Gene Ther Mol Biol Vol 4,
59-74. December 1999.
Glial cell line-derived neurotrophic factor (GDNF)
gene therapy in an aged rat model of Parkinson's disease
Review Article
Bronwen Connor and Martha C. Bohn
Children's Memorial Institute For Education and Research,
Department of Pediatrics, Northwestern University, Chicago, IL 60614.
______________________________________________________________________________________
Correspondence: Martha C. Bohn, Ph.D., Children's Memorial Institute For Education and
Research, 2300 Children's Plaza # 209, Chicago, IL 60614; Tel: (773)-868 8052;
Fax: (773)-868 8066; E-mail: m-bohn@nwu.edu
Abbreviations: GDNF,
glial cell line-derived neurotrophic factor; BDNF, brain-derived neurotrophic factor; DA, dopaminergic; 6-OHDA, 6-hydroxydopamine; Ad, adenoviral; SN, substantia nigra pars compacta; CNS, central nervous system; MFB, medial forebrain bundle; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP+, 1-methyl-4-phenyl pyridinium; MAO-B, monoamine oxidase; TH, tyrosine hydroxylase; AAV, adeno-associated virus; PSP, persephin; NTN, neurturin; IR,
immunoreactivity
Key Words:
Glial cell line-derived neurotrophic factor, GDNF, gene therapy, ex-vivo,
Parkinson's disease, neurotrophic factors, dopaminergic neurons, striatum,
6-OHDA, neurodegenerative disorders, brain-derived neurotrophic factor
Received:
22 September 1999; accepted: 29 September 1999
Summary
The chronic delivery of neuroprotective factors to
specific regions of the CNS via gene therapy offers a new strategy for the
treatment of neurodegenerative disorders. The neurotrophic factor, glial cell
line-derived neurotrophic factor (GDNF) is a potent dopaminergic (DA) trophic
factor that ameliorates the behavioral and histological consequences of lesioning
DA neurons in rodent and primate models of Parkinson’s disease. GDNF
gene therapy may therefore have potential use in the treatment of Parkinson's
disease. We have observed previously that an adenoviral (Ad) vector harboring
a GDNF minigene protects DA neurons from degeneration in the young rat brain.
However, as Parkinson’s disease occurs primarily in aged populations, we
have also studied the effects of GDNF gene delivery in an aged rat model of
Parkinson’s disease. In the aged (20 month) Fischer 344 rat, an Ad
vector was used to deliver GDNF either to the DA cell bodies in the SN or to
the DA terminals in the striatum. One week following gene delivery, the
neurotoxin 6-hydroxydopamine (6-OHDA) was injected unilaterally into the
striatum to cause progressive degeneration of DA neurons. Injection of GDNF
vector into either the striatum or SN provided significant cell protection
against 6-OHDA. However, only striatal injections of Ad GDNF protected against
the development of behavioral and neurochemical changes that occur in the
DA-depleted brain. The results of this study are reviewed here and the
behavioral and cellular effects of GDNF gene delivery into striatal versus
mesencephalic sites are discussed.
I. Introduction
While neurotrophic factors
are promising therapeutic agents in the treatment of neurodegenerative
disorders, the delivery of these factors to the central nervous system (CNS)
provides an interesting challenge. The identification of factors with potent
dopaminergic (DA) trophic activities in vitro led to the concept of using neurotrophic factors as
therapeutic agents for Parkinson’s disease. The most potent of these
factors is glial cell line-derived neurotrophic factor (GDNF) (Lin et al., 1993; Lin et al., 1994). There is much in vivo evidence demonstrating the effectiveness of GDNF
protein in ameliorating neurodegeneration and maintaining behavioral function
in animal models of Parkinson's disease. However, neurotrophic factors are
labile substances that are unable to cross the blood-brain barrier in
significant amounts. Therefore, the therapeutic use of factors such as GDNF in
the treatment of Parkinson's disease will require the development of methods
for delivering these factors to specific regions of the CNS in a continuous and
regulatable manner that is safe, minimally invasive and does not result in side
effects.
Parkinson’s disease is
a neurodegenerative disorder characterized by the progressive degeneration of
DA neurons in the substantia nigra pars compacta (SN) that innervate the
striatum. This degeneration results in the loss of DA terminals in the caudate
and putamen leading to the clinical symptoms of bradykinesia, rigidity and
resting tremor. Because Parkinson's disease is progressive, it is likely that
long-term trophic support for degenerating DA neurons will be required.
