[show abstract][hide abstract] ABSTRACT: The neuroprotective effect of the glial cell line-derived neurotrophic factor has been extensively studied in various toxic models of Parkinson's disease. However, it remains unclear whether this neurotrophic factor can protect against the toxicity induced by the aggregation-prone protein α-synuclein. Targeted overexpression of human wild-type α-synuclein in the nigrostriatal system, using adeno-associated viral vectors, causes a progressive degeneration of the nigral dopamine neurons and the development of axonal pathology in the striatum. In the present study, we investigated, using different paradigms of delivery, whether glial cell line-derived neurotrophic factor can protect against the neurodegenerative changes and the cellular stress induced by α-synuclein. We found that viral vector-mediated delivery of glial cell line-derived neurotrophic factor into substantia nigra and/or striatum, administered 2-3 weeks before α-synuclein, was inefficient in preventing the wild-type α-synuclein-induced loss of dopamine neurons and terminals. In addition, glial cell line-derived neurotrophic factor overexpression did not ameliorate the behavioural deficit in this rat model of Parkinson's disease. Quantification of striatal α-synuclein-positive aggregates revealed that glial cell line-derived neurotrophic factor had no effect on α-synuclein aggregation. These data provide the evidence for the lack of neuroprotective effect of glial cell line-derived neurotrophic factor against the toxicity of human wild-type α-synuclein in an in vivo model of Parkinson's disease. The difference in neuroprotective efficacy of glial cell line-derived neurotrophic factor seen in our model and the commonly used neurotoxin models of Parkinson's disease, raises important issues pertinent to the interpretation of the results obtained in preclinical models of Parkinson's disease, and their relevance for the therapeutic use glial cell line-derived neurotrophic factor in patients with Parkinson's disease.
[show abstract][hide abstract] ABSTRACT: Temporal lobe epilepsy patients remain refractory to available anti-epileptic drugs in 30% of cases, indicating a need for novel therapeutic strategies. In this context, glial cell line-derived neurotrophic factor (GDNF) emerges as a possible new agent for epilepsy treatment. However, a limited number of studies, use of different epilepsy models, and different methods of GDNF delivery preclude understanding of the mechanisms for the seizure-suppressant action of GDNF. Here we show that recombinant adeno-associated viral (rAAV) vector-based GDNF overexpression in the rat hippocampus suppresses seizures in two models of temporal lobe epilepsy. First, when rAAV-GDNF was injected before hippocampal kindling, the number of generalized seizures decreased, and the prolongation of behavioral convulsions in fully kindled animals was prevented. Second, injection of rAAV-GDNF after kindling increased the seizure induction threshold. Third, rAAV-GDNF decreased the frequency of generalized seizures during the self-sustained phase of status epilepticus. Our data demonstrate the complexity of mechanisms and the beneficial action of GDNF in epilepsy. Furthermore, we show that ectopic rAAV-mediated GDNF gene expression in the seizure focus is a feasible way to mitigate seizures and provides proof of principle that the neurotrophic factor-based gene therapy approach has the potential to be developed as alternative strategy for epilepsy treatment.
[show abstract][hide abstract] ABSTRACT: We investigated the feasibility of viral vector-mediated expression and axonal transport of the glial cell-line-derived neurotrophic factor, a potential antiepileptic agent, to the hippocampus and the piriform cortex, areas involved in the induction and spread of seizure activity. Glial cell-line-derived neurotrophic factor overexpression was induced by injections of recombinant vectors derived from serotype 2 adeno-associated virus or lentivirus. We found that recombinant adeno-associated viral vector was able to effectively transduce mitral cells of the olfactory bulb and pyramidal cells of CA1, resulting in transport of glial cell-line-derived neurotrophic factor to the piriform cortex and to the contralateral CA1 area, respectively. These data suggest that the recombinant adeno-associated viral vector vector system is an optimal alternative for therapeutic glial cell-line-derived neurotrophic factor gene transduction and transport of the protein to the epileptogenic brain areas.
