腫瘤壞死因子在中樞神經系統發育與再生之角色探討:腫瘤壞死因子調控神經幹母細胞分化之研究

全文

(1)行政院國家科學委員會專題研究計畫. 成果報告. 腫瘤壞死因子在中樞神經系統發育與再生之角色探討:腫瘤壞死因子調控神經幹母細胞分化之研究. 計畫類別：個別型計畫計畫編號： NSC91-2311-B-006-004執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日執行單位：國立成功大學生物學系（所）. 計畫主持人：曾淑芬. 報告類型：精簡報告處理方式：本計畫可公開查詢. 中. 華. 民. 國 92 年 10 月 25 日.

(2) 中文摘要關鍵詞：腫瘤壞死因子-α﹔脂多醣﹔前發炎組織介素﹔神經細胞﹔神經幹母細胞; 小神經膠細胞。腫瘤壞死因子-α (TNF-α) 是一種多功能的組織介素 (Cytokine)，在中樞神經系統受傷時扮演一個重要的角色。TNF-α 會與細胞表面的兩個接受器，腫瘤壞死因子接受器 1 (p55 TNFR1)及腫瘤壞死因子接受器 2 (p75 TNFR2) 作用而引起一連串的生物性反應。雖然在中樞神經系統受傷後，TNF-α 會正向調控神經細胞的死亡，但最近的研究顯示，TNF-α 能防止神經細胞的計劃性死亡且促進寡樹突膠質前驅細胞的增生及寡樹突膠質細胞 (oligodendrocytes) 的分化。TNF-α 也會高度表現於發育中的腦及神經細胞。本實驗室先期的研究亦發現，以 TNF-α 注射到成鼠的腦部會使次腦室/腦室區 (SVZ/VZ) 中的神經前驅細胞增生。這些研究猜測，TNF-α 在神經細胞的生成過程中扮演了一個重要的角色。. 脂多醣 (LPS) 是來自格蘭氏陰性菌細胞壁的內毒素的活性部分，會刺激小神經膠細胞 (microglia)製造大量的前發炎組織介素包括 TNF-α 及間白素-1β (IL-1β)。本實驗室先期的研究顯示，從胎鼠腦中分離出的神經幹母細胞及神經前驅細胞（在此統稱-神經幹母細胞）經培養後用脂多醣刺激微小神經膠細胞所產生的培養基刺激會促進神經樹 (neurites) 及寡樹突膠質細胞樹(processes)的生長。最近實驗結果指出 premature NSCs 以 TNF-α處理降低其神經細胞的生成，然而 TNF-α 對 immature NSCs 神經細胞的生成並沒有影響; 另外，前發炎因子 IL-18 對 premature NSCs 神經細胞的生成並無影響，但是增加 immature NSCs 神經細胞的生成。. 2.

(3) Abstract Key Words: Tumor necrosis factor-α; lipopolysaccharides; neural stem cells/neural progenitors; microglia. Tumor necrosis factor-α (TNF-α) is a pluripotent cytokine that has been believed to have a critical role in the pathogenesis of CNS injury. TNF-α induces a wide spectrum of biological signals by interacting with two cell surface receptors, TNF receptor 1 (p55 TNFR1) and 2 (p75 TNFR2). Although TNF-α has long been known to have positive regulatory role in neural cell death after CNS injury, recent studies have shown that TNF-α may prevent neuronal apoptosis, and promote the proliferation of oligodendroglial progenitors and oligodendroglial maturation. TNF-α is also highly expressed in developmental brains and neurons. In our previous findings, TNF-α administration into adult rat brains causes the proliferation of neural progenitors in the subventricular/ventricular zone (SVZ/VZ). These studies indicate a role for TNF-α in neurogenesis. Lipopolysaccharides (LPS), a major component of the cell wall of the gram- bacteria, stimulates microglia to highly produce proinflammatory cytokines including TNF-α and IL-1β. In our preliminary studies, the conditioned medium (CM) of microglia treated with LPS can enhance the growth of neurites and oligodendroglial processes in the culture of neural stem cells/neural progenitors (NSCs) isolated from embryonic rat brains. In contrast to our hypothesis, we found that TNF-α reduced the production of βIII-tubulin+ neurons in the premature NSC culture, while the molecule had no effect on the production of βIII-tubulin+ neurons in the immature NSC culture. On the other hand, IL-18, another proinflammatory cytokine, can increase the production of βIII-tubulin+ neurons in the immature NSC culture, whereas it had no effect on the premature NSC culture.. 3.

