Short Communication

Dopaminergic Trophism after Intrastriatal
Injection of Lentivirus-transferred GDNF in Parkinson Rat Model

SUN Bing^1,2, HUI Guo-Zhen², GUO Li-He¹*, REISER Jakob³

( 1Institutes of Biochemistry and Cell
Biology, the Chinese Academy of Sciences, Shanghai 200031, China; 2Department
of Neurosurgery, First Affiliated Hospital of Suzhou University, Suzhou 215006,
China; 3LSU Health Sciences Center, Louisana 70112, USA )

Abstract        To investigate the effects of lentivirus-mediated transfection of
GDNF on Parkinson’s disease (PD). The pNL-gdnf plasmid was constructed by
replacing the LacZ-coding region present in pNL-lacZ/CMV. Vector particles
involved a three-plasmid lentivirus expression system were co-transferred into
293T cells through calcium phosphate method. High-titer virus was collected
from infected 293T cells and injected into lesion-side striatum of PD rats, and
their apomorphine-induced rotations were assayed at day 14, 30 and 60,
respectively. GDNF protein was detected by Western blot analysis, and the
expression of lacZ and TH were detected by immunochemistry. Results showed that
behavioral recovery gradually appeared after transplantation, and a significant
reduction in the rotational response was observed at the 14th day. Meanwhile,
gdnf expression maintained for at least 60 d, which had dopaminergic trophism
to a certain degree, indicating that lentivirus-mediated transfection gdnf
could effectively improve the clinical function of PD rats, which provide a
potential attractive tool of gene therapy for PD.

Key
words     glial
cell line-derived neurotrophic factor; lentivirus vector; Parkinson’s disease;
gene therapy

Lentivirus was based
on oncoretrovirus but different fundamentally in that its replicative cycle
occurs independently of cell division[1]. Thus, lentiviral vector is powerful
tool for gene delivery in nondividing cells in vitro and in vivo[2,3]. Robust
transfer of marker and therapeutic gene has recently identified in the central
nervous system using a lentiviral vector, meanwhile, the transgene expression
is long-term and nontoxic[4,5]. Among candidate genes to treat Parkinson’s
disease (PD), glial cell line-derived neurotrophic factor (GDNF) is a potent
neurotrophic molecule for nigral dopaminergic neurons both in vitro and in
vivo, which is capable of alleviating on-going degeneration of the dopaminergic
neurons of the substantia nigra (SN)[6,7].

In the present
study, lentivirus-mediated gdnf was injected into the striatum of PD rat, and
the effects of expressed GDNF was further investigated on PD rat in vivo.

1    Materials and Methods

1.1   Plasmid constructs

The
three-plasmid lentivirus system (LSU Health Sciences Center,
Louisiana, USA)
is shown in Fig.1, including pNL-lacZ/CMV plasmid, pLTR-G plasmid encoding
VSV-G glycoprotein protein, pCD/NL-BH helper plasmid which is similar to
pC-Help. The pNL-gdnf plasmid was constructed by replacing the LacZ-coding
region in pNL-lacZ/CMV with gdnf.

Fig.1       Sketch
map of three-plasmids lentivirus system

(A) pNL-lacZ/CMV. (B) pLTR-G.
(C) pCD/NL-BH.

1.2   Production of lentivirus

Vector particles
involved in a three-plasmid expression system were transferred into 293T cells
by transient cotransfection[8]. Briefly, 293T cells were cultured in 6-well
plate (6×105
cells/well). 24 h later, 5 μg pNL-gdnf plasmid, the pCD/NL-BH helper plasmid and pLTR-G Env
plasmid were added to the culture per well. Calcium phosphate transfection with
the presence of 25 μmol/L chloroquine was carried out for 12－15 h and then the medium was
replaced. After 60－65 h post-transfection, the virus particles released into the medium
were harvested and concentrated by centrifugation at 25 000 r/min for 2 h at 15
℃. The final
virus titer was estimated to be 5.8×108 U/mL.

1.3   Establishment of rat PD model and
surgical graft

16 male
Sprague-Dawley rats weighing 200－220 g were purchased from Shanghai Laboratory Animal center, the
Chinese Academy of Sciences and equally divided into PD group and control
group. PD model was built up by application of 6-OHDA. 2 weeks after 4 μL 6-OHDA (2.5 g/L) injection into
the substantia nigra (SN), apomorphine (0.5 mg/kg) was injected
intrapertoneally to screen the usable subjects. Only rats exhibiting more than
7 rotations per min were used for experiments. All the rats were placed in the stereotaxic
frame, 2 μL lentivirus
(either lenti-gdnf or lenti-lacZ) suspended in PBS was injected into the
lesion-side striatum. Behavioral changes were assayed before and 14, 30, 60 d
after transplantation respectively.