Repeated injections of recombinant neurotrophic factors into the human brain
are unlikely to be practical, and are likely to elicit deleterious side effects
over the long-term. In contrast, genetic approaches are ideal for delivery of
neurotrophic factors to the CNS. Through the use of gene therapy techniques,
increased levels of neurotrophic factor biosynthesis in the CNS might prevent
neuronal cell death and enhance neuronal function. Furthermore, the
development of vectors harboring genes driven by specific cellular promoters
would provide the potential of limiting expression of a transgene to one
typically defined cell population in the CNS, as well as regulating transgene
expression.
Several research groups,
using adenoviral (Ad) or adeno-associated viral vectors expressing GDNF, have
reported that viral delivery of a GDNF gene protects DA neurons in the young
adult rat brain from 6-OHDA-induced degeneration (Bilang-Bleuel et al., 1997; Choi-Lundberg et al., 1997; Choi-Lundberg
et al., 1998; Mandel et al., 1997). However, Parkinson’s disease is
a neurodegenerative disorder that primarily affects the aging population.
Age-related reductions in neurotransmitter synthesis and alterations in
receptor levels may contribute to ongoing degenerative and behavioral deficits
in Parkinson's disease. Multiple functional changes of the nigrostriatal DA
system, similar to those observed in the Parkinsonian brain, have been
described during normal aging in both animals and humans (Felten et al., 1992; Hubble, 1998; Morgan and Finch,
1988; Roth and Joseph, 1994). Compensatory events may be observed in the
aged brain due to age-dependent degenerative changes occurring in the
nigrostriatal and mesolimbic pathways. Alternatively, the aged brain may be
unable to produce compensatory changes in response to either age-dependent or
lesion-induced neuronal degeneration, worsening lesion-induced degenerative
alterations in DA neuronal morphology and function in the aged brain (Demarest et al., 1980; Schallert, 1988; Unnerstall
and Long, 1996; Zigmond et al., 1993). Therefore, while Ad GDNF has been
observed to exhibit neuroprotective effects within the young rat brain, we
believed it to be important to examine whether chronic biosynthesis of GDNF,
achieved by Ad-mediated delivery of GDNF, is able to protect DA neurons and
maintain DA function in an aged rat model of Parkinson's disease.
Here, we review the use of
Ad to deliver a GDNF minigene to the aged (20 month) rat brain, prior to a
partial, progressive lesion of the nigrostriatal pathway. The results show
that Ad GDNF not only protects DA neurons from degeneration, but that
functional consequences in DA target neurons following a lesion of the
nigrostriatal pathway are prevented by Ad GDNF in a brain region specific
manner.
II. Animal models of Parkinson's disease
Since Parkinson's disease
does not occur naturally in animals, several well characterized models have
been developed for stimulating the neuropathological and neurological features
of Parkinson's disease in laboratory animals (reviewed in Bankiewicz et al., 1993). The most common animal
model of Parkinson's disease involves the intracerebral injection of the
catecholamine neurotoxin, 6-hydroxydopamine (6-OHDA), resulting in a reduction
of dopaminergic phenotypic markers (i.e. tyrosine hydroxylase) and the
selective death of DA neurons. 6-OHDA is a dopamine analog that is
specifically taken up by the high-affinity dopamine transporter. 6-OHDA
undergoes auto-oxidation, generating hydroxyl radical, hydrogen peroxide and
superoxide anion, which causes damage to various cellular components.
Injection of 6-OHDA into either the striatum, SN or the medial forebrain bundle
(MFB), which contains dopaminergic axons from the ventral tegmental area and
SN, results in specific loss of dopaminergic neurons and fibers. The
neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) also causes
Parkinsonian symptoms in humans, non-human primates and mice, following
systemic administration. MPTP is oxidized to the toxic molecule, MPP+
(1-methyl-4-phenyl pyridinium) by the monoamine oxidase system (MAO-B). MPP+
enters dopaminergic neurons by high-affinity through the dopamine transporter
and interferes with ATP production by inhibiting complex 1 of the mitochondrial
electron transport chain.
The standard animal model of
Parkinson's disease, based on unilateral injection of 6-OHDA into either the SN
or the MFB, has been used extensively in Parkinson's disease research.
Injection of 6-OHDA in either the SN or the MFB results in the rapid death of
dopaminergic neurons, within 48 hours. However, due to the rapid and near-complete
degeneration of nigral DA neurons, these lesion models do not closely reflect
the clinical picture in which DA neurons die over a prolonged time. These
models also are not optimal for studies on neuroprotection or regeneration of
the nigrostriatal system (reviewed in Bjorklund
et al., 1997). In contrast, intrastriatal delivery of 6-OHDA results in
the rapid destruction of dopaminergic terminals in the striatum, followed by
progressive degeneration of parent DA cell bodies in the SN over a period of
several weeks (Sauer and Oertel, 1994).
