||Investigation of the roles of CCAAT/enhancer binding protein delta (CEBPD) in astrogliosis after spinal cord injury
||Department of Pharmacology
spinal cord injury
脊髓損傷是一種常見且具破壞性的中樞神經系統疾病，其會導致脊髓中結構的瓦解並伴隨著有限的神經元再生乃至於後續的功能恢復降低的情形發生。脊髓損傷都會伴隨著星狀膠質細胞增生的產生，而星狀膠質數目大量的增加並活化為星狀膠質細胞增生的特徵。在嚴重的損傷之下，星狀膠質細胞增生會形成無法逆轉的神經膠質疤痕進而成為神經再生的屏障。因此，探討星狀膠質細胞如何被活化而形成神經膠質疤痕，將有助於脊髓損傷的治療。CEBPD是一個轉錄因子，其可受發炎因子TNF-α 和 IL-1β調控，而且在許多發炎相關疾病中，例如阿茲海默症病人中發現CEBPD在星狀膠質細胞有過度表現的現象。本研究發現在老鼠脊髓損傷的切片觀察到CEBPD會大量表現於神經膠質疤痕中。進一步地，我們對小鼠在脊髓損傷後星狀膠質細胞中存在CEBPD與否的影響進行行為分析，我們發現CEBPD的缺失有助於脊髓損傷小鼠的行動恢復。在分子機制探討中，星狀膠質細胞在IL-1β處理下，CEBPD的增加會經由抑制RhoA的路徑，而抑制了自身細胞的移動。有趣的是，在表現CEBPD的星狀膠質細胞會產生分泌型因子MMP3以促進星狀膠質細胞的移動。總結以上結果，我們推論出，在脊隨髓損傷區的星狀膠質細胞會因CEBPD的活化，而直接於損傷位置固著並釋放出促移行因子MMP3，而致使較遠端的未活化之星狀膠質細胞的移行至受損區，再被進一步激活並參與神經膠質疤痕的形成。
Spinal cord injury (SCI) is a common and devastating central nervous system (CNS) disease that results in disruption of cord microstructure and is followed by limited neuronal regeneration and functional recovery impairment. After SCI, astrocytes, the most abundant glial cells in the CNS, become reactive and hypertrophy. Astrogliosis is an increase in the number of astrocytes to above-normal levels that can be observed in all CNS injuries and neuroinflammatory diseases. In severe cases of injury, astrogliosis results in the formation of irreversible glia scarring that acts as a regeneration barrier. Thus, investigation targeting on astrocyte activation and glial scar formation could be useful for SCI therapy. Transcription factor CCAAT/enhancer binding protein delta (CEBPD) is responsive to inflammatory factors such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) and has been observed in many inflammation-related diseases including AD. In SCI mice, our results showed that CEBPD is expressed in reactive astrocyte border. Using animal behavior tests, we found that a better recovered effect was observed in injured Cebpd-deficient mice. Our results showed that increase of CEBPD in astrocytes inhibited their self-migration ability through the RhoA pathway upon IL-1β treatment. In addition, the conditioned medium of astrocyte expressing CEBPD could promote the migration of inactive astrocytes. We further identified matrix metalloproteinase-3 (MMP-3) was responsive to CEBPD in astrocytes through a transcriptional regulation. Taken together, the results suggested that the migration inactive astrocytes can be promoted by MMP3 secreted from the fixed activated astrocyte expressing CEBPD, which contributes to the formation of glial scar in SCI.