1.4   Western blot analysis

100 mg brain
tissue near the injection site were obtained and immediately placed in a
homogenizer containing 1 mL RIPA and 10 mL PMSF（100 mg/L）, incubated on ice for 10 min, and thoroughly homogenized. The
homogenate was centrifuged at 10 000 g 
for 30 min at 4 ℃ to remove the cell debris. The supernatant was collected and
quantified by protein assay kit. 30 μL extracted protein sample was separated by 15% SDS-PAGE and then
transferred to nitrocellulose membranes. The membrane was blocked overnight in
triethanolamine buffered saline containing 5% fat-free milk and 0.05% Tween-20
phosphate buffered saline. Western blot assay was performed using 1:1000
dilution of rabbit anti-GDNF antibody, followed by incubation with 1:5000
dilution of sheep anti-rabbit antibody, and detection with enhanced
chemiluminescence (ECL) reagents.

1.5   Immunohistochemistry

Rats were
trans-cardially perfused with 150 mL PBS followed by 500 mL ice-old 4%
paraformaledhyde in 0.1 mol/L phosphate buffer (PB), pH 7.2～7.4. After being dissected, brains
were fixed for 2 h in the same fixative at 4 ℃, then immersed in 25% sucrose in PBS for cryoprotection before
being stored. Coronal sections of brains were cut and collected in PBS for use.
The sections taken from control group were fixed for 10 min and incubated with
X-gal dye[15 mmol/L K2Fe(CN)6, 15 mmol/L K3Fe(CN)6, 1.5 mmol/L MgCl2, 1 g/L
X-gal]overnight at 37 ℃ to detect LacZ. For immunochemistry, endogenous peroxidase activity
was quenched by placing tissues into 3% H2O2 in 0.05 mol/L Tris-HCl buffer (pH
7.6) for 10 min. Then the sections taken from PD group were incubated in PBS
and 0.25% Triton X-100 with 3% normal goat serum for 1 h to block nonspecific
sites. Sections were incubated overnight with rabbit anti-tyrosine hydroxylase
(TH) (1:1000), followed by incubation with 1:200 dilution of sheep anti-rabbit
antibody (1:400). Then the expression of TH was detected by ABC kit.

2    Results

2.1   Behavioral recovery of
6-OHDA-lesioned rats

APO-induced
rotations on rats were assayed 14, 30, 60 d after transplantation,
respectively. A significant reduction in the rotational response was seen in
rats that receiving lenti-gdnf grafts for 14 d. Henceforth, rotations of rats
were gradually improved till 60 d (Fig.2).

Fig.2       Behaviral
recovery after transplantation in PD rats

*means
before transplantation.

2.2   Expression of GDNF protein in brain
tissue of rats

After
transplantation, GDNF proteins were detected in rat brain by Western blot
analysis. It was shown that GDNF protein maintained in brains of grafted rats
till 60 d, compared with control group (Fig.3).

Fig.3       Western
blot analysis of GDNF after grafts

1,
14 d after graft; 2, 30 d after graft; 3, 60 d after graft; 4, control 60 d.

2.3   Immunohistochemical detection of
target gene

X-gal dye showed
the distribution of expressed LacZ. which not only appeared in small vessels
near injection site but also in cortex of brain. TH  immunochemistry suggested that TH positive cells existed in
the vicinity of SN 30 d after transplantation and slightly increased along the
time. In contrast, only unorderly TH positive fiber was seen in control group
as shown in Fig.4.

Fig.4       Immunohistochemical
detection of LacZ and TH

(A) LacZ expression in small vessels.
(B) Cortex in control group. (C) Control of TH fiber 60 d. (D) TH-positive
cells in the vicinity of SN at 30 d after lenti-gdnf graft. (E) TH-positive
cells in the vicinity of SN at 60 d after lenti-gdnf graft.

3    Discussion

Various viral
vectors have been attempted in gene therapy for Parkinson’s disease, such as
retrovirus, adenovirus(AdV) and adeno-associated virus vectors (AAV). Previous
studies showed that ideal vectors should have characters as high efficiency of
gene delivery, integration into the host genome without inserting mutation,
high level of gene expression and long-term transgene expression. However, a
limitation of moloney murine leukemia virus (MoMLV)-based transduction approach
is a general lack of long-term expression, retrovirus is not suitable for PD.
Meanwhile, AdV has fatal side-effects and AAV’s capacity is not ideal. Thus, a
powerful vector tool is needed to facilitated gene therapy for PD. The present
study used the three-plasmids lentivirus system, constructed by Reiser et
al.[9], which includes pNL-lacZ/CMV plasmid, the pLTR-G plasmid encoding the
VSV-G glycoprotein protein, and pCD/NL-BH helper plasmid which is similar to pC-Help
but without the putative packaging signal of nucleotides 747－787 between the 5′ major splice donor site and the
beginning of the Gag coding region of the HIV-1 NL4-3 proviral DNA[10].

The improved
lentivirus vector has been identified to be suitable for long-term transgene
expression in cells of central nervous system. Its replicative cycle occurs
independently of cell division, which also makes it be an efficient gene
therapy tool for PD.

