger Y, Dynlacht BD (2004) A common set of gene regulatory networks links metabolism and growth inhibition. Molecular cell 16:399-411.
Challacombe JF, Snow DM, Letourneau PC (1996) Actin filament bundles are required for microtubule reorientation during growth cone turning to avoid an inhibitory guidance cue. Journal of cell science 109 ( Pt 8):2031-2040.
Chang WT, Chen HI, Chiou RJ, Chen CY, Huang AM (2005) A novel function of transcription factor alpha-Pal/NRF-1: increasing neurite outgrowth. Biochemical and biophysical research communications 334:199-206.
Conde C, Caceres A (2009) Microtubule assembly, organization and dynamics in axons and dendrites. Nature reviews. Neuroscience 10:319-332.
de la Torre-Ubieta L, Bonni A (2011) Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain. Neuron 72:22-40.
DeSimone SM, White K (1993) The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein. Molecular and cellular biology 13:3641-3649.
Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. The Journal of neuroscience 8:1454-1468.
Egile C, Rouiller I, Xu XP, Volkmann N, Li R, Hanein D (2005) Mechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions. PLoS biology 3:e383.
FitzGerald PC, Shlyakhtenko A, Mir AA, Vinson C (2004) Clustering of DNA sequences in human promoters. Genome research 14:1562-1574.
Gallo G (2011) The cytoskeletal and signaling mechanisms of axon collateral branching. Developmental neurobiology 71:201-220.
Geschwind DH, Levitt P (2007) Autism spectrum disorders: developmental disconnection syndromes. Current opinion in neurobiology 17:103-111.
Goldberg JL (2003) How does an axon grow? Genes & development 17:941-58.
Govek EE, Newey SE, Van Aelst L (2005) The role of the Rho GTPases in neuronal development. Genes & development 19:1-49.
Hernandez-Deviez DJ, Casanova JE, Wilson JM (2002) Regulation of dendritic development by the ARF exchange factor ARNO. Nature neuroscience 5:623-624.
Huo L, Scarpulla RC (2001) Mitochondrial DNA instability and peri-implantation lethality associated with targeted disruption of nuclear respiratory factor 1 in mice. Molecular and cellular biology 21:644-654.
Ikeuchi Y, Stegmuller J, Netherton S, Huynh MA, Masu M, Frank D, Bonni S, Bonni A (2009) A SnoN-Ccd1 pathway promotes axonal morphogenesis in the mammalian brain. The Journal of neuroscience 29:4312-4321.
Koh CG (2006) Rho GTPases and their regulators in neuronal functions and development. Neuro-Signals 15:228-237.
Kon S, Minegishi N, Tanabe K, Watanabe T, Funaki T, Wong WF, Sakamoto D, Higuchi Y, Kiyonari H, Asano K, Iwakura Y, Fukumoto M, Osato M, Sanada M, Ogawa S, Nakamura T, Satake M (2013) Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia. The Journal of clinical investigation 123:1123-1137.
Kon S, Tanabe K, Watanabe T, Sabe H, Satake M (2008) Clathrin dependent endocytosis of E-cadherin is regulated by the Arf6GAP isoform SMAP1. Experimental cell research 314:1415-1428.
McAllister AK (2000) Cellular and molecular mechanisms of dendrite growth. Cereb cortex 10:963-973.
Mukaetova-Ladinska EB, Arnold H, Jaros E, Perry R, Perry E (2004) Depletion of MAP2 expression and laminar cytoarchitectonic changes in dorsolateral prefrontal cortex in adult autistic individuals. Neuropathology and applied neurobiology 30:615-623.
Tanabe K, Kon S, Ichijo N, Funaki T, Natsume W, Watanabe T, Satake M (2008) A SMAP gene family encoding ARF GTPase-activating proteins and its implication in membrane trafficking. Methods in enzymology 438:155-170.
Tanabe K, Torii T, Natsume W, Braesch-Andersen S, Watanabe T, Satake M (2005) A novel GTPase-activating protein for ARF6 directly interacts with clathrin and regulates clathrin-dependent endocytosis. Molecular biology of the cell 16:1617-1628.
Tong CW, Wang JL, Jiang MS, Hsu CH, Chang WT, Huang AM (2013) Novel genes that mediate nuclear respiratory factor 1-regualted neurite outgrowth in neuroblastoma IMR-32 cells. Gene 515:62-70.
Xie X, Lu J, Kulbokas EJ, Golub TR, Mootha V, Lindblad-Toh K, Lander ES, Kellis M (2005) Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals. Nature 434:338-345.
Yang WK, Peng YH, Li H, Lin HC, Lin YC, Lai TT, Suo H, Wang CH, Lin WH, Ou CY, Zhou X, Pi H, Chang HC, Chien CT (2011) Nak regulates localization of clathrin sites in higher-order dendrites to promote local dendrite growth. Neuron 72:285-299.
Yaron A, Zheng B (2007) Navigating their way to the clinic: emerging roles for axon guidance molecules in neurological disorders and injury. Developmental neurobiology 67:1216-1231.
Ye B, Zhang Y, Song W, Younger SH, Jan LY, Jan YN (2007) Growing dendrites and axons differ in their reliance on the secretory pathway. Cell 130:717-729.
Yoshimura T, Arimura N, Kaibuchi K (2006) Signaling networks in neuronal polarization. The Journal of neuroscience 26:10626-10630.
Zheng Y, Wildonger J, Ye B, Zhang Y, Kita A, Younger SH, Zimmerman S, Jan LY, Jan YN (2008) Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons. Nature cell biology 10:1172-1180.

校內：2016-08-26公開
校外：2016-08-26公開

簡易檢索 / 詳目顯示

相關論文