[show abstract][hide abstract] ABSTRACT: Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in animal models of Parkinson's disease is, at least in part, dependent on the number of surviving dopaminergic neurons and the degree of graft-derived dopaminergic reinnervation of the host striatum. In the present study, we analyzed whether continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature dopaminergic grafts could further boost the functional outcome of widespread intrastriatal dopaminergic grafts. Rats with dopamine-denervating lesions received multiple intrastriatal transplants of fetal dopaminergic cells and graft-induced behavioral effects were analyzed in drug-induced and spontaneous motor behaviors. At three months after grafting, animals received intrastriatal injections of recombinant lentiviral vectors encoding for either human GDNF or the green fluorescent protein. Continuous exposure of GDNF to the grafts did not boost the functional recovery beyond what was observed in the control animals. Rather, in some of the spontaneous motor behaviors, animals in the GDNF-group showed deterioration as compared with control animals, and this negative effect of GDNF was associated with a down-regulation of the tyrosine hydroxylase enzyme. Based on these and our earlier results, we propose that intrastriatal administration of GDNF at the time of or shortly after grafting is highly effective in initially promoting the cell survival and fiber outgrowth from the grafts. However, once the grafts are mature, GDNF's ability to boost dopaminergic neurotransmission follows the same dynamics as for the native nigral dopaminergic neurons, which appears to be dependent on the concentration of GDNF. Since rather low doses of glial cell line-derived neurotrophic factor at nanogram levels appear to saturate these effects, it may be critical to adjust GDNF levels using tightly regulated gene expression systems.
[show abstract][hide abstract] ABSTRACT: Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped lentiviral vectors (RRV-LV)). The RRV is an alphavirus with an extremely broad host range, including the cells of the central nervous system. Previous studies have shown that lentiviral vectors can be efficiently pseudotyped with this envelope protein and have demonstrated promising features of such vectors, including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells.
[show abstract][hide abstract] ABSTRACT: Astrocytes are, as normal constituents of the brain, promising vehicles for ex vivo gene delivery to the central nervous system. In the present study, we have used a lentiviral vector encoding glial cell line-derived neurotrophic factor (GDNF) to transduce rat-derived primary astrocytes, in order to evaluate their potential for long-term transgene expression in vivo and neuroprotection in a rat model of Parkinson's disease. Following transplantation of GDNF-transduced astrocytes to the intact striatum, the level of released GDNF was 2.93 +/- 0.28 ng/mg tissue at 1 week post-grafting, reduced to 0.42 +/- 0.12 ng/mg tissue at 4 weeks, and thereafter was maintained at this level throughout the experiment (12 weeks; 0.53 +/- 0.068 ng/mg tissue). Similarly, grafting to the substantia nigra (SN) resulted in a significant overexpression of GDNF ( approximately 0.20 ng/mg tissue) at 1 week. Intact animals receiving transplants of GDNF-transduced astrocytes displayed an increased contralateral turning (5.39 +/- 1.19 turns/min) in the amphetamine-induced rotation test, which significantly correlated with the GDNF tissue levels measured in the striatum, indicating a stimulatory effect of GDNF on the dopaminergic function. Transplantation of GDNF-transduced astrocytes to the SN 1 week prior to an intrastriatal 6-hydroxydopamine lesion provided a significant protection of nigral tyrosine hydroxylase-positive cells. By contrast, when the cells were transplanted to the striatum, the level of released GDNF was not sufficient to rescue the striatal fibers and, hence, to protect the nigral dopaminergic neurons. Overall, our results suggest that genetically modified astrocytes expressing GDNF can provide neuroprotection in a rat model of Parkinson's disease following transplantation to the SN.
European Journal of Neuroscience 01/2006; 22(11):2755-64. · 3.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) for Parkinson's disease is likely to depend on sustained delivery of the appropriate amount to the target areas. Recombinant adeno-associated viral vectors (rAAVs) expressing GDNF may be a suitable delivery system for this purpose. The aim of this study was to define a sustained level of GDNF that does not affect the function of the normal dopamine (DA) neurons but does provide anatomical and behavioral protection against an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in the common marmoset. We found that unilateral intrastriatal injection of rAAV resulting in the expression of high levels of GDNF (14 ng/mg of tissue) in the striatum induced a substantial bilateral increase in tyrosine hydroxylase protein levels and activity as well as in DA turnover. Expression of low levels of GDNF (0.04 ng/mg of tissue), on the other hand, produced only minimal effects on DA synthesis and only on the injected side. In addition, the low level of GDNF provided approximately 85% protection of the nigral DA neurons and their projections to the striatum in the 6-OHDA-lesioned hemisphere. Furthermore, the anatomical protection was accompanied by a complete attenuation of sensorimotor neglect, head position bias, and amphetamine-induced rotation. We conclude that when delivered continuously, a low level of GDNF in the striatum (approximately threefold above baseline) is sufficient to provide optimal functional outcome.