GDNF has potent
trophic effects on dopaminergic nigral neuron, suggesting that it might be
neuroprotective for PD. In this study, we examined whether lentiviral-mediated
transfection of gdnf could improve the clinical function and the extent of
neuroprotection in rat PD model damaged by 6-OHDA. The results showed that
functional recovery increased significant along with the expression of
transferred gdnf, suggesting the trophism of GDNF was positively correlated
with its protein concentration. Moreover, lentivirus-transferred lacZ was
expressed not only in needle site but also in cortex of brain, which maintained
at least 60 d. It was previously reported that virus-mediated GDNF could rescue
the dopaminergic neuron[11]. In our study, the rescue of the striatal
TH-positive innervation was also observed. Though no sprouting of lesioned axon
was detectable, TH-positive cells could be found in the vicinity of SN 30 d
after transplantation and the number slightly increased along the time,
suggesting lenti-gdnf has effective dopaminergic trophism. Therefore,
lentivirus-transferred gdnf is an effective approach for gene therapy of PD.

References

1     Lewis
PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not
for the human immunodeficiency virus. J Virol, 1994, 68(1): 510－516

2     Vigna
E, Naldini L. Lentiviral vectors: Excellent tools for experimental gene
transfer and promising candidates for gene therapy. J Gene Med, 2000, 2(5): 308－316

3     Trono
D. Lentiviral vector: Turning a deadly foe into a therapeutic agent. Gene Ther,
2000, 7(1): 20－23

4     Naldini
L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM et al. In vivo gene
delivery and stable transduction of nondividing cells by a lentiviral vector.
Science, 1996, 272(5259): 263－267

5     Déglon N, Tseng JL, Bensadoun J-C,
Zurn AD, Arsenijevic Y, de Almeida LP, Zufferey R et al. Self-inactivating
lentiviral vectors with enhanced transgene expression as potential gene
transfer system in Parkinson’s disease. Hum Gene Ther, 2000, 11(1): 179－190

6     Hou
JG, Lin LF, Mytilineou C. Glial cell line-derived neurotrophic factor exerts
neurotrophic effects on dopaminergic neurons in vitro and promotes their
survival and regrowth after damage by 1-methyl-4-phenylpyridinium. J Neurochem,
1996, 66(1): 74－82

7     Sauer
H, Rosenblad C, Bjorklund A. Glial cell line-derived neurotrophic factor but
not transforming growth factor β3 prevents delayed degeneration of nigral
dopaminergic neurons following striatal 6-hydroxydopamine lesion. Proc Natl
Acad Sci USA, 1995, 92(19): 8935－8939

8     Mochizuki
H, Schwartz JP, Tanaka K, Brady RO, Reiser J. High-titer human immunodeficiency
virus type-1 based vector systems for gene delivery into nondividing cells. J
Virol, 1998, 72(11): 8873－8883

9     Reiser
J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M. Transduction
of nondividing cells using pseudotyped defective high-titer HIV type 1
particles. Proc Natl Acad Sci USA, 1996, 93(26): 15266－15271

10    Adachi
A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA. Production
of acquired immunodeficiency syndrome-associated retrovirus in human and
nonhuman cells transfected with an infectious molecular clone. J Virol, 1986,
59(2): 284－291

11    Mandel
RJ, Spratt SK,
Snyder RO, Leff SE. Midbrain injection of recombinant adeno-associated virus encoding
rat glial cell line-derived neurotrophic factor protects nigral neurons in a
progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease
in rats. Proc Natl Acad Sci USA, 1997, 94(25): 14083－14088

______________________________________

Received: June
3, 2003  Accepted: July
9, 2003

This work was supported by a grant from the
National Natural Science Foundation of China (Key Project) (No. 39993430)

*Corresponding author: Tel, 86-21-54921392;
Fax, 86-21-54921391; e-mail, [email protected]

Updated at: 2003-10-09

慢病毒介导GDNF对帕金森病的多巴胺能神经营养作用

孙兵^1,2           惠国桢²         郭礼和¹*        REISER
Jakob³

( 1中国科学院上海生命科学研究院生物化学与细胞生物学研究所, 上海 20031; 2苏州大学附属第一医院神经外科, 苏州 215006; 3路易斯安那健康研究中心, 路易斯安那州 70112 )

摘要       探讨慢病毒介导GDNF 对帕金森病的治疗作用。 将gdnf片段替代pNL-lacZ/CMV质粒中的LacZ编码区， 构建pNL-gdnf 质粒。 采用磷酸钙转染方法, 将慢病毒系统中三个质粒瞬时共转染293T细胞， 并收集病毒粒子。 用立体定位仪将高滴度病毒注射入PD大鼠的纹状体中。 分别在治疗后14, 30, 60 天检测阿朴吗啡（APO）诱导旋转反应的变化； 用Western 印迹方法测定蛋白质的表达； 用免疫组织化学方法检测lacZ和TH的表达； 移植治疗后PD鼠的行为学逐渐有了改善， 尤以治疗后14天明显。 GDNF 蛋白在大鼠脑内至少表达了60天， 对多巴胺神经有一定的神经营养作用。
因此， 慢病毒介导GDNF能显著改善PD鼠的行为学表现， 是治疗帕金森病的有效方法之一。

关键词   胶质细胞源性神经生长因子; 慢病毒（lentivirus）载体; 帕金森病; 基因治疗