Abstract in Chinese III
Chapter 1 Introduction 1
1.1 Spinal cord injury 1
1.2 Inﬂammation following spinal cord injury 2
1.3 Astrogliosis and glial scar formation 3
1.4 CCAAT/enhancer-binding protein delta (CEBPD) 3
1.5 Matrix metalloproteinases (MMPs) family 4
1.6 Ras homolog gene family, member A (RhoA) 5
1.7 Motivation 5
Chapter 2 Materials and methods 7
2.1 Materials 7
2.2 Methods 7
Cell culture and isolation of primary mouse astrocytes 7
Quantitative PCR (Q-PCR) 8
Western blot analysis 8
Luciferase reporter assay 9
Lentiviral knockdown 9
Immunofluorescence analysis 10
Cell migration assays 11
Spinal cord injury mouse model 11
Assessment of recovery 11
Open-field locomotion. 12
Footprint analysis. 12
Luxol fast blue assay 12
Chapter 3 Results 14
3.1 CEBPD expression in astrocytes associates with glial scar formation after SCI 14
3.2 Loss of Cebpd improves behavioral recovery in SCI mice 14
3.3 Loss of CEBPD shows a decreased glial scar formation and increased neuronal regeneration 15
3.4 CEBPD has no effect on the proliferation of astrocytes in glial scar formation after SCI 15
3.5 Increasing of CEBPD expression in astrocytes attenuates self-migration through RhoA inhibition 16
3.6 MMP3 contributes to promote the migration of inactive astrocytes in conditioned medium of astrocyte expressing CEBPD 17
3.7 Conclusions 18
Chapter 4 Discussion 20
4.1 CNS injury and CEBPD 20
4.2 Glial scar formation and CEBPD 21
4.3 Glial scar formation and MMP3 23
4.4 CEBPD for SCI novel therapeutic target 24
Figure 1. CEBPD expression in astrocytes associates with glial scar formation in SCI mice. 36
Figure 2. Loss of CEBPD shows a recovery effect in SCI mice 38
Figure 3. Loss of CEBPD attenuates glial scar formation and is more permeable for axon passing through in SCI mice. 40
Figure 4. Cebpd has no effect on the proliferation of astrocytes. 42
Figure 5. Cebpd contributes to IL-1β-inhibited astrocytes migration. 43
Figure 6. Cebpd represses RhoA transcription in IL-1β-treated primary astrocytes. 44
Figure 7. Increase of CEBPD down-regulates RhoA expression in astrocytes in SCI mice. 45
Figure 8. Conditioned media from astrocytes expressing CEBPD induces the migration of inactive astrocytes. 46
Figure 9. CEBPD positively regulates Mmp3 transcription in primary astrocytes 48
Figure 10. Mmp3 expression was attenuated in the astrocytes of spinal cord injured-Cebpd-/- mice. 49
Figure 11. Conditioned media from MMP3-knockdowned astrocytes inhibits the migration of inactive astrocytes. 50
Araque A, Navarrete M (2010) Glial cells in neuronal network function. Philosophical transactions of the Royal Society of London Series B, Biological sciences 365:2375-2381.
Asher RA, Morgenstern DA, Fidler PS, Adcock KH, Oohira A, Braistead JE, Levine JM, Margolis RU, Rogers JH, Fawcett JW (2000) Neurocan is upregulated in injured brain and in cytokine-treated astrocytes. The Journal of neuroscience : the official journal of the Society for Neuroscience 20:2427-2438.
Barnabe-Heider F, Goritz C, Sabelstrom H, Takebayashi H, Pfrieger FW, Meletis K, Frisen J (2010) Origin of new glial cells in intact and injured adult spinal cord. Cell stem cell 7:470-482.
Barrett CP, Guth L, Donati EJ, Krikorian JG (1981) Astroglial reaction in the gray matter lumbar segments after midthoracic transection of the adult rat spinal cord. Experimental neurology 73:365-377.
Basso DM, Fisher LC, Anderson AJ, Jakeman LB, McTigue DM, Popovich PG (2006) Basso mouse scale for locomotion detects differences in recovery after spinal cord in ury in five common mouse strains. Journal of neurotrauma 23:635-659.
Bjorklund M, Koivunen E (2005) Gelatinase-mediated migration and invasion of cancer cells. Biochimica et biophysica acta 1755:37-69.
Bourguignon LY, Gilad E, Peyrollier K, Brightman A, Swanson RA (2007) Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes. Journal of neurochemistry 101:1002-1017.
Bracken MB (2002) Steroids for acute spinal cord injury. The Cochrane database of systematic reviews:CD001046.
Bradbury EJ, Moon LD, Popat RJ, King VR, Bennett GS, Patel PN, Fawcett JW, McMahon SB (2002) Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416:636-640.
Bush TG, Puvanachandra N, Horner CH, Polito A, Ostenfeld T, Svendsen CN, Mucke L, Johnson MH, Sofroniew MV (1999) Leukocyte infiltration, neuronal degeneration, and neurite outgrowth after ablation of scar-forming, reactive astrocytes in adult transgenic mice. Neuron 23:297-308.
Cardinaux JR, Allaman I, Magistretti PJ (2000) Pro-inflammatory cytokines induce the transcription factors C/EBPbeta and C/EBPdelta in astrocytes. Glia 29:91-97.
Chang C, Werb Z (2001) The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends in cell biology 11:S37-43.
Cregg JM, DePaul MA, Filous AR, Lang BT, Tran A, Silver J (2014) Functional regeneration beyond the glial scar. Experimental neurology 253:197-207.
Davies SJ, Fitch MT, Memberg SP, Hall AK, Raisman G, Silver J (1997) Regeneration of adult axons in white matter tracts of the central nervous system. Nature 390:680-683.