(4) Introduction The neural cells in the central nervous system (CNS) can be divided into neurons and glia. Glial populations consist of oligodendroglia, astroglia and microglia. The CNS network is formed by neurons that respond to stimuli (such as excitatory and inhibitory AAs, neurotransmitters, etc.) with an electrical discharge. In this way, signals can be transmitted in milliseconds from one cell to another cell. Oligodendroglia produce myelin that functions as an electrical insulator and facilitates conduction whereas the functional roles of astroglia in the CNS are controversial. The presence of astroglia is generally thought to be critical during CNS development and after CNS injuries. Microglia act as the CNS scavenger during development and after injury, and initiate a series of inflammatory reactions after injury. During embryogenesis, neuroepithelial cells in the neural tube undergo proliferation and give rise to neural progenitors that generate neurons, oligodendroglia and astroglia at different stages of development (Gage, 2000; Svendsen and Caldwell, 2000). Traditional notions are that neural stem cells/neural progenitors (NSCs) are depleted when the development terminates. Yet, many studies now indicate that NSCs exist in adult, and preserve their potential for proliferation and differentiation (Cameron and McKay, 1998). NSCs may function at the lower rate in adult than in development because the adult CNS environment may lack constructive factors, such as growth factors and neurotrophic factors. Growing evidence also show that bFGF and EGF are mitogenic to neural progenitors in vivo and in vitro (Gensburger et al., 1981; Calof, 1995; Craig et al., 1996; Kuhn et al., 1997). Neurotrophic factors, bFGF, BDNF, GDNF, and NT3/4 , not only are survival factors for neurons, but also may act as factors for the differentiation of neural precursor cells (Thoenen et al., 1993; Averbuch-Heller et al., 1994; Lindsay et al., 1994; Ghosh and Greenberg, 1995; Lewin and Barde, 1996). In our laboratory, the NSC culture isolated from embryonic day 14-15 rat brains has been established. In consistence with observations in other laboratories, NSCs aggregate to clones referred as neurospheres and express nestin, a NSC cell marker (Section of Preliminary Data). They show a normal growth rate, and are able to differentiate into neurons, astroglia and oligodendroglia. TNF-α, a pleiotropic cytokine, is developmentally expressed in the CNS (Pan et al., 1997; Munoz-Fernandez and Fresno, 1998). Its expression is undetectable in adult rodent and human brains (Masson et al., 1998). The TNFα expressing cell populations in the developmental CNS include neurons and glia. Functionally, it is widely accepted that TNF-α mediates apoptosis during CNS maturation. However, several in vitro studies show TNF-α-stimulated differentiation of neuronal cell lines (Obregon et al., 1997; 1999), indicating the dual biological activity of TNF-α in the CNS.. 4.

(5) After injuries to the CNS, TNF-α is produced as a proinflammatory cytokines that mediates inflammation, demyelination, and cytotoxicity. Therefore, it is considered to be a cytotoxic cytokine in many neurodegenerative diseases, such as cerebral ischemia, multiple sclerosis, Alzheimer's disease, and human immunodeficiency virus (HIV) encephalopathy. TNFα signals its effects via binding to distinct receptors, the TNFR1 (p55 TNF receptor) and the TNFR2 (p75 TNF receptor). TNFR1 and TNFR2 are ubiquitously coexpressed on glial, neuronal, and endothelial cells of the CNS (Vandenbeele et al., 1995; Dopp et al.,1997). This TNF ligand/receptor system has been known to trigger oligodendglial death (Selmaj and Raine, 1988; D'Souza et al., 1995). In fact, most biological responses to TNF-α are mediated by the TNFR1 that belongs to the death receptor family and mediates cell apoptosis by activation of NFκB (Wallach et al., 1997). In contrast, TNFR2 is thought to suppress TNF-α mediated inflammatory responses in vivo (Peschon et al., 1998). In general, the two TNF receptors have distinct roles in the CNS and can mediate neuropathology either through an apoptotic pathway or through an inflammatory cascade (Probert and Akassoglou, 2001). Based on these fruitful findings, the role of TNF-α ligand/receptor system in the induction of apoptosis and inflammation is well accepted. Recently, Arnett et al. (2001) have reported that TNF-α promotes oligodendroglial progenitor proliferation and remyelination through TNFR2 signaling. Moreover, deletion of TNFR decreases nuclear factor-κB (NF-κB) activation and causes a poorer neurological outcome after spinal cord injury (Kim et al., 2001). NF-κB activation has been thought to enhance or reduce apoptosis (Mattson et al., 2000). However, recent studies have also addressed that NFκB activated by TNF-α ligand/receptor system may be involved in anti-apoptosis (Tamatani et al., 1999; Chen et al., 2000; Foehr et al., 2000). These findings indicate the beneficial role of TNFR1 and TNFR2 in CNS. In our previous studies, well differentiated neurons were observed when NSCs were treated with in the conditioned medium (CM) of activated microglia by LPS. Because treatment of microglia with LPS dramatically increased the amounts of TNF-α in microglia CM. Thus, we continued to study the role of TNF-α in neuronal differentiation. Materials and Methods NSC culture preparation NSCs were prepared from embryonic day 14/15 rat brains using the method described in Tzeng (2002) with little modification. Briefly, tissues were chopped in DMEM/F-12 medium with 0.01% (w/v) DNase, and continuously triturated several times, passed through a 40 µm nylon mesh. After centrifugation at 1500 rpm for 10 min, the pellet was resuspended with the growth medium that consisted of DMEM/F-12, N2, bFGF (20 ng/ml), and EGF (20 ng/ml). Culture medium were changed every 2 days. After 1-2 weeks, neurospheres were generated and floated in the culture. Neurospheres were collected and replated onto PDL-coated petri dishes. Cultures were incubated in the neural basal medium plus serum supplement-B27 (differentiation 5.

