Abreviaturas
Abreviaturas
10 Abreviaturas
ADAMTS5: aggrecanase-2
AH: ácido hialurônico
AS: ácido siálico
ASP: asparagina
BDNF: brain-derived neurotrophic factor
C4S ou CS-A: condroitim 4-sulfato
C6S ou CS-C: condroitim 6-sulfato
CS-D: condroitim 2,6-sulfato
CS-E: condroitim 4,6-sulfato
CETAVLON: brometo de cetiltrimetilamônio
CHO: células ovarianas de hamster chinês
CHO-K1: células ovarianas de hamster chinês selvagem
CREB: cAMP response element binding protein
CS: condroitim sulfato
CSSTs: condroitim sulfato sulfotransferases
DAPI: 4´,6-diamidino-2-fenilindol dihidrocloreto
DMEM: Dulbeco’s Modified Eagle Medium
DS ou CS-B: dermatam sulfato
EBSS: solução salina balanceada de Earle
EDTA: etilenodiamino tetracetato
EGF: epidermal growth factor
F98: células de glioblastoma de rato
FITC: fluoroisotiocianato
FGF: fibroblast growth factor
Gal: galactose
GalNAc: N-acetilgalactosamina
GAP-43: growth-associated protein 43
GDNF: glial cell line-derived neurotrophic factor
GFAP: glial acidic fibrillary protein
GFP: proteína verde fluorescente
129
Abreviaturas
GlcA: ácido glucurônico
GlcNAc: N-acetilglucosamina
GPI: glicosilfosfatidilinositol
Hep: heparina
HB-EGF: heparin-binding EGF-like growth factor
HGF/SF: hepatocyte growth factor/scatter factor
HS: heparam sulfato
IL-4: Interleucina-4
IFN-γ: Ιnterferon-γ
IP-10: quimocina chemokine interferon-gamma-inducible protein-10
kD: kilodaltons
KS: queratam sulfato
LB: meio Luria Bertani
LINGO-1: leucine rich repeat transmembrane protein
MAG: myelin-associated glycoprotein
Man: manose
MB: membrana basal
MCP-1: monocyte chemotactic protein-1
MEC: matriz extracelular
MHC: major histocompatibility complex
MT3-MMP: membrane-type 3 matrix metalloproteinase
NeuN: neuron-specific nuclear protein
NGF: nerve growth factor
NgR: Nogo receptor
OMgp: oligodendrocyte myelin glycoprotein
OPC: células precursoras de oligodendrócitos
p75: p75 nerve growth factor receptor
PBS: phosphate buffered saline
PCR: reação da DNA polimerase em cadeia
PDA: tampão 1,3-diaminopropano acetato
PDGF: platelet derived growth factor receptor
130
Abreviaturas
PGCS: proteoglicano de condroitim sulfato
PGKS: proteoglicanos de queratam sulfato
PKC: proteína kinase C
PMSF: fluoreto de fenilmetilsulfonila
Pro-MMP-2: pro-matrix metalloproteinase-2
RPTPβ: receptor-type protein-tyrosine phosphatase β
SC: superfície celular
SDF-1b: quimocina Stromal cell-derived factor 1
Sema 3A: Semaforina 3A
SER: serina
SFB: soro fetal bovino
SLC: quimocina secondary lymphoid-tissue
SNC: sistema nervoso central
SNP: sistema nervoso periférico
TAE: tampão Tris-acetato 40mM; EDTA 1mM
TCE: Traumatismo crânio-encefálico
TNF-α: tumor necrosis factor-α
Tris: tris(hidroximetil)-aminometano
TSG-6: necrosis factor-α stimulated gene-6
WISP-1: wnt-1 induced secreted protein 1
Xil: xilose
μ: micro
9L: células de gliosarcoma de rato
131
Curriculum vitae resumido
Curriculum vitae resumido
11 Curriculum vitae resumido
Formação Acadêmica/Titulação
2004 - 2008
Doutorado em Ciências Biológicas (Biologia Molecular).
Universidade Federal de São Paulo, UNIFESP, Sao Paulo, Brasil
Título: Estudo da regeneração de lesões no sistema nervosa central por
terapia celular combinada à terapia gênica.
Orientador: Marimélia A Porcionatto
Bolsista do(a): Conselho Nacional de Desenvolvimento Científico e
Tecnológico
2001 - 2003
Mestrado em Ciências Biológicas (Biologia Molecular).
Universidade Federal de São Paulo, UNIFESP, Sao Paulo, Brasil
Título: Possível interação entre proteoglicano de heparam sulfato de
superfície celular e catepsina B, Ano de obtenção: 2003
Orientador: Prof Dr. Ivarne L. S. Tersariol
Bolsista do(a): Fundação de Amparo à Pesquisa do Estado de São Paulo
1997 - 2000
Graduação em Ciencias Biologicas Modalidade Medica.
Universidade Federal de São Paulo, UNIFESP, Sao Paulo, Brasil
Título: Possível interação entre catepsina B e proteoglicano de heparam
sulfato de superfície celular
Orientador: Proa Dra. Marimélia A. Porcionatto
Bolsista do(a): Conselho Nacional de Desenvolvimento Científico e
Tecnológico
________________________________________________________________________
Prêmios e Títulos
2005
Prêmio SBBq Melhor poster na àrea de neuroquímica, SBBq
________________________________________________________________________
Produção bibliográfica
Artigos completos publicados em periódicos
1. COULSON-THOMAS, Y. M., COULSON-THOMAS, V. J., FILIPPO, T. R.,
MORTARA, R. A., SILVEIRA, R. B. da, NADER, H. B., PORCIONATTO, M. A.