DeVivo MJ (1997) Causes and costs of spinal cord injury in the United States. Spinal cord 35:809-813.
Erturk A, Mauch CP, Hellal F, Forstner F, Keck T, Becker K, Jahrling N, Steffens H, Richter M, Hubener M, Kramer E, Kirchhoff F, Dodt HU, Bradke F (2012) Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nature medicine 18:166-171.
Esposito E, Cuzzocrea S (2011) Anti-TNF therapy in the injured spinal cord. Trends in pharmacological sciences 32:107-115.
Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology. Nature 420:629-635.
Faulkner JR, Herrmann JE, Woo MJ, Tansey KE, Doan NB, Sofroniew MV (2004) Reactive astrocytes protect tissue and preserve function after spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 24:2143-2155.
Garcia-Alias G, Barkhuysen S, Buckle M, Fawcett JW (2009) Chondroitinase ABC treatment opens a window of opportunity for task-specific rehabilitation. Nature neuroscience 12:1145-1151.
Gasche Y, Copin JC, Sugawara T, Fujimura M, Chan PH (2001) Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 21:1393-1400.
Gijbels K, Masure S, Carton H, Opdenakker G (1992) Gelatinase in the cerebrospinal fluid of patients with multiple sclerosis and other inflammatory neurological disorders. Journal of neuroimmunology 41:29-34.
Giulian D, Woodward J, Young DG, Krebs JF, Lachman LB (1988) Interleukin-1 injected into mammalian brain stimulates astrogliosis and neovascularization. The Journal of neuroscience : the official journal of the Society for Neuroscience 8:2485-2490.
Goldshmit Y, Frisca F, Pinto AR, Pebay A, Tang JK, Siegel AL, Kaslin J, Currie PD (2014) Fgf2 improves functional recovery-decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury. Brain and behavior 4:187-200.
Hellal F, Hurtado A, Ruschel J, Flynn KC, Laskowski CJ, Umlauf M, Kapitein LC, Strikis D, Lemmon V, Bixby J, Hoogenraad CC, Bradke F (2011) Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 331:928-931.
Hsu JY, Bourguignon LY, Adams CM, Peyrollier K, Zhang H, Fandel T, Cun CL, Werb Z, Noble-Haeusslein LJ (2008) Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord. The Journal of neuroscience : the official journal of the Society for Neuroscience 28:13467-13477.
Hu R, Zhou J, Luo C, Lin J, Wang X, Li X, Bian X, Li Y, Wan Q, Yu Y, Feng H (2010) Glial scar and neuroregeneration: histological, functional, and magnetic resonance imaging analysis in chronic spinal cord injury. Journal of neurosurgery Spine 13:169-180.
Hunanyan AS, Garcia-Alias G, Alessi V, Levine JM, Fawcett JW, Mendell LM, Arvanian VL (2010) Role of chondroitin sulfate proteoglycans in axonal conduction in Mammalian spinal cord. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:7761-7769.
Ito Z, Sakamoto K, Imagama S, Matsuyama Y, Zhang H, Hirano K, Ando K, Yamashita T, Ishiguro N, Kadomatsu K (2010) N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:5937-5947.
Jensen TS, Madsen CS, Finnerup NB (2009) Pharmacology and treatment of neuropathic pains. Current opinion in neurology 22:467-474.
John GR, Chen L, Rivieccio MA, Melendez-Vasquez CV, Hartley A, Brosnan CF (2004) Interleukin-1beta induces a reactive astroglial phenotype via deactivation of the Rho GTPase-Rock axis. The Journal of neuroscience : the official journal of the Society for Neuroscience 24:2837-2845.
Kinoshita S, Akira S, Kishimoto T (1992) A member of the C/EBP family, NF-IL6 beta, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proceedings of the National Academy of Sciences of the United States of America 89:1473-1476.
Ko CY, Wang WL, Wang SM, Chu YY, Chang WC, Wang JM (2014) Glycogen synthase kinase-3beta-mediated CCAAT/enhancer-binding protein delta phosphorylation in astrocytes promotes migration and activation of microglia/macrophages. Neurobiology of aging 35:24-34.
Ko CY, Chang LH, Lee YC, Sterneck E, Cheng CP, Chen SH, Huang AM, Tseng JT, Wang JM (2012a) CCAAT/enhancer binding protein delta (CEBPD) elevating PTX3 expression inhibits macrophage-mediated phagocytosis of dying neuron cells. Neurobiology of aging 33:422.e411-425.
Ko CY, Chang LH, Lee YC, Sterneck E, Cheng CP, Chen SH, Huang AM, Tseng JT, Wang JM (2012b) CCAAT/enhancer binding protein delta (CEBPD) elevating PTX3 expression inhibits macrophage-mediated phagocytosis of dying neuron cells. Neurobiology of aging 33:422 e411-425.