(6) medium) for 3 d or 5 d, and subsequently treated with LPS-microglial CM or recombinant TNF-α for 3 d or 5 d. Primary rat microglial culture to produce LPS-microglial CM Primary mixed glial cultures were prepared from cerebral cortices of 1-2 d SD rats by the method of McCarthy and de Vellis (1980). Briefly, cerebral cortices were mechanically minced, digested by acetylated trypsin, and subsequently filtered through Nitex 130 and 35. After 7-8 d, culture were shaken at 100 rpm for 1 h. After shaking, medium was collected and centrifuged. Cell pellet was resuspended with DMEM/F12 plus 10% fetal calf serum (FCS) medium. Microglial cells were plated onto 35mm petri dishes. Next day, culture was treated with LPS (10, and/or 100 ng/ml) for 24 h. LPS-microglial CM was collected, and stored at -80oC until use. Immunocytochemstry The culture was fixed in PBS containing 4% paraformaldehyde for 15 min, and then incubated with PBS containing 0.1% Triton X-100 and 5% normal goat serum (NGS) for 20 min. Subsequently, cells were incubated at room temperature (RT) with primary antibodies at the appropriate dilution for 1 h followed by biotinylated secondary antibodies (1:200) for 1 h. Streptavidin-conjugated alkaline phosphatase was added to the culture for 20 min. Staining was observed by adding the substrate of alkaline phosphatase (BCIP/NBT) to the culture. The staining was visualized under a Nikon E-800 microscope. Results When NSCs were cultured in the microglial CM without LPS stimulation for 72 h, βIII-tubulin+ neurons appeared in the culture and mostly showed the bipolar morphology (Fig. 1A-C). When NSCs were treated with the CM of microglia activated by LPS at 10 ng/ml, the elongation of neurites was observed (Fig. 1D-F). Moreover, the CM of microglia activated with LPS at 100 ng/ml resulted in branching of neurites.. 6.

(7) Figure 1. βIII-tubulin immunostaining in NSC cultures treated with microglial CM (A-C), CM of microglia activated by LPS, 10 ng/ml (D-F), and CM of microglia activated by LPS, 100 ng/ml (G-I). Scale bar: 25 µm. Treatment of NSCs with the CM of LPS-treated microglia caused a reduction in the production of βIII-tubulin+ neurons (Fig. 2). Moreover, its effect was restored when LPS-activated microglia were treated with pentoxifylline (PEN), an inhibitor for TNF-α production.. Figure 2. The CM of LPS-treated microglia affects the production of βIII-tubulin+ neurons. Microglia were treated with LPS at the amounts of 10 or 100 ng/ml. At 24 h, the microglial CM was collected, and then added to NSC cultures which were maintained in the differentiation medium for 3 days (so-called premature NSC culture here). At 5 days after CM treatment, the culture were harvested and fixed in 4 % paraformaldehyde for immunocytochemical staining. Alternatively, microglia were treated with LPS at 10 or 100 ng/ml in the presence of 500 µM PEN. At 24 h, the CM was collected and added to NSC cultures. We found that PEN restored the production of βIII-tubulin+ neurons in NSC culture. Indeed, we found that TNF-α reduced the production of βIII-tubulin+ neuron in premature NSC culture (Fig. 3). However, IL-18, another proinflammatory cytokines, showed no effect on the production of βIII-tubulin+ neuron in premature NSC culture. Interestingly, TNF-α had no influence on the production of βIII-tubulin+ neuron in immature NSC culture (Fig. 4) that were maintained in the differentiation medium for 5 days before treatment. In addition, TNF-α did not affect the action of IL-18 on the production of βIII-tubulin+ neuron in immature NSC culture.. 7.