This degeneration is preceded and accompanied by cellular atrophy and a
partial loss of tyrosine hydroxylase (TH) expression. This progressive lesion
model yields an animal model which closely resembles the early stages of
Parkinson's disease in humans, in which a portion of the nigrostriatal
projection remains intact. These remaining neurons serve as a substrate for
regeneration and functional recovery. The progressive lesion model is an ideal
model for studying the neuroprotective or restorative properties of trophic
factors (Bilang-Bleuel et al., 1997;
Choi-Lundberg et al., 1997; Choi-Lundberg et al., 1998; Connor et al., 1999;
Horger et al., 1998; Mandel et al., 1997; Milbrandt et al., 1998; Rosenblad et
al., 1999; Rosenblad et al., 1998; Sauer et al., 1995; Shults et al., 1996).
Unilateral physical and
chemical lesions of the nigrostriatal pathway result in an imbalance in the
level of DA and DA receptors between the two striatae, producing impairment in
DA-dependent behavioral function. Lesion-induced behavioral impairment and the
effects of experimental interventions can be quantified by several means
(reviewed in (Schallert, 1995; Schwarting
and Huston, 1996). Specifically, unilaterally lesioned animals exhibit
rotational behavior in response to amphetamine or DA agonists, such as
apomorphine, that is readily quantifiable (Schwarting
and Huston, 1996; Ungerstedt and Arbuthnott, 1970; Figure 1A). Specifically, in animals with a unilateral lesion
of the nigrostriatal DA system, the injection of drugs that act to release DA,
such as amphetamine, will induce rotational behavior towards the denervated
striatum due to an imbalance in striatal DA levels. Lesioned animals will turn
away from the hemisphere where there is greater amphetamine-stimulated DA
release and greater DA receptor stimulation (Ungerstedt,
1971). In contrast, injection of DA agonists, such as apomorphine, in
animals exhibiting a < 90% loss of DA in the lesioned hemisphere, will
induce rotational behavior away from the denervated striatum due to
compensatory upregulation and supersensitivity of DA receptors in the
unlesioned striatum (Ungerstedt and
Arbuthnott, 1970). In addition, unilaterally lesioned animals exhibit
deficits in contralateral limb use in several spontaneous behaviors (Olsson et al., 1995; Schallert, 1995; Figure
1B).
Figure 1A. - Diagram representing the behavioral and neurochemical
changes that occur in the DA-depleted brain. In rats with a unilateral lesion
of the nigrostriatal DA system, injection of amphetamine will induce rotational
behavior towards the lesioned hemisphere. The lesioned rat turns away from the
hemisphere where there is greater amphetamine-stimulated DA release and greater
DA receptor stimulation. In the unlesioned striatum, injection of amphetamine
also results in the induction of the transcription factor, c-fos in striato-nigral cells. However in the lesioned
striatum, due to a loss of functional DA terminals and a reduction in striatal
DA levels, amphetamine-induced Fos expression is reduced. Dashed lines
indicates the degeneration and loss of the nigrostriatal pathway connecting the
SN and the ST. Hatched area represents a loss of DA.
Figure 1B. - Rat exhibiting preferential use of the ipsilateral
forelimb following a unilateral 6-OHDA-induced lesion of the nigrostriatal
pathway. Spontaneous exploratory forelimb use is a non-drug induced test of
forelimb locomotor function which has been shown to correlate with DA depletion
in the lesioned hemisphere. Following a unilateral 6-OHDA lesion of the
nigrostriatal pathway, rats preferentially use the forelimb ipsilateral to the
side of the lesion to initiate and terminate weight-shifting movements during
rearing and exploration along vertical surfaces.