Journal of Neuroscience 02/2005; 25(4):769-77. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Functional effects of ventral mesencephalic (VM) cell transplants in models of Parkinson's disease are limited by the survival and fiber outgrowth of the grafted cells. In a recent study, we used a recombinant lentiviral vector (rLV) encoding glial cell line-derived neurotrophic factor (GDNF) for gene delivery to the chronically dopamine (DA)-depleted striatum, in order to test whether long-term GDNF support could increase the cell survival, fiber density innervation and thereby also the functional benefits from intrastriatal VM grafts (Georgievska et al., EJN 20(11):3121-3130, 2004). Grafting of VM cells into the GDNF-overexpressing striatum provided a 2-fold increase in the number of surviving DA neurons at 4 weeks, however, the initially protected cells failed to survive at 6 months post-grafting, despite the continued presence of GDNF. In addition, GDNF induced a selective downregulation of tyrosine hydroxylase (TH) in the graft-derived fibers and the GDNF treatment failed to provide any additional recovery in spontaneous motor performance. The aim of the present study was to evaluate whether continuous long-term expression of GDNF could improve the functional efficacy of maturated VM grafts. For this purpose, chronically 6-hydroxydopamine (6-OHDA) lesioned rats first received multiple intrastriatal VM transplants, followed 3 months later by injections of rLV-GDNF into the striatum. Thus, GDNF treatment of the grafts was initiated at 3 months after transplantation, i.e. at a time when the grafts are fully integrated into the host brain. Stereological quantification of the number of TH+ cells in the grafts at 6 months after transplantation (i.e. after 3 months of GDNF exposure) showed a similar cell survival in both groups (GDNF vs. GFP). Striatal fiber innervation was estimated using densitometry analyses on sections immunostained for TH and the vesicular monoamine transporter-2 (VMAT2). In non-grafted lesioned animals the TH+ and VMAT2+ fiber innervation was 10% and 20% of control side, respectively. The TH+ fiber innervation density was lower in the GDNF-treated grafted animals (22% of normal) compared to control (GFP) grafts (39% of normal), whereas the VMAT2+ fiber density was equal in both groups, suggesting a selective downregulation of TH in the graft-derived DA fibers. Behavioral analyses at 6 months after transplantation demonstrated that control grafted animals improved to about 40-60% of normal performance in the stepping and cylinder test. By contrast, GDNF-treated animals failed to show any significant improvement in these spontaneous motor tests. Our data suggest that GDNF treatment of mature DA grafts impairs their ability to provide functional recovery in parkinsonian rats.
[show abstract][hide abstract] ABSTRACT: The present study was designed to analyse whether continuous overexpression of glial cell line-derived neurotrophic factor (GDNF) in the striatum by a recombinant lentiviral vector can provide improved cell survival and additional long-term functional benefits after transplantation of fetal ventral mesencephalic cells in Parkinsonian rats. A four-site intrastriatal 6-hydroxydopamine lesion resulted in an 80-90% depletion of nigral dopamine cells and striatal fiber innervation, leading to stable motor impairments. Histological analysis performed at 4 weeks after grafting into the GDNF-overexpressing striatum revealed a twofold increase in the number of surviving tyrosine hydroxylase (TH)-positive cells, as compared with grafts placed in control (green fluorescent protein-overexpressing) animals. However, in animals that were allowed to survive for 6 months, the numbers of surviving TH-positive cells in the grafts were equal in both groups, suggesting that the cells initially protected at 4 weeks failed to survive despite the continued presence of GDNF. Although cell survival was similar in both grafted groups, the TH-positive fiber innervation density was lower in the GDNF-treated grafted animals (30% of normal) compared with animals with control grafts (55% of normal). The vesicular monoamine transporter-2-positive fiber density in the striatum, by contrast, was equal in both groups, suggesting that long-term GDNF overexpression induced a selective down-regulation of TH in the grafted dopamine neurons. Behavioral analysis in the long-term grafted animals showed that the control grafted animals improved their performance in spontaneous motor behaviors to approximately 50% of normal, whereas the GDNF treatment did not provide any additional recovery.
