Abstract
Thymosin β4 (Tβ4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tβ4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tβ4 are associated with L1 functions. In the present study, we hypothesized that Tβ4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tβ4 ranging from 0.1 to 10 μg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tβ4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, β-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tβ4. Furthermore, the number of β-tubulin III-positive cells and neurite lengths were measured. We found that Tβ4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tβ4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tβ4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 μg/ml Tβ4, respectively, whereas the effects of Tβ4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment. These above results indicate that the promotive effect of Tβ4 on the survival and neurite outgrowth of cultured spinal cord neurons might be mediated, at least in part via a stimulation of the production of L1 in the neurons.
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Yu FX, Lin SC, Morrsion-Bogorad M, Atkinson MA, Yin HL (1993) Thymosin beta 10 and thymosin beta 4 are both actin monomer sequestering proteins. J Biol Chem 268:502–509
Molitoris BA (1997) Putting the actin cytoskeleton into perspective: pathophysiology of ischemic alterations. Am J Physiol 272:430–433
Huff T, Muller CS, Otto AM, Netzker R, Hannappel E (2001) beta-Thymosins, small acidic peptides with multiple functions. Int J Biochem Cell Biol 33:205–220
Kobayashi T, Okada F, Fujii N, Tomita N, Ito S, Tazawa H, Aoyama T, Choi SK, Shibata T, Fujita H, Hosokawa M (2002) Thymosin-beta4 regulates motility and metastasis of malignant mouse fibrosarcoma cells. Am J Pathol 160:869–882
Roy P, Rajfur Z, Jones D, Marriott G, Loew L, Jacobson K (2001) Local photorelease of caged thymosin beta4 in locomoting keratocytes causes cell turning. J Cell Biol 153:1035–1048
Gomez-Marquez J, Pedrares JI, Otero A, Anadon R (1993) Prominent expression of the actin-sequestering peptide Fx gene in the hippocampal region of rat brain. Neurosci Lett 152:41–44
Bambury JR, Bernstein BW (1991) Actin and actin-binding proteins in neurons. In: Burgoyne R (ed) The neuronal cytoskeleton. Wiley-liss, New York, pp 121–160
Fifkova E (1985) Actin in nervous sytem. Brain Res 356:187–215
Border BG, Lin SC, Griffin WS, Pardue S, Morrison-Bogorad M (1993) Alterations in actin-binding beta-thymosin expression accompany neuronal differentiation and migration in rat cerebellum. J Neurochem 61:2104–2114
Otero A, Bustelo XR, Pichel JG, Freire M, Gomez-Marquez J (1993) Transcript levels of thymosin beta 4, an actin-sequestering peptide, in cell proliferation. Biochim Biophys Acta 1176:59–63
Anadon R, Rodriguez Moldes I, Carpintero P, Evangelatos G, Livianou E, Leondiadis L, Quintela I, Cervino MC, Gomez-Marquez J (2001) Differential expression of thymosins beta(4) and beta(10) during rat cerebellum postnatal development. Brain Res 894:255–265
Carpinterio P, Anadon R, Del Amo FF, Gomez-Marquez J (1995) The thymosin beta 4 gene is strongly activated in neural tissues during early postimplantation mouse development. Neurosci Lett 184:63–66
Gomez-Marquez J, Anadon R (2002) The beta-thymosins, small actin-binding peptides widely expressed in the developing and adult cerebellum. Cerebellum 1:95–102
Carpintero P, Anadon R, Diaz-Regueira S, Gomez-Marquez J (1999) Expression of thymosin beta4 messenger RNA in normal and kainate-treated rat forebrain. Neuroscience 90:1433–1444
Vartiainen N, Pyykonen I, Hokfelt T, Koistinaho J (1996) Induction of thymosin beta (4) mRNA following focal brain ischemia. Neuroreport 7:1613–1616
Yu FX, Lin SC, Morrison-Bogorad M, Yin HL (1994) Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells. Cell Motil Cytoskeleton 27:13–25
Matsuoka Y, Li X, Bennett V (1998) Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin–actin complexes and occurs in many cells, including dendritic spines of neurons. J Cell Biol 142:485–497
Akiyama H, Tooyama I, Kondo H, Ikeda K, Kimura H, McGeer EG, McGeer PL (1994) Early response of brain resident microglia to kainic acid-induced hippocampal lesions. Brain Res 635:257–268