III. Neurotrophic factors for dopaminergic neurons
The discovery and
characterization of neurotrophic factors that promote the survival, neurite
outgrowth and phenotypic differentiation of DA neurons has been an area of
intense research in recent years. Early studies of DA neurons in culture
showed glia from different brain regions and glial cell lines produce factors
that influence the survival and differentiation of embryonic DA neurons in
culture (Engele and Bohn, 1991; Engele et
al., 1991; Rousselet et al., 1988). These studies prompted the search for
specific DA neurotrophic factors. To date, more than 20 neurotrophic factors
have been identified for DA neurons (Table 1). These include members of several growth factor
families, operating through different intracellular signalling mechanisms,
including the TGF-b superfamily, the neurotrophins, cytokines and
mitogenic growth factors. The first neurotrophic factor shown to act directly
on DA neurons was brain-derived neurotrophic factor (BDNF) (Hyman et al., 1991). Subsequently, GDNF was
purified from the glial cell line, B49, and shown to be a very potent DA factor
that enhances survival and neurite outgrowth of embryonic DA neurons in
vitro (Lin et al., 1993; Lin et al., 1994). Recently, three additional
members of the GDNF family have been identified - neurturin (Kotzbauer et al., 1996), persephin (Milbrandt et al., 1998) and artemin (Baloh et al., 1998). These factors have been
shown to exhibit various degrees of trophic support to DA neurons.
A wealth of in vivo evidence has been accumulated in animal models of
Parkinson's disease, supporting the potential therapeutic use of several DA
neurotrophic factors, in particular GDNF, in the treatment of Parkinson's
disease. A summary of these studies is shown in Table 2. GDNF has been shown to ameliorate the behavioral
and pathological consequences of lesioning DA neurons in rodent and primate
models of Parkinson's disease when administered to the adult nigrostriatal DA
system (reviewed in Bjorklund et al.,
1997; Gash et al., 1998). Specifically, in animal models of Parkinson's
disease, single or continuous injection of recombinant GDNF protein has been
shown to rescue injured or axotomized DA neurons when administered before or
shortly after insult, and to preserve injured atrophic DA neurons during
chronic neurodegeneration (Beck et al.,
1995; Bowenkamp et al., 1995; Gash et al., 1996; Hoffer et al., 1994; Kearns
and Gash, 1995; Lapchak et al., 1997; Sauer et al., 1995; Tomac et al., 1995).
In addition, GDNF stimulates regenerative growth or axonal sprouting after
partial lesions of the DA system and stimulates metabolism and function of
lesioned DA neurons (Lindner et al.,
1995; Rosenblad et al., 1998; Shults et al., 1996).
Table 1. Dopaminergic Neurotrophic
Factors (updated from Bohn and
Choi-Lundberg, 1997)
TGF-b Superfamily
GDNF
Family
GDNF (Lin et al., 1993; Lin et al., 1994)
Neurturin (Kotzbauer et al., 1996)
Persephin (Milbrandt et al., 1998)
Artemin (Baloh et al., 1998)
Others
TGF-b-1 (Krieglstein et al., 1995)
TGF-b-2,
3 (Krieglstein et al., 1995; Poulsen et al., 1994)
GDF-5 (Krieglstein et al., 1995)
Activin
A (Krieglstein et al., 1995)
Neurotrophins
BDNF (Hyman et al., 1991)
NT-3 (Hyman et al., 1994)
NT-4/5 (Hyman et al., 1994)
Mitogenic
Growth Factors
TGF-a (Alexi and Hefti, 1993)
aFGF and bFGF (Beck et al., 1993; Engele and Bohn, 1991; Ferrari et
al., 1989; Otto and Unsicker, 1990)
EGF (Casper et al., 1991)
Insulin
(Knusel et al., 1990)
IGF-I (Beck et al., 1993)
IGF-2 (Liu and Lauder, 1992)
PDGF (Othberg et al., 1995)
Midkine (Kikuchi et al., 1993)
Cytokines
CNTF (Hagg and Varon, 1993; Magel et al., 1993)
IL-1b (Akaneya et al., 1995)
IL-6 (Hama et al., 1991; von Coelln et al., 1995)
IL-7 (von Coelln et al., 1995)
Cardiotrophin-1 (Pennica et al., 1995)
Comparable findings for BDNF
have been reported in both the 6-OHDA lesioned rat and the MPTP primate model
in which BDNF improved DA levels or DA-dependent behavior in the absence of an
effect on the density of DA fibers in the striatum (Altar et al., 1994; Galpern et al., 1996; Levivier et
al., 1995; Lucidi-Phillipi et al., 1995; Tsukahara et al., 1995; Yoshimoto et
al., 1995). In addition, both neurturin and persephin have been reported
to protect mature DA neurons from cell death induced by 6-OHDA in the absence
of striatal reinnervation and DA-dependent behavioral improvement (Akerud et al., 1999; Horger et al., 1998; Milbrandt
et al., 1998; Rosenblad et al., 1999; Tseng et al., 1998).