European Journal of Neuroscience 01/2005; 20(11):3121-30. · 3.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3-11 pg/mg tissue), and doxycycline-induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2-8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.
Human Gene Therapy 11/2004; 15(10):934-44. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effects of continuous glial cell line-derived neurotrophic factor (GDNF) overexpression in the intact nigrostriatal dopamine (DA) system was studied using recombinant lentiviral (rLV) vector delivery of GDNF to the striatum or substantia nigra (SN) in the rat. Intrastriatal delivery of rLV-GDNF resulted in significant overexpression of GDNF in the striatum (2-4 ng/mg tissue) and anterograde transport of GDNF protein to the SN. Striatal rLV-GDNF delivery initially induced an increase in DA turnover (1-6 weeks), accompanied by significant contralateral turning in response to amphetamine, suggesting an enhancement of the DA system on the injected side. Starting 6 weeks after continuous GDNF delivery, we observed a selective downregulation of tyrosine hydroxylase (TH) protein (approximately 70%) that was maintained until the end of the experiment (24 weeks). A similar effect was observed when rLV-GDNF was injected into the SN. The magnitude of TH downregulation was related to the level of GDNF expression and was most pronounced in animals in which the striatal GDNF level exceeded 0.7 ng/mg tissue. The decreased TH protein levels were associated with similar reductions in the in vitro TH enzyme activity (approximately 70%); however, in vivo L-3,4-dihydroxyphenylalanine production rate and DA tissue levels were maintained at normal levels. The results indicate that downregulation of TH protein reflects a compensatory effect in response to continuous GDNF stimulation of the DA neurons mediated by a combination of overactivity at the DA synapse and a direct GDNF-induced action on TH gene expression. This compensatory mechanism is proposed to maintain long-term DA neuron function within the normal range.
Journal of Neuroscience 08/2004; 24(29):6437-45. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability to regulate transgene expression will be crucial for development of gene therapy to the brain. The most commonly used systems are based on a transactivator in combination with a drug, e.g. the tetracycline-regulated system. Here we describe a different method of transgene regulation by the use of the human glial fibrillary acidic protein (GFAP) promoter. We constructed a lentiviral vector that directs transgene expression to astrocytes. Using toxin-induced lesions we investigated to what extent transgene expression could be regulated in accordance with the activation of the endogenous GFAP gene. In animals receiving excitotoxic lesions of the striatum we detected an eightfold increase of green fluorescent protein (GFP)-expressing cells. The vast majority of these cells did not divide, suggesting that the transgene was indeed regulated in a similar fashion as the endogenous GFAP gene. This finding will lead to the development of lentiviral vectors with autoregulatory capacities that may be very useful for gene therapy to the brain.
European Journal of Neuroscience 03/2004; 19(3):761-5. · 3.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Te n years ago, a glial cell line-derived neurotrophic factor (GDNF) that has prominent actions on nigral dopaminergic neurons, both in vitro and in animal models of Parkinson disease (PD), was discov- ered. A recently published open-label clinical trial now reports that long-term intracerebral delivery of GDNF may also markedly improve symptoms in patients with PD. Here we review the experi- mental data underlying the current clinical trial and discuss the types of structural and functional changes induced by GDNF that may pro- vide symptomatic benefit in PD patients. Data obtained in rodent and primate models of PD highlight the importance of how and where the factor is administered, supporting the view that GDNF has to be delivered locally in the brain parenchyma, at the receptive target site, to provide therapeutic benefit in PD patients. The cardinal symptoms of PD, including a difficulty in initiating movement, slowness of movement and stiffness and shaking at rest, are to a large extent caused by the progressive degeneration of the dopamine-producing neurons in the substantia nigra. Nigral cell loss proceeds over many years, during the early symptomatic stage, during manifest PD and during severe, end-stage disease. At the onset of dis- ease, about 50% of dopaminergic