Adult bone marrow-derived mononuclear cells expressing chondroitinase AC transplanted
into CNS injury sites promote local brain chondroitin sulphate degradation. Journal of
Neuroscience Methods, v.171, p.19 - 29, 2008.
Comunicações e Resumos Publicados em Anais de Congressos ou Periódicos (resumo)
1. COULSON-THOMAS, Y. M., FILIPPO, T. R., TOBARUELLA, F. S., COULSONTHOMAS, V. J., PORCIONATTO, M. A.
Construção de um vetor contendo o gene da condroitinase AC de Flavobacterium
132
Curriculum vitae resumido
heparinum para expressão em células-tronco de medula óssea In: II Simpósio
Multidisciplinar sobre Células-Tronco, 2007, São Paulo.
Anais do II Simpósio Multidisciplinar sobre Células-Tronco, 2007. v.1. p.45 - 45
2. COULSON-THOMAS, Y. M., FILIPPO, T. R., MOREIRA, C. M., TOBARUELLA, F.
S., NAFFAH-MAZZACORATTI, M. G., PORCIONATTO, M. A.
Construction of a chondroitinase AC vector for expression in bone marrow stem cells In:
5th International Conference on Proteoglycans, 2007, ClubMed Rio das Pedras.
Program and abstract book, 2007. v.1. p.63 - 63
3. COULSON-THOMAS, Y. M., AGUIAR, J. A. K., MICHELACCI, Y. M., NAFFAHMAZZACORATTI, M. G., PORCIONATTO, M. A.
Construction of a chondroitinase AC-EGFP vector for expression in bone marrow stem
cells In: II Simpósio do Instituto Internacional de Neurociências de Natal, 2007, Natal.
Anais do II Simpósio do Instituto Internacional de Neurociências de Natal, 2007. v.1.
p.18 - 18
4. FILIPPO, T. R., COULSON-THOMAS, Y. M., JULIANO, M. A., JULIANO, L.,
PORCIONATTO, M. A.
Migration of cerebellar neuronal precursors is stimultated by a synthetic peptide analogous
to SDF-1/CXCL12 N-terminal In: Progress in Motor Control VI, 2007, Santos.
The international journal for the multidisciplinary study of voluntary movement,
2007. v.11. p.S30 - S30
5. COULSON-THOMAS, Y. M., AGUIAR, J. A. K., MICHELACCI, Y. M., NAFFAHMAZZACORATTI, M. G., PORCIONATTO, M. A.
Construção de um vetor contendo o gene da condroitinase AC de Flavobacterium
heparinum para expressão em células-tronco de medula óssea In: I Simpósio
Multidisciplinar sobre células-tronco, 2006, São Paulo.
Anais do I Simpósio Multidisciplinar sobre células-tronco, 2006. v.1.
6. COULSON-THOMAS, Y. M., AGUIAR, J. A. K., MICHELACCI, Y. M.,
PORCIONATTO, M. A.
Construction of a vector containing Flavobacterium heparinum chondroitinase AC gene for
expression in eukaryotes. In: IV International Symposium on Extracellular Matrix/ IX
Simpósio Brasileiro de Matriz Extracelular, 2006, Búzios.
Livro resumo SBBC SIMEC 2006, 2006. v.1. p.118 - 118
7. COULSON-THOMAS, Y. M., MORAES, J. R., PORCIONATTO, M. A.
Molecular response to neuronal injury: identification of molecules involved in axonal
regeneration in gastropods In: XXXIV Reunião anual Sociedade Brasileira de Bioquímica e
Biologia Molecular, 2005, Águas de Lindóia.
Programa e índices, 2005. v.1. p.81 8. COULSON-THOMAS, Y. M., LOBÃO, V. L., PORCIONATTO, M. A.
Glycosaminoglycans from snail cerebral ganglia. In: XXXIII Sociedade Brasileira de
Bioquímica e Biologia Molecular, 2004, Caxambu.
133
Curriculum vitae resumido
Livro de resumos, 2004. v.1. p.69 - 69
9. COULSON-THOMAS, Y. M., LOBÃO, V. L., SMAILI, S. S., PORCIONATTO, M. A.
Proteoglycans and proteins involved in the response to injuries caused to neurons:
Development of a study model using gastropod cerebral ganglia In: III Simpósio
Internacional sobre Matriz Extracelular, 2004, Angra dos Reis.
Program and abstract book, 2004. p.107 10. COULSON-THOMAS, Y. M., NASCIMENTO, F. D., CHAGAS, J. R., ALMEIDA, P.
C., TRINDADE, E. S., NADER, H. B., PORCIONATTO, M. A., TERSARIOL, I. L. S.
Possible role of cell surface heparan sulphate proteoglycans in cell trafficking of cathepsin
B In: XXXII Sociedade Brasileira de Bioquímica e Biologia Molecular, 2003, Caxambu.
XXXII Reunião Anual Programa e Resumos, 2003.
11. COULSON-THOMAS, Y. M., STAQUICINI, Fernanda I, NASCIMENTO, F. D.,
CHAGAS, J. R., ALMEIDA, P. C., LOPES, José D, NADER, H. B., TERSARIOL, I. L.
S., PORCIONATTO, M. A.