While many studies have demonstrated the efficacy of
DA neurotrophic factors, such as GDNF, in animal models of Parkinson's disease,
these studies have utilized infusion of protein into the striatum, near the SN
or into the lateral ventricles. In addition, large quantities of proteins are
infused in these studies (typically 10mg or more), and repeated injections are typically
required to maintain effects. Repeated injections of recombinant neurotrophic
factors into the human brain are unlikely to be practical, and are likely to
elicit deleterious side effects over the long term. In contrast, genetic
approaches are ideal for delivering GDNF to the CNS.
Several laboratories have
studied viral vector mediated GDNF gene delivery in animal models of
Parkinson's disease (Table 2). In
young rats, we have observed that injection of an Ad vector harboring human
GDNF into the SN one week prior to a progressive 6-OHDA lesion significantly
protects DA neurons in the lesioned SN (Choi-Lundberg
et al., 1997; Choi-Lundberg et al., 1998). In addition, intrastriatal
injection of Ad GDNF in this model was effective in protecting DA cell bodies
and also prevented the onset of DA-dependent behaviors that occurred in control
rats as a consequence of the unilateral 6-OHDA lesion (Choi-Lundberg et al., 1998). Similar effects of
GDNF gene delivery have been confirmed by other groups using either adenoviral
or adeno-associated viral vectors (Table 2). In addition, our laboratory has recently observed that injection of
Ad GDNF into the SN 1 week after 6-OHDA induced damage has commenced, not only
rescues DA neurons, but also protects against alterations in DA target cells in
the striatum (Kozlowski et al, Submitted).
Table 2 Delivery
of neurotrophic factors in animal models of Parkinson’s disease (selected
references)
|
Paradigm
|
Delivery
site, Factor
|
Biological
Effects
|
Reference
|
Neurotrophic
factor protein infusion
|
6-OHDA
- complete
|
Intranigral,
GDNF
|
Ú
in nigral DA levels
Improvement
in behavioral function
|
Hoffer
et al., 1994
|
|
6-OHDA
- partial striatal
|
Intranigral,
BDNF / NT-3
|
Ú
in striatal DA metabolites
Improvement
in behavioral function
No
change in striatal DA levels
No
reinnervation of lesioned striatum
|
Altar
et al., 1994
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
|
Ú
survival of DA cell bodies in SN
Improvement
in behavioral function
No
reinnervation of lesioned striatum
|
Bowenkamp
et al., 1995
|
|
MPTP
- mouse model
|
Intrastriatal,
GDNF
Intranigral,
GDNF
|
Ú
in nigral and striatal DA / metabolite levels
Protection
of striatal innervation (intrastriatal only)
|
Tomac
et al., 1995
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
|
Protection
of DA cell bodies in SN
Improvement
in behavioral function
|
Beck
et al., 1995
|
|
MPTP
- monkey model
|
Intrathecal,
BDNF
|
Protection
of DA cell bodies in SN
Improvement
in behavioral function
|
Tsukahara
et al., 1995
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
Intraventricular,
GDNF
|
Ú
in striatal DA / metabolite levels
Protection
of DA uptake sites
Improvement
in behavioral function
|
Opacka-Juffry
et al., 1995
|
|
6-OHDA
- complete or partial
|
Intranigral,
GDNF
|
Protection
of DA cell bodies in SN
|
Kearns
and Gash, 1995
|
|
6-OHDA
- partial striatal
|
Intranigral
,GDNF
|
Protection
of DA cell bodies in SN
|
Sauer
et al., 1995
|
|
6-OHDA
- partial striatal
|
Intrastriatal
,GDNF
|
Protection
of DA cell bodies in SN
Protection
of striatal innervation
Improvement
in behavioral function
|
Shults
et al., 1996
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
|
Protection
of DA cell bodies in SN
No
reinnervation of lesioned striatum
No
improvement in behavioral function
|
Winkler
et al., 1996
|
|
MPTP
- monkey model
|
Intracerebral,
GDNF
|
Ú
DA cell body size
Ú
fiber density
Ú
in nigral and striatal DA levels
Improvement
in behavioral function
|
Gash
et al., 1996
|
|
6-OHDA
- complete
|
Intraventricular,
GDNF
|
Ú
in nigral DA / metabolite levels
Protection
of DA cell bodies in SN
Improvement
in behavioral function
|
Bowenkamp
et al., 1997
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