(8) Fig. 3. TNF-α and IL-18 affect the production of βIII-tubulin+ neurons in premature NSC culture.. Fig. 3. TNF-α and IL-18 affect the production of βIII-tubulin+ neurons in premature NSC culture.. Summary 1. The CM of LPS-treated microglia reduced neuron production in NSC culture although well-differentiated neurons were observed in NSC culture treated with the CM. 2. The inhibitor of TNF-α production can restore the reduction in neuron production induced by the CM of LPS-treated microglia. 8.

(9) 3. TNF-α inhibited neuron production in the premature NSC culture, but had no effect on the immature NSC culture. 4. In contrast, IL-18 increased neuron production in the immature NSC culture, but did not affect neuron production in the premature NSC culture. References Arnett HA, Mason J, Marino M, Suzuki K, Matsushima GK, Ting JP. 2001. TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nat Neurosci., 4:1116-1122. Averbuch-Heller, L., Pruginin, M., Kahane, N., Tsoulfas, P., Parada, L., Rosenthal, A., Kalcheim, C. 1994. Neurotrophin 3 stimulates the differentiation of motoneurons from avian neural tube progenitor cells. Proc. Natl. Acad. Sci. USA., 91:3247-3251. Baud V, Karin M. 2001. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol, 11: 372-377. Calof, A.L. 1995. Intrinsic and extrinsic factors regulating vertebrate neurogenesis. Curr. Opin. Neurobiol., 5: 19-27. Cameron HA, McKay R. 1998. Stem cells and neurogenesis in the adult brain. Curr Opin Neurobiol., 8:677-680. Chen C, Edelstein LC, Gelinas C. 2000. The Rel/NF- B family directly activates expression of the apoptosis inhibitor Bcl-xL. Mol Cell Biol 20:2687-2695. Craig CG, Tropepe V, Morshead CM, Reynolds BA, Weiss S, van der Kooy D. 1996. In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. J. Neurosci. 16:2648-2658. Dignam JD, Lebovitz RM, Roeder RG (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11:1475-1489. Dopp JM, Mackenzie-Graham A, Otero GC, Merrill JE. 1997. Differential expression, cytokine modulation, and specific functions of type-1 and type-2 tumor necrosis factor receptors in rat glia. J Neuroimmunol., 75:104-112.. 9.

(10) Dotti CG, Sullivan CA, Banker GA. 1998. The establishment of polarity of hippocampal neurons in culture. J. Neurosci. 8:1454-1468. D'Souza S, Alinauskas K, McCrea E, Goodyer C, Antel JP. 1995.. Differential susceptibility of. human CNS-derived cell populations to TNF-dependent and independent immune-mediated injury. J Neurosci, 15:7293-300. Foehr ED, Lin X, O'Mahony A, Geleziunas R, Bradshaw RA, Greene WC. 2000.. NF-kappa. B signaling promotes both cell survival and neurite process formation in nerve growth factor-stimulated PC12 cells. J Neurosci, 20:7556-7563. Gage FH. 2000. Mammalian neural stem cells. Science, 287:1433-1438. Gary DS, Bruce-Keller AJ, Kindy MS, Mattson MP. 1998. Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J Cereb Blood Flow Metab, 18:1283-1287. Gensburger, C., Labourdette, G., Sensenbrenner, M. 1981. Brain bFGF stimulates the proliferation of rat neuronal precursor cells in vitro. FEBS Lett., 217:1-5. Ghosh, A., Greenberg, M. 1995. Distinct roles for bFGF and NT3 in the regulation of cortical neurogenesis. Neuron. 15:89-103.. Kim GM, Xu J, Xu J, Song SK, Yan P, Ku G, Xu XM, Hsu CY. 2001.Tumor necrosis factor receptor deletion reduces nuclear factor-kappaB activation, cellular inhibitor of apoptosis protein 2 expression, and functional recovery after traumatic spinal cord injury. J Neurosci, 21:6617-6625. Kuhn, H.G., Winkler, J., Kempermann, G., Thal, L.J., Gage, F.H. 1997. Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J. Neurosci., 17: 5820-5829.. Lewin, G.R., Barde, Y.A. 1996. Physiology of the neurotrophins. Annu. Rev. Neurosci., 19: 289-317.. Lindsay, R., Wiegand, S., Altar, A., DiStefano, P. 1994. Neurotrophic factors: from molecule to man. Trends Neurosci. 17:182-190. 10.