neurons have been lost, and there is on average a further loss of 45% within the first decade, accompanied by a profound striatal dopaminergic denervation. It is this slow and pro- tracted degenerative process that creates opportunities for disease inter- vention, such as blocking nigral cell loss and promoting recovery by improved function—and possibly by inducing regeneration and sprout- ing—of the surviving nigral dopaminergic neurons. Results obtained in animal models of PD indicate that GDNF may possess the desired prop- erties to be used as a disease-modifying therapeutic factor for PD. Neurotrophic factors, by virtue of their neuroprotective properties, have attracted considerable interest as potential therapeutic agents in neurodegenerative diseases. Attempts to apply these factors clinically, however, have so far been disappointing because of their poor efficacy and induction of troublesome side effects. In these clinical trials, the recombinant protein was delivered either systemically or into the cerebrospinal fluid (intraventricularly or intrathecally) in patients suffering from amyotrophic lateral sclerosis, peripheral neuropathy, PD or Alzheimer disease 1,2 .R esults from these studies indicate that the neurotrophic factors, whose receptors are widely distributed, are prone to inducing pronounced side effects when delivered by these routes. The poor penetration across the blood-brain barrier, as well as the limited passage of proteins from the cerebrospinal fluid into the brain tissue, has made it necessary to administer the factors at doses that are likely to induce side effects. These effects may not be so evi- dent in small-sized experimental animals. For this reason, they may have gone unnoticed in the preclinical studies and may have become apparent in some cases only at the phase II/III stage of the clinical trails when larger numbers of patients were included. The therapeutic value of neurotrophic-factor delivery, therefore, may not be possible to achieve unless the factors are delivered locally at the receptive target sites within the central nervous system. Steven Gill and collaborators 3 have, for the first time, tested this mode of delivery in patients with advanced PD using continuous intracere- bral infusion of GDNF. Although quite promising, the results of this initial open-label trial should be interpreted cautiously because the study was based on a small number of patients who were monitored over a relatively short follow-up period. Nevertheless, the data reported indicate that pronounced clinical benefit, in the absence of any serious side effects, may be possible to obtain by GDNF using intrastriatal delivery. Neuroprotective effects of GDNF in animal models of PD
[show abstract][hide abstract] ABSTRACT: Sustained neurotrophic factor treatment in neurodegenerative disorders such as Parkinson's disease is likely to affect both degenerating and intact neurons. To investigate the effect of long-term glial cell line-derived neurotrophic factor (GDNF) overexpression on intact nigrostriatal dopamine neurons, we injected a recombinant lentiviral vector encoding GDNF, or green fluorescent protein, in the right striatum of young adult rats. Thirteen months after viral injection GDNF levels were 4.5 ng/mg tissue in the striatum and 0.9 ng/mg in the substantia nigra as measured by ELISA, representing a 25-100-fold increase above control vector- or nontransduced tissue. GDNF overexpression significantly reduced tyrosine hydroxylase mRNA levels (by 39-72%) in the substantia nigra and ventral tegmental area neurons, and the optical density of tyrosine hydroxylase-immunoreactive innervation in the striatum was reduced by 25-52% with the most prominent reductions appearing caudally. No significant reduction was seen in striatal vesicular monoamine transporter 2-immunoreactivity or [3H]mazindole binding autoradiography to dopamine uptake sites, two other presynaptic markers in dopamine axon terminals. The striatal D1 and D2 receptor binding as determined by [3H]SCH23390 and [3H]spiperone binding, respectively, was unaltered relative to the intact side in both treatment groups. Preproenkephalin mRNA levels in postsynaptic striatal neurons, which increase upon removal of striatal dopamine, were also unaffected by the GDNF treatment. Taken together our findings indicate that sustained GDNF administration to intact nigrostriatal dopamine neurons selectively reduces tyrosine hydroxylase expression, without altering striatal dopamine transmission to the extent that compensatory changes in several other components related to dopamine storage and signalling occur.