Dusart I, Marty S, Peschanski M (1992) Demyelination, and remyelination by Schwann cells and oligodendrocytes after kainate-induced neuronal depletion in the central nervous system. Neuroscience 51:137–148
Marty S, Dusart I (1991) Glial changes following an excitotoxic lesion in the CNS-II. Astrocytes. Neuroscience 45:541–549
Sosne G, Szliter EA, Barrett R, Kernacki KA, Kleinman H, Hazlett LD (2002) Thymosin beta 4 promotes corneal wound healing and decreases inflammation in vivo following alkali injury. Exp Eye Res 74:293–299
Sosne G, Hafeez S, Greenberry AL, Kurpakus-Wheater M (2002) Thymosin beta4 promotes human conjunctival epithelial cell migration. Curr Eye Res 24:268–273
Sosne G, Xu L, Prach L, Mrock LK, Kleinman HK, Letterio JJ, Hazlett LD, Kurpakus-Wheater M (2004) Thymosin beta 4 stimulates laminin-5 production independent of TGF-beta. Exp Cell Res 293:175–183
Sosne G, Chan CC, Thai K, Kennedy M, Szliter EA, Hazlett LD, Kleinman HK (2001) Thymosin beta 4 promotes corneal wound healing and modulates inflammatory mediators in vivo. Exp Eye Res 72:605–608
Brummendorf T, Kenwrick S, Rathjen F (1998) Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations. Curr Opin Neurobiol 8:87–97
Chen S, Mange A, Dong L, Lehmann S, Schachner M (2003) Prion protein as trans-interacting partner for neurons is involved in neurite outgrowth and neuronal survival. Mol Cell Neurosci 22:227–233
Kamiguchi H, Hlavin ML, Yamasaki M, Lemmon V (1998) Adhesion molecules and inherited diseases of the human nervous system. Annu Rev Neurosci 21:97–125
Hortsch M (1996) The L1 family of neural cell adhesion molecules: old proteins performing new tricks. Neuron 17:587–593
Schachner M (1997) Neural recognition molecules and synaptic plasticity. Curr Opin Cell Biol 9:627–634
Lindner J, Rathjen FG, Schachner M (1983) L1 mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum. Nature 305:427–430
Lindner J, Zinser G, Werz W, Goridis C, Bizzini B (1986) Schachner experimental modification of postnatal cerebellar granule cell migration in vitro. Brain Res 377:298–304
Hoffman S, Friedlander DR, Chuong CM, Grumet M, Edelman GM (1986) Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions. J Cell Biol 103:145–158
Honig MG, Petersen GG, Rutishauser US, Camilli SJ (1998) In vitro studies of growth cone behavior support a role for fasciculation mediated by cell adhesion molecules in sensory axon guidance during development. Dev Biol 204:317–326
Fischer G, Kunemund V, Schachner M (1986) Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1. J Neurosci 6:605–512
Rathjen FG, Wolff JM, Frank R, Bonhoeffer F, Rutishauser U (1987) Membrane glycoproteins involved in neurite fasciculation. J Cell Biol 104:343–353
Bastmeyer M, Ott H, Leppert CA, Stuermer CA (1995) Fish E587 glycoprotein, a member of the L1 family of cell adhesion molecules, participates in axonal fasciculation and the age-related order of ganglion cell axons in the goldfish retina. J Cell Biol 130:969–976
Stallcup WB, Beasley L (1985) Involvement of the nerve growth factor-inducible large external glycoprotein (NILE) in neurite fasciculation in primary cultures of rat brain. Proc Natl Acad Sci USA 82:1276–1280
Lagennaur C, Lemmon V (1987) An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc Natl Acad Sci USA 84:7753–7757
Lemmon V, Farr KL, Lagenaur C (1989) L1-mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron 2:1597–1603
Williams EJ, Furness J, Walsh FS, Doherty P (1994) Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13:583–594
Kuhn TB, Stoeckli ET, Condrau MA, Rathjen FG, Sonderegger P (1991) Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4). J Cell Biol 115:1113–1126
Volkmer H, Leuschner R, Zacharias U, Rathjen FG (1996) Neurofascin induces neurites by heterophilic interactions with axonal NrCAM while NrCAM requires F11 on the axonal surface to extend neurites. J Cell Biol 135:1059–1069
Yang H, Liang Z, Bao X, Ju G (1997) A study on improving call yield and viability of spinal cord neurons in vitro. Chinese J Anat 20:235–242