Lambertsen KL, Biber K, Finsen B (2012) Inflammatory cytokines in experimental and human stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 32:1677-1698.
Li R, Strohmeyer R, Liang Z, Lue LF, Rogers J (2004) CCAAT/enhancer binding protein delta (C/EBPdelta) expression and elevation in Alzheimer's disease. Neurobiology of aging 25:991-999.
Matrisian LM (1992) The matrix-degrading metalloproteinases. BioEssays : news and reviews in molecular, cellular and developmental biology 14:455-463.
McDonald JW, Sadowsky C (2002) Spinal-cord injury. Lancet 359:417-425.
Morawietz C, Moffat F (2013) Effects of locomotor training after incomplete spinal cord injury: a systematic review. Archives of physical medicine and rehabilitation 94:2297-2308.
Myer DJ, Gurkoff GG, Lee SM, Hovda DA, Sofroniew MV (2006) Essential protective roles of reactive astrocytes in traumatic brain injury. Brain : a journal of neurology 129:2761-2772.
Narumiya S, Tanji M, Ishizaki T (2009) Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion. Cancer metastasis reviews 28:65-76.
Nishimura S, Yasuda A, Iwai H, Takano M, Kobayashi Y, Nori S, Tsuji O, Fujiyoshi K, Ebise H, Toyama Y, Okano H, Nakamura M (2013) Time-dependent changes in the microenvironment of injured spinal cord affects the therapeutic potential of neural stem cell transplantation for spinal cord injury. Molecular brain 6:3.
Noble LJ, Donovan F, Igarashi T, Goussev S, Werb Z (2002) Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. The Journal of neuroscience : the official journal of the Society for Neuroscience 22:7526-7535.
Ramos-DeSimone N, Hahn-Dantona E, Sipley J, Nagase H, French DL, Quigley JP (1999) Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion. The Journal of biological chemistry 274:13066-13076.
Ridet JL, Malhotra SK, Privat A, Gage FH (1997) Reactive astrocytes: cellular and molecular cues to biological function. Trends in neurosciences 20:570-577.
Rosenberg GA, Dencoff JE, McGuire PG, Liotta LA, Stetler-Stevenson WG (1994) Injury-induced 92-kilodalton gelatinase and urokinase expression in rat brain. Laboratory investigation; a journal of technical methods and pathology 71:417-422.
Sanford DC, DeWille JW (2005) C/EBPdelta is a downstream mediator of IL-6 induced growth inhibition of prostate cancer cells. The Prostate 63:143-154.
Schachtrup C, Ryu JK, Helmrick MJ, Vagena E, Galanakis DK, Degen JL, Margolis RU, Akassoglou K (2010) Fibrinogen triggers astrocyte scar formation by promoting the availability of active TGF-beta after vascular damage. The Journal of neuroscience : the official journal of the Society for Neuroscience 30:5843-5854.
Sekhon LH, Fehlings MG (2001) Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26:S2-12.
Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nature reviews Neuroscience 5:146-156.
Sofroniew MV (2005) Reactive astrocytes in neural repair and protection. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 11:400-407.
Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annual review of cell and developmental biology 17:463-516.
Takeji M, Kawada N, Moriyama T, Nagatoya K, Oseto S, Akira S, Hori M, Imai E, Miwa T (2004) CCAAT/Enhancer-binding protein delta contributes to myofibroblast transdifferentiation and renal disease progression. Journal of the American Society of Nephrology : JASN 15:2383-2390.
Tator CH (1995) Update on the pathophysiology and pathology of acute spinal cord injury. Brain pathology 5:407-413.
Wang SM, Lee YC, Ko CY, Lai MD, Lin DY, Pao PC, Chi JY, Hsiao YW, Liu TL, Wang JM (2014) Increase of Zinc Finger Protein 179 in Response to CCAAT/Enhancer Binding Protein Delta Conferring an Antiapoptotic Effect in Astrocytes of Alzheimer's Disease. Molecular neurobiology.
Wells JE, Rice TK, Nuttall RK, Edwards DR, Zekki H, Rivest S, Yong VW (2003) An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice. The Journal of neuroscience : the official journal of the Society for Neuroscience 23:10107-10115.
Yong VW, Power C, Forsyth P, Edwards DR (2001) Metalloproteinases in biology and pathology of the nervous system. Nature reviews Neuroscience 2:502-511.
Yuan YM, He C (2013) The glial scar in spinal cord injury and repair. Neuroscience bulletin 29:421-435.