Cell surface heparan sulphate proteoglycans and cathepsin B: possible role of heparan
sulphate proteoglycans in cell trafficking of cathepsin B. In: II Simpósio Internacional
sobre Matriz Extracelular, 2002, Angra dos Reis.
Livro de resumos, 2002.
12. COULSON-THOMAS, Y. M., NASCIMENTO, F. D., CHAGAS, J. R., ALMEIDA, P.
C., TRINDADE, E. S., NADER, H. B., PORCIONATTO, M. A., TERSARIOL, I. L. S.
Possible interaction between cell surface heparan sulfate proteoglycan and cathepsin B In:
XXXI Sociedade Brasileira de Bioquímica e Biologia Molecular, 2002, Caxambu.
Programa e Resumos da XXXI Reunião Anual, 2002.
13. COULSON-THOMAS, Y. M., NASCIMENTO, F. D., CHAGAS, J. R., ALMEIDA, P.
C., TRINDADE, E. S., NADER, H. B., PORCIONATTO, M. A., TERSARIOL, I. L. S.
Possible interaction between cell surface heparan sulphate proteoglycan and cathepsin B In:
XXX Sociedade Brasileira de Bioquímica e Biologia Molecular, 2001, Caxambu.
Programa e resumos da XXX reunião anual, 2001.
14. COULSON-THOMAS, Y. M., NASCIMENTO, F. D., NADER, H. B., TERSARIOL,
I. L. S., PORCIONATTO, M. A.
Estudo do possível papel do proteoglicano de heparam sulfato nos processos de secreção e
internalização de catepsina B. In: VIII Congresso de iniciação científica, 2000, São Paulo.
Anais Pibic, 2000. p.32 15. COULSON-THOMAS, Y. M., STAQUICINI, Fernanda I, NASCIMENTO, F. D.,
CHAGAS, J. R., ALMEIDA, P. C., LOPES, José D, NADER, H. B., TERSARIOL, I. L.
S., PORCIONATTO, M. A.
Possible interaction between heparan sulfate proteoglycan and cathepsin B In: I Simpósio
Internacional sobre Matriz Extracelular, 2000, Angra dos Reis.
Program and Abstract Book, 2000.
134
Curriculum vitae resumido
Supervisões concluídas
Iniciação científica
1. Flávia S Tobaruella. Inibição da expressão de versicam em lesões do SNC. 2007.
Iniciação científica (Ciencias Biologicas Modalidade Medica) - Universidade Federal de
São Paulo
Supervisões em andamento
Iniciação científica
1. Caroline Mônaco Moreira. Construção de retrovírus utilizando sistema RevTet On
para a expressão controlada da enzima condroitinase AC em lesões no sistema
nervoso central. 2007. Iniciação científica (Ciencias Biologicas Modalidade Medica) Universidade Federal de São Paulo
Eventos
Participação em eventos
1. Apresentação de Poster / Painel no(a) II Simpósio Multidisciplinar sobre CélulasTronco, 2007. (Simpósio)
Construção de um vetor contendo o gene da condroitinase AC de Flavobacterium
heparinum para expressão em células-tronco de medula óssea.
2. Apresentação de Poster / Painel no(a) 5th International Conference on Proteoglycans,
2007. (Congresso)
Construction of a chondroitinase AC vector for expression in bone marrow stem cells.
3. Apresentação de Poster / Painel no(a) II Simpósio do Instituto Internacional de
Neurociências de Natal, 2007. (Simpósio)
Construction of a chondroitinase AC-EGFP vector for expression in bone marrow stem
cells.
4. Apresentação de Poster / Painel no(a) Progress in Motor Control VI, 2007.
(Congresso)
Migration of cerebellar neuronal precursors is stimulated by a synthetic peptide analogous
to SDF-1/CXCL12 N-terminal.
5. Curso Modelos pré-clínicos de terapia celular e reparo tecidual em neurociências,
135
Curriculum vitae resumido
2007. (Outra)
6. Apresentação de Poster / Painel no(a) I Simpósio Multidisciplinar sobre célulastronco, 2006. (Simpósio)
Construção de um vetor contendo o gene da condroitinase AC de Flavobacterium
heparinum para expressão em células-tronco de medula óssea.
7. Apresentação de Poster / Painel no(a) SIMEC, 2006. (Simpósio)
IV Simpósio Internacional sobre matriz extracelular.
8. Laboratório de células-tronco, sinalização e possiblidades terapêuticas, 2006.
(Outra)
9. Apresentação de Poster / Painel no(a) XXXIV Reunião anual Sociedade Brasileira de
Bioquímica e Biologia Molecular, 2005. (Congresso)
XXXIV Reunião anual Sociedade Brasileira de Bioquímica e Biologia Molecular.
10. Apresentação de Poster / Painel no(a) International Symposium on Extracellular
Matrix, 2004. (Outra)
Curso: Extracellular Matrix and Bone Regeneration.
11. Apresentação de Poster / Painel no(a) III Simpósio Internacional sobre Matriz
Extracelular, 2004. (Simpósio)
III Simpósio Internacional sobre Matriz Extracelular.
12. Apresentação de Poster / Painel no(a) XXXIII Reunião anual Sociedade Brasileira
de Bioquímica e Biologia Molecular, 2004. (Congresso)
XXXIII Reunião anula Sociedade Brasileira de Bioquímica e Biologia Molecular.
13. Curso Bases celulares e moleculares da migração celular, 2004. (Oficina)
14. Apresentação de Poster / Painel no(a) XXXII Reunião anual Sociedade Brasileira de
Bioquímica e Biologia Molecular, 2003. (Congresso)
XXXII Reunião anual Sociedade Brasileira de Bioquímica e Biologia Molecular.