|
Protection
of DA cell bodies in SN
Did
not restore TH-IR in nigral neurons
|
Lu
and Hagg, 1997
|
|
6-OHDA
- complete
|
Intranigral
,GDNF
|
Partial
restoration of nigral DA levels
Improvement
in behavioral function
|
Hoffman
et al., 1997
|
|
6-OHDA
- complete
|
Intraventricular,
GDNF
Intranigral,
GDNF
|
Ú
expression of TH in SN
Improvement
in behavioral function
Intranigral
GDNF prevented lesion-induced increase in dynorphin A
|
Lapchak
et al., 1997
|
|
6-OHDA
- partial striatal
|
Intranigral,
NTN
|
Protection
of DA cell bodies in SN
|
Horger
et al., 1998
|
|
6-OHDA
- complete
|
Intranigral,
GDNF
Intraventricular,
GDNF
|
Prevented
loss of DA re-uptake sites
Prevented
loss of striatal DA / metabolites
Prevented
loss of DA cell bodies in SN
Improvement
in behavioral function
|
Sullivan
et al., 1998
|
|
6-OHDA
- partial striatal
|
Intranigral,
PSP
|
Protection
of DA cell bodies in SN
|
Milbrandt
et al., 1998
|
|
MPTP
- mouse model
|
Intrastriatal
,GDNF
|
Protection
of nigral and striatal DA levels
Improvement
in behavioral function
|
Cheng
et al., 1998
|
|
6-OHDA
- partial striatal
|
Intrastriatal,
GDNF
|
Restored
DA uptake sites
Protection
of DA cell bodies in SN
Improvement
in behavioral function
|
Rosenblad
et al., 1998
|
|
6-OHDA
- partial striatal
|
Intrastriatal,
GDNF / NTN
Intraventricular,
GDNF / NTN
|
GDNF
- Protection of DA cell bodies in SN
- No reinnervation of lesioned striatum
- No improvement in behavioral function
NTN - Partial protection of DA cell
bodies in SN (intrastriatal only)
- No reinnervation of lesioned striatum
- No improvement in behavioral function
|
Rosenblad
et al., 1999
|
|
MPTP
- monkey model
|
Intraventricular,
GDNF
|
Ú
in nigral DA / metabolite levels
Improvement
in behavioral function
|
Gerhardt
et al., 1999
|
Ex vivo gene delivery
|
MPP+
- rat model
|
Fibroblasts,
BDNF
|
Protection
of DA cell bodies in SN
|
Frim
et al., 1994
|
|
6-OHDA
- complete
|
BHK,
GDNF
|
TH-IR
fiber ingrowth to BHK-GDNF capsules
No
improvement in behavioral function
|
Lindner
et al., 1995
|
|
6-OHDA
- partial SN
|
Astrocytes,
BDNF
|
No
effect on TH-IR fibers in ipsilateral striatum
Improvement
in behavioral function
|
Yoshimoto
et al., 1995
|
|
6-OHDA
- partial striatal
|
Fibroblasts,
BDNF
|
Prevented
loss of nerve terminals
Protection
of DA cell bodies in SN
|
Levivier
et al., 1995
|
|
6-OHDA
- complete
|
Fibroblasts,
BDNF
|
Ú
in nigral DA / metabolite levels
Did
not induce fiber sprouting
No
improvement in behavioral function
|
Lucidi-Phillipi
et al., 1995
|
|
MPP+
- rat model
|
Fibroblasts,
BDNF
|
Ú
in nigral DA levels
|
Galpern
et al., 1996
|
|
6-OHDA
- complete
|
BHK,
GDNF
|
Protection
of DA cell bodies in SN
Improvement
in behavioral function
Did
not prevent loss of striatal DA
|
Tseng
et al., 1997
|
|
6-OHDA
- complete
|
BHK,
NTN
|
Protection
of DA cell bodies in SN
No
improvement in behavioral function
|
Tseng
et al., 1998
|
|
6-OHDA
- complete
|
BHK,
GDNF
BHK,
NTN
|
Both
GDNF and NTN protected DA cell bodies in SN
GDNF
induced TH-IR, sprouting or hypertrophy of DA neurons
|
Akerud
et al., 1999
|
In vivo gene delivery
|
6-OHDA
- partial striatal
|
Intranigral,
Ad GDNF
|
Protection
of DA cell bodies in SN
|
Choi-Lundberg
et al., 1997
|
|
6-OHDA
- partial striatal
|
Intrastriatal,
Ad GDNF
|
Protection
of DA cell bodies in SN
Protected
innervation of striatum
Improvement
in behavioral function
|
Bilang-Bleuel
et al., 1997
|
|
6-OHDA
- complete
|
Intranigral,
Ad GDNF
|
Restored
nigral DA levels
Improvement
in behavioral function
|
Lapchak
et al., 1997
|
|
MPTP
- mouse model
|
Intrastriatal,
Ad GDNF
|
Ú
in striatal DA levels
|
Kojima
et al., 1997
|
|
6-OHDA
- partial striatal
|
Intranigral,
AAV GDNF
|
Protection
of DA cell bodies in SN
|
Mandel
et al., 1997
|
|
6-OHDA
- partial striatal
|
Intrastriatal,
Ad GDNF
|
Protection
of DA cell bodies in SN
Improvement
in behavioral function
|
Choi-Lundberg
et al., 1998
|
IV. The differential effects of GDNF gene delivery in the striatum and
SN of the aged Parkinsonian rat
In order to extend our
initial observations in the young rat, we recently examined whether Ad GDNF was