European Journal of Neuroscience 02/2003; 17(2):260-70. · 3.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effects of sustained (up to 9 months) striatal overexpression of glial cell line derived neurotrophic factor (GDNF) on lesioned nigrostriatal dopamine (DA) neurons was studied using a recombinant lentiviral (rLV) vector to deliver GDNF into the striatum 4 weeks prior to the creation of an intrastriatal 6-hydroxydopamine lesion. The results of the amphetamine-induced rotation suggested an initial partial protection followed by a complete recovery, whereas the spontaneous motor behaviors remained impaired. There was a clear protection of the nigral tyrosine hydroxylase (TH)-positive neurons in the rLV-GDNF group compared to rats injected with the control vector encoding green fluorescent protein (GFP) (70 and 20% of the intact side, respectively). However, the striatal TH+ fiber density was equally reduced (to 20% of the intact side) in both groups. Further morphological analyses indicated that the nigrostriatal projections of the DA neurons were indeed preserved in the GDNF group. The axonal projections were visualized using two independent methods: First, retrograde labeling of the nigral cell bodies by intrastriatal Fluoro-Gold injections showed that the majority of rescued cells in the GDNF group had preserved axonal projections to striatum. Second, injections of a recombinant adeno-associated viral vector expressing GFP into the nigra was used to anterogradely fill the DA neurons and their projections with GFP protein. GFP immunostaining clearly demonstrated that the fibers of the nigral DA cells were preserved along the nigrostriatal pathway and innervated large parts of the striatum, but did not express TH at detectable levels. In addition, fiber sprouting was observed in the globus pallidus, entopeduncular nucleus, and substantia nigra, corresponding to areas where GDNF protein was released. The lack of functional recovery in the spontaneous motor behaviors may, at least in part, be explained by this extensive aberrant fiber sprouting in the downstream striatal target nuclei and/or decreased synthesis of dopamine in the striatum.
[show abstract][hide abstract] ABSTRACT: Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol/mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6-hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.
Proceedings of the National Academy of Sciences 05/2002; 99(7):4708-13. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: We used a recombinant lentiviral vector (rLV) for gene delivery of GDNF to the striatum, and assessed its neuroprotective effects in the intrastriatal 6-hydroxydopamine (6-OHDA) lesion model. The level of GDNF expression obtained with the rLV-GDNF vector was dose-related and ranged between 0.9-9.3 ng/mg tissue in the transduced striatum, as determined by ELISA, and 0.2-3.0 ng/mg tissue were detected in the ipsilateral substantia nigra (SN), due to anterograde transport of the GDNF protein. GDNF expression was apparent at 4 days and maintained for > 8 months after injection. Striatal delivery of rLV-GDNF efficiently protected the nigral dopamine (DA) neurons and their projection, against the 6-OHDA lesion (65-77% of intact side). Sprouting of the lesioned axons was observed along the nigrostriatal pathway, precisely corresponding to the areas containing anterogradely transported GDNF.
[show abstract][hide abstract] ABSTRACT: Here we studied the effects of glial cell line-derived neurotrophic factor (GDNF) in a rat model that represents the symptomatic stages of Parkinson's disease. GDNF was infused starting 2 weeks after an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in order to halt the ongoing degeneration of the nigrostriatal dopaminergic neurons. GDNF or vehicle was infused in the striatum or the lateral ventricle via an osmotic minipump over a total 4-week period (2-6 weeks postlesion). Motor function was evaluated by the stepping, paw reaching and drug-induced motor asymmetry tests before the pump infusion was initiated, and was repeated once during (5 weeks postlesion) and twice after the withdrawal of the minipumps (7 and 11 weeks postlesion). We found that within two weeks following the lesion approximately 40% of the nigral TH-positive neurons were lost. In the vehicle infusion groups there was an additional 20% cell loss between 2 and 12 weeks after the lesion. This latter cell loss occurred mainly in the caudal part of the SN whereas the cell loss in the rostral SN was almost complete within the first two weeks. Ventricular GDNF infusion completely blocked the late degenerating neurons in the caudal SN and had long lasting behavioural effects on the stepping test and amphetamine rotation, extending to 6 weeks after withdrawal of the factor. Striatal infusion affected the motor behaviour transiently during the infusion period but the motor performance of these animals returned to baseline upon cessation of the GDNF delivery, and the delayed nigral cell loss was marginally affected. We conclude that intraventricular GDNF can successfully block the already initiated degenerative process in the substantia nigra, and that the effects achieved via the striatal route, when GDNF is given acutely after the lesion, diminish as the fibre terminal degeneration proceeds.
European Journal of Neuroscience 05/2001; 13(8):1589-99. · 3.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.
Brain Research 01/2001; 886(1-2):82-98. · 2.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson’s disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3–6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson’s disease.
Brain Research - BRAIN RES. 01/2000; 886(1):82-98.