15. Apresentação de Poster / Painel no(a) II Simpósio Internacional sobre Matriz
Extracelular, 2002. (Simpósio)
II Simpósio Internacional sobre Matriz Extracelular.
16. Apresentação (Outras Formas) no(a) Simpósio Internacional Novas Abordagens
Clínicas e Moleculares voltadas para o Câncer, 2002. (Simpósio)
Novas abordagens clínicas e moleculares voltadas para o câncer.
17. Apresentação (Outras Formas) no(a) Simpósio Internacional Melatonina: ritmos
circadianos e reprodução., 2002. (Simpósio)
Simpósio Internacional Melatonina: ritmos circadianos e reprodução.
136
Curriculum vitae resumido
18. Apresentação de Poster / Painel no(a) XXXI Reunião anual Sociedade Brasileira de
Bioquímica e Biologia Molecular, 2002. (Congresso)
XXXI Reunião anual Sociedade Brasileira Bioquímica e Biologia Molecular.
19. Apresentação (Outras Formas) no(a) IV Workshop Temático do CAT/CEPID, 2001.
(Oficina)
Coagulação e Fibrinólise: respostas celulares a agentes exógenos.
20. Apresentação de Poster / Painel no(a) XXX Reunião anual Sociedade Brasileira de
Bioquímica e Biologia Molecular, 2001. (Congresso)
XXX Reunião anual Sociedade Brasileira de Bioquímica e Biologia Molecular.
21. Apresentação de Poster / Painel no(a) I Simpósio Internacional sobre Matriz
Extracelular, 2000. (Simpósio)
I Simpósio Internacional sobre Matriz Extracelular.
22. Apresentação de Poster / Painel no(a) VIII Congresso de Iniciação Científica, 2000.
(Congresso)
VIII Congresso de Iniciação Científica.
137
Anexo 1
Journal of Neuroscience Methods 171 (2008) 19–29
Adult bone marrow-derived mononuclear cells expressing chondroitinase
AC transplanted into CNS injury sites promote local brain
chondroitin sulphate degradation
Yvette M. Coulson-Thomas a , Vivien J. Coulson-Thomas a , Thais R. Filippo a , Renato A. Mortara b ,
Rafael B. da Silveira a,1 , Helena B. Nader a , Marimélia A. Porcionatto a,∗
b
a Department of Biochemistry, UNIFESP, São Paulo 04044-020, Brazil
Department of Microbiology, Immunology and Parasitology, UNIFESP, São Paulo 04023-901, Brazil
Received 14 December 2007; received in revised form 29 January 2008; accepted 30 January 2008
Abstract
Injury to the CNS of vertebrates leads to the formation of a glial scar and production of inhibitory molecules, including chondroitin sulphate
proteoglycans. Various studies suggest that the sugar component of the proteoglycan is responsible for the inhibitory role of these compounds in
axonal regeneration. By degrading chondroitin sulphate chains with specific enzymes, denominated chondroitinases, the inhibitory capacity of
these proteoglycans is decreased. Chondroitinase administration involves frequent injections of the enzyme at the lesion site which constitutes a
rather invasive method. We have produced a vector containing the gene for Flavobacterium heparinum chondroitinase AC for expression in adult
bone marrow-derived cells which were then transplanted into an injury site in the CNS. The expression and secretion of active chondroitinase
AC was observed in vitro using transfected Chinese hamster ovarian and gliosarcoma cells and in vivo by immunohistochemistry analysis which
showed degraded chondroitin sulphate coinciding with the location of transfected bone marrow-derived cells. Immunolabelling of the axonal
growth-associated protein GAP-43 was observed in vivo and coincided with the location of degraded chondroitin sulphate. We propose that bone
marrow-derived mononuclear cells, transfected with our construct and transplanted into CNS, could be a potential tool for studying an alternative
chondroitinase AC delivery method.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Chondroitinase AC; Chondroitin sulphate; Bone marrow-derived cells; Injury; Nervous system
1. Introduction
Spontaneous regeneration does not occur in the CNS of
vertebrates. Injury to the CNS is followed by the influx of
glial cells to the injury site (Fawcett and Asher, 1999; Silver
and Miller, 2004), which then attracts oligodendrocyte precursors, astrocytes, meningeal cells and microglia, constituting
the glial scar. These cells produce high levels of axon growth
inhibitory molecules, including chondroitin sulphate proteoglycans (CSPGs) (Fawcett and Asher, 1999; Asher et al., 2001;
Garwood et al., 2001; Moreau-Fauvarque et al., 2003; Silver
and Miller, 2004).
∗
Corresponding author. Tel.: +55 11 5576 4442; fax: +55 11 5573 6407.
E-mail address: [email protected] (M.A. Porcionatto).
1 Present address: Department of Structural Biology, Molecular Biology and
Genetics, UEPG, Paraná 84030-900, Brazil.
The CSPGs neurocan, versican, brevican, NG2 and phosphacan are up-regulated after injury to the vertebrate CNS (McKeon
et al., 1995; Stichel et al., 1995; Fitch and Silver, 1997; Lemons
et al., 1999; Asher et al., 2000; Jones et al., 2002; Tang et al.,
2003). These proteoglycans have been shown to inhibit neurite
outgrowth by binding to the growth-promoting molecule laminin
preventing it from interacting with its receptor integrin located
in growth cones (Burg et al., 1996; Sanes and Jessell, 2000;
Morgenstern et al., 2002).