able to restore compensatory events, protect DA neurons and maintain DA
function in the aged rat brain following a partial lesion of the nigrostriatal
pathway. It has also become apparent that the site of GDNF administration is
important in determining the degree of functional recovery observed in the
damaged or degenerating nigrostriatal DA system. Previous studies have
demonstrated that, while repeated injections of GDNF near cell bodies in the SN
following a partial 6-OHDA lesion of the nigrostriatal pathway protects or
restores DA neuronal function, intranigral injection of GDNF does not prevent
impairment of behavioral function in the unilaterally lesioned Parkinsonian
animal (Sauer et al., 1995; Winkler et
al., 1996). This suggests that long-lasting functional recovery in the
intrastriatal 6-OHDA lesion model may require reinnervation and restoration of
DA neurotransmission in the denervated striatum. Supporting this proposal, several
studies examining the effect of intrastriatal injections of GDNF have reported
that, in addition to preventing the loss of dopaminergic neurons in the
lesioned SN, GDNF injected into the striatum also induces partial axonal
regeneration or reinnervation of the denervated striatum and prevents
behavioral impairment caused by 6-OHDA-induced depletion of striatal DA (Bilang-Bleuel et al., 1997; Choi-Lundberg et al.,
1998; Rosenblad et al., 1998; Shults et al., 1996).
In order to fully examine
the differential effect of injecting GDNF either near DA cell bodies in the SN
or at dopaminergic terminals in the striatum, we compared the behavioral and
cellular effects of GDNF gene delivery into striatal and mesencephalic sites in
aged (20 month) Fischer-344 rats with progressive lesions of the nigrostriatal
pathway (Connor et al., 1999). In
this study, a subpopulation of DA neurons was prelabelled by bilateral
intrastriatal injection of the retrograde tracer fluorogold (FG) so that the
fate of those DA neurons that projected specifically to the lesion site could
be assessed without having to rely on DA phenotypic markers. Following FG
injection, one group of rats was injected unilaterally in the striatum, while a
second group of rats was injected unilaterally into the SN with Ad vectors
encoding either GDNF, a mutant form of GDNF lacking bioactivity (muGDNF) or
LacZ. An additional group of rats underwent surgery, but received no vector
injection into either the striatum or the SN. One week later, rats received a
unilateral intrastriatal injection of 6-OHDA on the same side as the vector
injection and at the same coordinates as the FG injection.
Thirty-five days after
lesioning, we observed that injections of Ad GDNF into either the striatum or
the SN provided significant cell protection against 6-OHDA (Connor et al., 1999). As shown in Figure 2, Ad GDNF injected in the SN protected an average of
about 55% of the FG labeled DA neurons while Ad GDNF injected in the striatum
protected an average of about 65% of FG labeled neurons. In contrast, only an
average of about 30% of the neurons remained in the control groups (Connor et al., 1999). Overall, injections of Ad
GDNF into either the striatum or the SN were similarly effective in protecting
DA neurons in the lesioned SN of the aged rat brain. This indicates that the
site of GDNF injection (striatum versus SN) does not affect the degree of
neuronal protection seen in the lesioned SN.
Figure 2A: Injection of Ad GDNF in either the striatum or the SN
prevents the degeneration of FG-positive DA neurons in the SN five weeks after
6-OHDA. - Five weeks following intrastriatal injection of 6-OHDA many large
FG-positive cells (i.e.: DA neurons - arrows) remain in the SN on the
unlesioned side (A & B) and on the lesioned side in rats injected with Ad
GDNF in either the striatum (E & F) or the SN (G & H). In contrast,
fewer large FG-positive neurons, but many small secondarily labeled FG-positive
cells (microglia and other non-neuronal cells - *) were observed in the
lesioned SN in control rats (C & D). Scale bars: A, C, E & G = 100mm;
B, D, F & H = 50mm. Reprinted with permission from Connor et al, 1999.