Chen YK, Yang H, Lu F, Pu Q, Li RD, Zhao ZL (2002) Cloning expression in E. coli and biological activity of human thymosin β4. Acta Biochim Biophys Sin 34:502–505
Doherty P, Williams E, Walsh FS (1995) A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth. Neuron 14:54–63
Mohajeri MH, Bartsch U, Van der Putten H, Sansig G, Mucke L, Schachner M (1996) Neurite outgrowth on non-permissive substrates in vitro is enhanced by ectopic expression of the neural adhesion molecule L1 by mouse astrocytes. Eur J Neurosci 8:1085–1097
Stoscheck CM (1990) Quantitation of protein. Methods Enzymol 182:50–68
Inatani M, Honjo M, Otori Y, Oohira A, Kido N, Tano Y, Honda Y, Tanihara H (2001) Inhibitory effects of neurocan and phosphacan on neurite outgrowth from retinal ganglion cells in culture. Invest Ophthalmol Vis Sci 42:1930–1938
Mitchell PJ, Hanson JC, Quets-Nguyen AT, Bergeron M, Smith RC (2007) A quantitative method for analysis of in vitro neurite outgrowth. J Neurosci Methods 164:350–362
Roth LW, Borman P, Bonnet A, Reinhard E (1999) Beta-thymosin is required for axonal tract formation in developing zerbrafish brain. Development 126:1365–1374
Malinda KM, Sidhu GS, Mani H, Banaudha K, Maheshwari RK, Goldstein AL, Kleinman HK (1999) Thymosin beta4 accelerates wound healing. J Invest Dermatol 113:364–368
Gomez-Marquez J, Franco del Amo F, Carpintero P, Anadon R (1996) High levels of mouse thymosin beta4 mRNA in differentiating P19 embryonic cells and during development of cardiovascular tissues. Biochim Biophys Acta 1306:187–193
Girardi M, Sherling MA, Renata FB, Filler RB, Shires J, Theodoridis E, Hayday AC, Tigelaar RE (2003) Anti-inflammatory effects in the skin of thymosin-β4 splice-variants. Immunology 109:1–7
Sosne G, Siddiqi A, Kurpakus-Wheater M (2004) Thymosin beta4 inhibits corneal epithelial cell apoptosis after ethanol exposure in vitro. Invest Ophthalmol Vis Sci 45:1095–1100
Malinda KM, Sidhu CS, Kleinman HK (1997) Thymosin beta 4 stimulates directional migration of human umbilical vein endothelial cells. FASEB 11:474–481
Fields RD, Itoh K (1996) Neural cell adhesion molecules in activity-dependent development and synaptic plasticity. Trends Neurosci 19:473–480
Haspel J, Grumet M (2003) The L1-CAM extracellular region: a multi-domain protein with modular and cooperative binding modes. Front Biosci 8:1210–1225
Hortsch M (2000) Structural and functional evolution of the L1 family: are four adhesion molecules better than one? Mol cell Neurosci 15:1–10
Edehnan GM (1993) Jones FS outside and downstream of the homenbox. J Biol Chem 268:20683–20686
Mitchison T, Kirschner M (1988) Cytoskeletal dynamics and nerve growth. Neuron 1:761–772
Lin CH, Espreafico EM, Mooseker MS, Forscher P (1996) Myosin drives retrograde F-actin flow in neuronal growth cones. Neuron 16:769–782
Forscher P, Smith SJ (1988) Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol 107:1505–1516
Bray D, Chapman K (1985) Analysis of microspike movements on the neuronal growth cone. J Neurosci 5:3204–3213
Okabe S, Hirokawa N (1991) Actin dynamics in growth cones. J Neurosci 11:1918–1929
Theriot JA, Mitchison TJ (1992) Comparison of actin and cell surface dynamics in motile fibroblasts. J Cell Biol 118:367–377
Theriot JA, Mitchison TJ (1991) Actin microfilament dynamics in locomoting cells. Nature 352:126–131
Turrini P, Tirassa P, Vigneti E, Aloe L (1998) A role of the thymus and thymosin-α1 in brain NGF levels and NGF receptor expression. J Neuroimmunol 82:64–78
Tanaka EM, Kirschner MW (1991) Microtubule behavior in the growth cones of living neurons during axon elongation. J Cell Biol 115:345–363
Acknowledgments
We thank Dr. Zhe Ling from Emory University for critical reading of the manuscript. This work was supported by the National Basic Research Program of China (2003CB515301) and National Nature Science Foundation of China (30270450).
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Yang, H., Cheng, X., Yao, Q. et al. The Promotive Effects of Thymosin β4 on Neuronal Survival and Neurite Outgrowth by Upregulating L1 Expression. Neurochem Res 33, 2269–2280 (2008). https://doi.org/10.1007/s11064-008-9712-y
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DOI: https://doi.org/10.1007/s11064-008-9712-y