Various studies suggest that the glycosaminoglycan chains
attached to the CSPGs are responsible for inhibiting axonal
regeneration (McKeon et al., 1995; Smith-Thomas et al., 1995;
Powell and Geller, 1999; Grimpe and Silver, 2004). The enzymatic removal of these CS chains using specific enzymes
denominated chondroitinases reduces the inhibitory effect of
CSPGs, promoting axonal regeneration and functional recovery (Lemons et al., 1999; Krekoski et al., 2001; Moon et al.,
2001; Bradbury et al., 2002; Jones et al., 2002; Yick et al., 2003;
0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jneumeth.2008.01.030
138
20
Y.M. Coulson-Thomas et al. / Journal of Neuroscience Methods 171 (2008) 19–29
Caggiano et al., 2005; Fouad et al., 2005; Groves et al., 2005;
Steinmetz et al., 2005; Houle et al., 2006).
Chondroitinases are of bacterial origin and are identified by their substrates. Chondroitinase AC (EC 4.2.2.5)
cleaves chondroitin-4-sulphate (C4S) and chondroitin-6sulphate (C6S), whereas chondroitinase ABC (EC 4.2.2.4) also
cleaves dermatan sulphate (Yamagata et al., 1968; Michelacci
and Dietrich, 1975; Gu et al., 1995). Chondroitinase AC is
expressed by Flavobacterium heparinum, a Gram-negative,
non-pathogenic soil bacterium (Payza and Korn, 1956).
Regarding cell therapy, the adult CNS is capable of incorporating stem cells or progenitor cells transplanted into an injury
site or close to it (Gage et al., 1995; Lundberg et al., 1997). Transplantation of neural stem/progenitor cells into injured spinal
cord contributes to the repair of injured spinal cord in adult
rats (Ogawa et al., 2002; Okano et al., 2003; Watanabe et al.,
2004) and non-human primates (Iwanami et al., 2005). Ikegami
et al. (2005) suspected that CSPG deposition in glial scars after
spinal cord injury could diminish the beneficial effects of neural
stem/progenitor cell transplantation. The authors observed that
chondroitinase ABC pre-treatment promoted the migration of
transplanted neural stem/progenitor cells and the outgrowth of
GAP-43-positive fibres. Unfortunately, chondroitinase delivery
is a prolonged invasive method, and several protocols involve
repeated injections of the enzyme into the lesion site.
A prolonged non-invasive delivery method could be achieved
by a single injection into a lesion site of adult bone marrowderived cells transfected with the gene encoding chondroitinase
AC, so that this enzyme would be continuously expressed and
secreted. Our goal was to construct a vector containing the
gene encoding the bacterial chondroitinase AC and analyse the
expression and secretion of the active enzyme by mammalian
cells in vitro and in vivo.
2. Materials and methods
All surgical interventions and animal care procedures were
approved by the University’s Ethics Research Committee (protocol number 0735/05).
2.1. Cloning
Flavobacterium heparinum was suspended in sterile water,
vortexed, centrifuged and the supernatant that contained
DNA was used as the template. The gene for chondroitinase AC was PCR amplified (5 -GGGGTACCATGGGGAAATTATTTGTAACCT-3 ; 3 -CGTTGCCAACTTGACTTTATCCTTAAGG-5 ). The forward primer was constructed so as to
incorporate a KpnI restriction site and the reverse primer an
EcoRI restriction site. A Kozak (1987, 1990, 1991) sequence
was also included in the forward primer as recommended by
the pcDNA manufacturers (Invitrogen). The gene product was
cloned into the KpnI–EcoRI sites of pcDNA3.1(+).
2.2. Cell culture
Wild-type Chinese hamster ovarian (CHO-K1) cells were
maintained in F12 medium (GibcoBRL)/10% foetal bovine
serum (FBS, Cultilab), 10 units/ml penicillin and 10 ␮g/ml
streptomycin, at 37 ◦ C in 2.5% CO2 . Rat gliosarcoma 9L cells
(ATCC) were maintained in DMEM/10% FBS, 30 units/ml penicillin and 30 ␮g/ml streptomycin, at 37 ◦ C in 5% CO2 .
Bone marrow-derived mononuclear cells were obtained
from 3-month-old female C57BL/6-Tg(act-EGFP)C14-Y01FM131Osb (“green mice”) or wild-type C57BL/6 mice.
The C57BL/6 mice were supplied by INFAR/UNIFESP
Animal Facility and the green mice were maintained at
CEDEME/UNIFESP. Animal care procedures were in accordance with Guidelines for the Care and Use of Mammals in
Neuroscience and Behavioural Research (2003). Bone marrow was collected from the long bones (two femurs and
two tibias) of each mouse in DMEM and mononuclear cells
were isolated using Ficoll-Paque (1.077 g/ml density, Amersham Biosciences). Cells were maintained in DMEM/20% FBS,
30 units/ml penicillin and 30 ␮g/ml streptomycin, at 37 ◦ C in 5%
CO2 .
2.3. Cell transfection
Twenty-four hours before transfection, 2.5 × 105 CHO-K1
and 5 × 105 9L cells were seeded in 35 mm2 dishes. Cells
(90% confluent) were transfected with 1.5 ␮g of DNA/4 ␮l
Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s protocol.