Copyright 1999 Stockton Press.
Figure 2B: (Cont.) - A significant increase in the percentage of
FG-positive DA neurons was observed in the lesioned SN in rats injected with Ad
GDNF in either the striatum or the SN compared to control groups (* p £ 0.01). There was no significant difference in the
percentage of FG-positive neurons between rats injected with Ad GDNF in the
striatum versus the SN (p > 0.05). Reprinted with permission from Connor et
al, 1999. Copyright 1999 Stockton Press.
Figure 3: Striatal injections of Ad GDNF prevents the development
of amphetamine-induced ipsilateral rotational behavior observed in control rats
after 6-OHDA lesioning. Rats were injected with dl-amphetamine (5mg/kg i.p),
placed in a rotation chamber and their behavior recorded for 60 minutes. Baseline
amphetamine rotation tests were performed 7 days before lesioning (-7) and the
results used to assign the side of 6-OHDA lesioning. Five weeks after
lesioning, a significant reduction in ipsilateral rotational behavior was
observed in rats injected with Ad GDNF in the striatum compared to control
groups and rats injected with Ad GDNF in the SN (* p £ 0.05). In contrast, rats injected with Ad GDNF into
the SN exhibited a significant increase in ipsilateral rotational behavior 35
days after lesioning compared to control groups (** p £ 0.05). Reprinted with permission from Connor et al,
1999. Copyright 1999 Stockton Press.
The differential effects of
the vector placed into these two sites became evident however, when we examined
DA dependent behaviors and cellular changes in DA target neurons. Only
striatal injections of Ad GDNF protected against the development of behavioral
impairment characteristic of unilateral DA-dependent deficits (Connor et al., 1999). Thirty-five days after
lesioning, rats injected with Ad GDNF in the striatum exhibited a significant
reduction in amphetamine-induced rotational asymmetry compared to control
groups and rats injected with Ad GDNF in the SN (Figure 3, Connor et al.,
1999). In addition, rats injected with Ad GDNF in striatum exhibited a
significant reduction in the preferential use of the ipsilateral forelimb 21
days and 28 days after 6-OHDA lesioning compared to control groups and rats
injected with Ad GDNF in the SN (Connor
et al., 1999). Maintenance of DA-dependent behavioral function requires
the presence of functional DA terminals and/or preservation of striatal DA
levels. Our observations suggest that when GDNF biosynthesis is increased near
the DA terminals prior to 6-OHDA delivery, the terminals are protected against
degeneration and striatal DA levels persist. In contrast, when GDNF
biosynthesis is increased near DA cell bodies in the aged rat brain, levels are
sufficient to inhibit cell death, but insufficient to increase striatal DA
levels or to stimulate terminal sprouting. This apparently results in the
development of behaviors indicative of unilateral DA deficiency.
These behavioral
observations are further supported by morphological data on the response of
striatal target neurons and DA fiber density. In the unlesioned striatum,
injection of indirect DA agonists, such as amphetamine results in the
expression of Fos protein (Figure 1A; Graybiel et al., 1990; Robertson et al., 1989).
Amphetamine is thought to induce Fos in the striatum primarily by its ability
to release endogenous DA which, via activation of DA D1 receptors, triggers a
transduction cascade culminating in the expression of the c-fos gene in striato-nigral cells (Berretta et al., 1992; Konradi et al., 1994; Paul et
al., 1992; Robertson et al., 1992). Therefore in the striatum,
amphetamine-induced Fos expression requires the presence of functional DA
terminals and is used as a marker of DA-mediated postsynaptic responses (Cenci et al., 1992; Labandeira-Garcia et al., 1996;
Morgan and Curran, 1991). We observed that striatal, but not SN,
injections of Ad GDNF increased amphetamine-induced Fos expression in the
lesioned striatum above that in the unlesioned contralateral striatum,
indicating protection of DA terminals (Connor
et al., 1999). As shown in Figure 4, a reduction in the percentage of Fos-immunopositive neurons,
reflecting a decrease in functional DA terminals was seen in both control
groups and rats injected with Ad GDNF in the SN at the lesion site and 700mm posterior to the lesion site (Connor
et al., 1999). In contrast, rats injected with Ad GDNF in the striatum
exhibited a signi