(11) Masson R, Regnier CH, Chenard MP, Wendling C, Mattei MG, Tomasetto C, Rio MC. 1998. Tumor necrosis factor receptor associated factor 4 (TRAF4) expression pattern during mouse development. Mech Dev., 71:187-191.. Mattson MP, Culmsee C, Yu Z, Camandola S. 2000. Roles of nuclear factor- B in neuronal survival and plasticity. J Neurochem 74:443-456. McCarthy KD, de Vellis J. 1980. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85:890-902.. Munoz-Fernandez MA, Fresno M. 1998. The role of tumour necrosis factor, interleukin 6, interferon-gamma and inducible nitric oxide synthase in the development and pathology of the nervous system. Prog Neurobiol 56:307-340. Obregon E, Punzon MC, Gonzalez-Nicolas J, Fernandez-Cruz E, Fresno M, Munoz-Fernandez MA. 1997. Induction of adhesion/differentiation of human neuroblastoma cells by tumor necrosis factor-alpha requires the expression of an inducible nitric oxide synthase. Eur J Neurosci., 9:1184-1193. Obregon E, Punzon C, Fernandez-Cruz E, Fresno M, Munoz-Fernandez MA. 1999. HIV-1 infection induces differentiation of immature neural cells through autocrine tumor necrosis factor and nitric oxide production. Virology, 261:193-204. Pan W, Zadina JE, Harlan RE, Weber JT, Banks WA, Kastin AJ. 1997. Tumor necrosis factor-alpha: a neuromodulator in the CNS. Neurosci Biobehav Rev, 21:603-613. Peschon JJ, Torrance DS, Stocking KL, Glaccum MB, Otten C, Willis CR, Charrier K, Morrissey PJ, Ware CB, Mohler KM. 1998. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J Immunol., 160:943-952. Probert L, Eugster HP, Akassoglou K, Bauer J, Frei K, Lassmann H, Fontana A. 2000. TNFR1 signaling is critical for the development of demyelination and the limitation of T-cell responses during immune-mediated CNS disease. Brain, 123:2005-2019.. 11.

(12) Selmaj KW, Raine CS. 1988. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol., 23:339-346. Svendsen CN, Caldwell MA. 2000. Neural stem cells in the developing central nervous system: implications for cell therapy through transplantation. Prog Brain Res 127:13-34. Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, Mizuno T, Tohyama M. 1999. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF- B activation in primary hippocampal neurons. J Biol Chem 274:8531-8538 Thoenen, H., Huges, R.A., Sendtner, M. (1993) Trophic support of motoneurons: Physiological, pathophysiological, and therapeutic implications. Exp. Neurol., 124:47-55. Tzeng SF. 2002. Neural progenitors isolated from newborn rat spinal cords differentiate into neurons and astroglia. J Biomed Sci. 9:10-16.. Vandenabeele P, Declercq W, Beyaert R, Fiers W. 1995. Two tumour necrosis factor receptors: structure and function. Trends Cell Biol 5: 392-399. Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin Jr AS. 1998.. NF- κB. anti-apoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 281:1680 –1683. Wallach D, Boldin M, Varfolomeev E, Beyaert R, Vandenabeele P, Fiers W. 1997. Cell death induction by receptors of the TNF family: towards a molecular understanding. FEBS Lett. 410:96-106. Wu JP, Kuo JS, Liu YL, Tzeng SF. 2000. Tumor necrosis factor-alpha modulates the proliferation of neural progenitors in the subventricular/ventricular zone of adult rat brain. Neurosci Lett , 292:203-206.. 12.

(13)