For the transfection of bone marrow-derived cells with
pcDNA3.1(+) or the construct pcDNA3.1(+)-chondroitinase
AC, 2 × 106 mononuclear cells were isolated from 3-month-old
female green mice as described above. As soon as the cells were
obtained, they were transfected in suspension with 1.5 ␮g of
DNA/4 ␮l Lipofectamine 2000 Reagent according to the manufacturer’s protocol. After a 5-h transfection period, the cells were
centrifuged, suspended in DMEM and injected into a lesion site
in the primary motor cortex of C57BL/6 mice, using a Hamilton
syringe.
The transfection protocols were tested and optimized by
tranfecting CHO-K1, 9L and wild-type C57BL/6 bone marrowderived cells with pEGFP-N1 (CLONTECH) followed by in vivo
fluorescent microscopy using a Zeiss LSM510 inverted scanning
confocal microscope.
2.4. RNA extraction
Total RNA was isolated from cells 24 h post-transfection
using Trizol Reagent (Invitrogen). First strand cDNA was
reverse transcribed using 1 ␮g of total RNA. PCR amplification was performed on 1 ␮l of RT product with chondroitinase
AC and ␤-actin specific primers. The primer combinations used
for chondroitinase AC were: forward: 5 -AGCAATGCCCCTGAAAAC-3 ; reverse: 3 -CAAGACCAACAACGTGCTAC5 (162 bp product) and forward: 5 –GGGGTACCATGGGGAAATTATTTGTAACCT-3 ; reverse: 3 -CGTTGCCAACTTGACTTTATCCTTAAGG-5 (2103 bp product). The primer combination used for rat ␤-actin was: forward: 5 -AAGCAGGAGTATGACGAGTCCG-3 ; reverse: 3 -GGTTGAACTCTACATACTTCCG-5 (590 bp product) and for mouse ␤-actin
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was: forward: 5 -ACTCTTCCAGCCTTCCTTC-3 ; reverse: 3 CTGTGCTACGTCTTCCTCAT-5 (200 bp product).
2.5. Metabolic labelling of chondroitin sulphate with
[35 S]-sulphate
Glycosaminoglycans and proteoglycans were labelled by
adding 5.55MBq [35 S]-sulphate (CNEN, Brazilian Nuclear
Energy National Council) to the culture medium supplemented
with dialyzed FBS of transfected cells and by incubating for 6 h
at 37 ◦ C, followed by extraction and analysis by agarose gel electrophoresis as previously described (Dietrich and Dietrich, 1976;
Porcionatto et al., 1998). Radioactive glycosaminoglycans were
visualized and quantified after being exposed to a radioactivity sensitive screen using the Cyclone Storage Phosphor System
(Packard Instrument Company).
2.6. Lesion to murine CNS and cell transplantation
Adult female C57BL/6 mice were used for all groups, and
each group consisted of two animals. All surgeries were performed under anaesthesia with intraperitoneal administration of
ketamine chloridrate (Dopalen, Vetbrands) and the protocol used
was based on the one described by Chiba et al. (2004). Briefly, a
metal needle was chilled using isopentane on dry ice and inserted
four times into the motor cortex (M1) (stereotaxic coordinates:
anteroposterior: +0.198 mm; lateral: +0.175 mm; dorsoventral:
−0.15 mm; Paxinos and Franklin, 2001). Two million nontransfected, pcDNA3.1(+) or pcDNA3.1(+)-chondroitinase AC
transfected bone marrow-derived cells were injected into the
lesion using a Hamilton syringe, immediately after the injury
was caused, using the same stereotaxic coordinates under the
same anaesthesia. Two weeks later, the animals were anaesthetized by an intraperitoneal injection of sodium thiopental
(Tiopentax, Cristália) and then intracardially perfused with 4%
paraformaldehyde in 0.1 M phosphate-buffered saline (PBS).
The brain was removed and immersed in 4% paraformaldehyde
for 24 h at 4 ◦ C, placed in 30% sucrose in PBS for 24 h at 4 ◦ C,
embedded in Tissue Freezing Medium (Electron Microscopy
Sciences) and frozen using isopentane on dry ice. The embedded tissue was sliced into 20 ␮m sagittal sections using a cryostat
(Leica). The tissue slices were collected on silanized slides for
immunofluorescence staining.
Chronic injuries were obtained using the same protocol
described above with the difference that animals were maintained for 4 weeks before being transplanted with 2 × 106
non-transfected, pcDNA3.1(+) or pcDNA3.1(+)-chondroitinase
AC transfected bone marrow-derived cells into each lesion,
using a Hamilton syringe. The same stereotaxic coordinates
used to cause the injuries were used to transplant the cells.
One and two weeks later, the animals were anaesthetized by an
intraperitoneal injection of sodium thiopental and then intracardially perfused with 4% paraformaldehyde in 0.1 M PBS. The
brain was removed and immersed in 4% paraformaldehyde for
24 h at 4 ◦ C, placed in 30% sucrose in PBS for 24 h at 4 ◦ C,
embedded in Tissue Freezing Medium and frozen using isopen-
21
tane on dry ice. The embedded tissue was sliced into 10 ␮m
sagittal sections which were collected on silanized slides for
immunofluorescence staining.
2.7. Immunofluorescence
For immunofluorescence staining, the sections were incubated overnight at 4 ◦ C with anti-GFP (1:500, Molecular
Probes/Invitrogen) or anti-GAP-43 (1:500, Chemicon International). After washing with PBS, the sections were incubated
at room temperature for 4 h with anti-chondroitin-6-sulphate
MAB2035 (1:100, Chemicon International), anti-GFAP (1:500,
Chemicon International) or anti-␤III tubulin (1:500, Chemicon
International). Anti-chondroitin-6-sulphate labels the stubs that
remain after CS digestion by chondroitinase and not the intact
molecule. The slices were incubated with appropriate secondary
antibodies (Alexa FluorTM 488-conjugated goat anti-mouse
1:100, Molecular Probes, Alexa FluorTM 594-conjugated goat
anti-rabbit 1:100, Molecular Probes, and Fluorescein conjugated
rabbit anti-chicken 1:100, Chemicon International). Nuclei were
stained by DAPI (1:500, Molecular Probes). After immunoand DAPI staining, glass slides were mounted using Fluoromount G (2:1 in PBS, Electron Microscopy Sciences). The
fluorescently labelled tissue slices were then analysed using
either a Zeiss LSM510 scanning confocal inverted microscope
(GFP, GFAP and -Di6S) or a BioRad 1024-UV confocal
system attached to a Zeiss Axiovert 100 microscope, using
a 40× N.A. 1.2 Plan-Apochromatic DIC water immersion
objective and images were collected by Kalman averaging
at least 15 frames (512 × 512 pixels), using an aperture (pinhole) of 2.0 mm maximum (for ␤III-tubulin and GAP-43).
Co-localization images were generated using ImageJ software
(http://rsb.info.nih.gov/ij/).
3. Results
3.1. Cloning of chondroitinase AC from Flavobacterium
heparinum
We cloned the gene encoding chondroitinase AC from F. heparinum since there are no mammalian homologs. The primers
used for PCR amplification of the gene were designed based on
the sequence obtained by Tkalec et al. (2000). The mature chondroitinase AC protein lacks amino acids 1–22 which implies that
this sequence is a signal peptide (Gu et al., 1995). We used the
PSORT II program (www.psort.org) to analyse possible sorting of the enzyme if the 22 amino acid signal peptide was to
be maintained when cloning chondroitinase AC and expressing
the enzyme in eukaryotic cells. Results of the k-NN prediction
were: 66.7% extracellular, 11.1% vacuolar, 11.1% endoplasmic
reticulum and 11.1% mitochondrial.
Also, the program SignalP 3.0 Server (www.cbs.dtu.dk/
services/SignalP) was used to check the probability of this signal peptide being recognized in eukaryotic cells. The prediction
results were a 0.999 probability of it being identified as a signal peptide and the cleavage site probability was 0.891 between
positions 22 and 23. Therefore, the first 66 nucleotides were
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Fig. 1. The gene encoding chondroitinase AC was PCR amplified from F. heparinum genomic DNA (A, arrow) and cloned into pcDNA3.1(+). The construct
was transformed into E. coli DH5␣ cells and colonies were screened by PCR
amplification (B). The arrow in B indicates the colony that was selected for
restriction digestion and sequencing to confirm incorporation of the gene.
maintained when the gene for chondroitinase AC from F. heparinum was cloned.
The Kozak sequence contains a start codon followed by a
guanine nucleotide, consequently, the cloned chondroitinase AC
gene codes for glycine as the second amino acid instead of the
usual lysine. The F. heparinum gene encoding for chondroitinase AC was amplified by PCR and the product was analysed
by agarose gel electrophoresis (Fig. 1A). The chondroitinase
AC gene has an open reading frame of 2,103 bp (Tkalec et al.,
2000) and a single band was observed between the standard
markers 2322 and 2027 bp. The PCR product was cloned into
the KpnI–EcoRI sites of the expression vector pcDNA3.1(+)
and transformed into chemically competent E. coli DH5␣ cells.
Colonies were screened by PCR amplification using the same
primers described in Section 2 (Fig. 1B). The positive colony
indicated by the arrow in Fig. 1B was selected and incorporation of the chondroitinase AC gene was confirmed by restriction
digestion and sequencing.
3.2. Recombinant expression of chondroitinase AC
To determine whether recombinant chondroitinase AC would
be expressed in mammalian cells, rat gliosarcoma 9L cells were
transfected with the construct pcDNA-chondroitinase AC. Total
RNA was extracted and PCR amplification was performed on the
RT product with chondroitinase AC and ␤-actin specific primers.
The products were analysed by agarose gel electrophoresis
(Fig. 2). The expected chondroitinase AC product band was
observed only for cells transfected with the construct pcDNAchondroitinase AC (9L/pcDNA-cAC). Samples obtained from
cells transfected with empty plasmid (9L/pcDNA) or cells that
were not transfected (9L) showed no PCR amplification for
chondroitinase AC.
3.3. Secretion of active chondroitinase AC
Since the expression of chondroitinase AC mRNA in mammalian cells was observed, the secretion of active enzyme was
investigated using 9L cells and also CHO cells, which are routinely used to study proteoglycan expression.
Fig. 2. PCR amplification of chondroitinase AC from non-transfected (9L),
pcDNA3.1(+) (9L/pcDNA) or pcDNA3.1(+)-chondroitinase AC (9L/pcDNAcAC) transfected 9L rat gliosarcoma cells total RNA.
The amount of chondroitin sulphate present in the conditioned medium of cells transfected with the construct
pcDNA-chondroitinase AC and of cells transfected with empty
plasmid were compared (Fig. 3). Fig. 3A shows the agarose
gel electrophoresis of labelled 9L glycosaminoglycans. Quantification of CS shows that cells transfected with the construct
pcDNA-chondroitinase AC have an average of 40% less CS
in the conditioned medium compared to cells transfected with
empty plasmid (Fig. 3B). The experiment was carried out twice,
each time in duplicate, and Student’s t test shows that this
decrease is significant (p = 0.012).
CHO cells transfected with the construct pcDNAchondroitinase AC had an average of 22% less CS in the
conditioned medium compared to cells transfected with the
empty plasmid (Fig. 3C). The experiment was carried out four
times and each time in duplicate. Student’s t test shows this
decrease is significant (p = 0.008).
3.4. Expression of chondroitinase AC by bone
marrow-derived mononuclear cells
Considering CHO-K1 and gliosarcoma 9L cells expressed
and secreted active enzyme, we ventured to transfect adult
bone marrow-derived cells with the construct. Mononuclear
cells were transfected with the construct pcDNA-chondroitinase
AC or empty plasmid. Total RNA was isolated from cells 24 h
post-transfection and first strand cDNA was reverse transcribed.
A PCR amplification product corresponding to chondroitinase
AC was observed only in cells transfected with pcDNAchondroitinase AC (BM/pcDNA-cAC, Fig. 4).
3.5. Secretion of active chondroitinase AC at a lesion site
of murine CNS
For in vivo assays, mononuclear cells were obtained from
green mice since all cells from these animals express GFP and
can therefore be located in tissue slices after being injected into
a lesion site in murine CNS. We aimed to investigate whether
mononuclear cells transfected with the construct pcDNA3.1(+)chondroitinase AC secrete active chondroitinase AC in both
acute and chronic injury models. In the acute injury protocol,
non-transfected, pcDNA3.1(+) or pcDNA3.1(+)-chondroitinase
AC-transfected bone marrow-derived cells were injected into
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23
the lesions as soon as they were caused in adult female
C57BL/6 mice and the animals were subjected to euthanasia two
weeks later. In the chronic injury model, bone marrow-derived
cells transfected with empty plasmid or with the construct
pcDNA3.1(+)-chondroitinase AC were transplanted into the
lesions using the same stereotaxic coordinates a month after the
injuries were caused and the animals were subjected to euthanasia either one or two weeks later. Brain slices were analysed for
GFP labelling, indicating the location of the injected mononuclear cells, and for degraded chondroitin-6-sulphate labelling,
indicating chondroitinase AC activity. GFP(+) cells injected into
the acute injury and analysed two weeks later can be observed
at the lesion area along with degraded chondroitin sulphate
(Fig. 5). When bone marrow-derived cells transfected with the
construct pcDNA3.1(+)-chondroitinase AC were injected into a
chronic injury and given a week to migrate, GFP(+) cells are
observed both in the lesion area and migrating inwards, and
degraded chondroitin sulphate can be observed in the lesion area
(Fig. 6A). However, cells that were injected into a chronic injury
and analysed two weeks later not only migrated inwards, but
also tended to migrate towards the left lateral ventricle and left
a trail of degraded chondroitin sulphate (Fig. 6B). The extent
of cell migration did not vary between bone marrow-derived
cells transfected with empty plasmid and transfected with the
construct pcDNA3.1(+)-chondroitinase AC (data not shown).
3.6. Distribution of reactive glia
Fig. 3. Glycosaminoglycan expression by 9L cells, transfected with empty
plasmid (9L/pcDNA) or the construct pcDNA3.1(+)-chondroitinase AC
(9L/pcDNA-cAC) were metabolically labelled with [35 S]-sulphate (5.55MBq)
and analysed by agarose gel electrophoresis (A). Chondroitin sulphate secreted
to the conditioned medium by 9L (B, *p = 0.012) and CHO cells (C, *p = 0.008)
was quantified. 9L: rat gliosarcoma cells; CHO: Chinese hamster ovarian cells;
CS: chondroitin sulphate; HS: heparan sulphate.
Once we observed that bone marrow-derived cells transfected
with our construct expressed active chondroitinase AC and that
the cells were capable of migrating from the injury site, we
ventured to investigate other effects the cells could have on the
CNS in the lesion area. We analysed the distribution of reactive
glia around the lesion sites using GFAP as a marker. In Fig. 7, we
can observe more intense GFAP labelling around lesions with
chondroitinase AC-expressing cells when compared to lesions
with cells transfected with the empty plasmid.
3.7. Growth-associated protein-43 immunolabelling
Brain slices obtained from animals with chronic injury
and sacrificed a week after the injection of transfected bone
marrow-derived cells were analysed for GAP-43 labelling
since this protein is a specific marker for axonal regeneration.
Limited GAP-43 labelling was observed around lesions with
bone marrow-derived cells transfected with the empty plasmid
(Fig. 8A). On the other hand, considerable GAP-43 labelling was
observed at lesion sites with cells expressing chondroitinase AC,
and coincided with degraded chondroitin sulphate (Fig. 8C) as
well as co-localized with ␤III tubulin (Fig. 8D, arrowheads).
4. Discussion
Fig. 4. PCR amplification of chondroitinase AC from non-transfected (BM),
pcDNA3.1(+) (BM/pcDNA) or pcDNA3.1(+)-chondroitinase AC (BM/pcDNAcAC) transfected bone marrow-derived mononuclear cells total RNA. BM: bone
marrow-derived mononuclear cells.
Chondroitin sulphate proteoglycans are upregulated after
injury to the vertebrate CNS (McKeon et al., 1995; Stichel et
al., 1995; Fitch and Silver, 1997; Lemons et al., 1999; Asher
et al., 2000; Jones et al., 2002; Tang et al., 2003) and have
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