Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.     

Effect of AdGDNF on dopaminergic neurotransmission in the striatum of 6-OHDA-treated rats

We have previously observed that the delivery of an adenoviral vector encoding for glial cell line-derived neurotrophic factor (AdGDNF) into the substantia nigra (SN) 7 days after intrastriatal administration of 6-hydroxydopamine (6-OHDA) protects dopamine (DA)-dependent behaviors, tyrosine hydroxylase immunoreactive (TH+) cells in SN, and amphetamine-induced c-fos induction in striatum. In the present study, we sought to determine if the behavioral protection observed in 6-OHDA-treated rats receiving AdGDNF was associated with an increase in DA availability in the striatum as measured by microdialysis. Rats received intrastriatal 6-OHDA (16 microg/2.8 microl) or vehicle followed 7 days later by intranigral AdGDNF (3.2x10(7) pfu/2 microl), AdLacZ (3.2 x 10(7) pfu/2 microl), or phosphate buffered saline (PBS). Three weeks later, microdialysis samples were collected from the same striatal region under basal conditions, following KCl (100 mM) or amphetamine (250 microM) administered via the striatal microdialysis probe, or amphetamine administered systemically (6.8 mg/kg i.p). Animals given 6-OHDA followed by either PBS or AdLacZ showed a decrease in basal extracellular striatal DA levels to 24% of control. In contrast, basal extracellular DA in 6-OHDA-lesioned rats with a nigral injection of AdGDNF was almost 3-fold higher than 6-OHDA-vehicle treated animals, 65% of control DA levels. Moreover, although KCl and amphetamine produced no increase in striatal DA release in 6-OHDA-treated rats that subsequently were given either PBS or AdLacZ, these manipulations increased DA levels significantly in 6-OHDA-treated rats later given AdGDNF. Thus, DA neurotransmission within the striatum of 6-OHDA treated rats appears to be enhanced by increased expression of GDNF in the nigra.     

Behavioral and Cellular Protection of Rat Dopaminergic Neurons by an Adenoviral Vector Encoding Glial Cell Line-Derived Neurotrophic Factor

Previously, we observed that an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF), injected near the rat substantia nigra (SN), protects SN dopaminergic (DA) neuronal soma from 6-hydroxydopamine (6-OHDA)-induced degeneration. In the present study, the effects of Ad GDNF injected into the striatum, the site of DA nerve terminals, were assessed in the same lesion model. So that effects on cell survival could be assessed without relying on DA phenotypic markers, fluorogold (FG) was infused bilaterally into striatae to retrogradely label DA neurons. Ad GDNF or control treatment (Ad mGDNF, encoding a deletion mutant GDNF, Ad lacZ, vehicle, or no injection) was injected unilaterally into the striatum near one FG site. Progressive degeneration of DA neurons was initiated 7 days later by unilateral injection of 6-OHDA at this FG site. At 42 days after 6-OHDA, Ad GDNF prevented the death of 40% of susceptible DA neurons that projected to the lesion site. Ad GDNF prevented the development of behavioral asymmetries which depend on striatal dopamine, including limb use asymmetries during spontaneous movements along vertical surfaces and amphetamine-induced rotation. Both behavioral asymmetries were exhibited by control-treated, lesioned rats. Interestingly, these behavioral protections occurred in the absence of an increase in the density of DA nerve fibers in the striatum of Ad GDNF-treated rats. ELISA measurements of transgene proteins showed that nanogram quantities of GDNF and lacZ transgene were present in the striatum for 7 weeks, and picogram quantities of GDNF in the SN due to retrograde transport of vector and/or transgene protein. These studies demonstrate that Ad GDNF can sustain increased levels of biosynthesized GDNF in the terminal region of DA neurons for at least 7 weeks and that this GDNF slows the degeneration of DA neurons and prevents the appearance of dopamine dependent motor asymmetries in a rat model of Parkinson's disease (PD). GDNF gene therapy targeted to the striatum, a more surgically accessible site than the SN, may be clinically applicable to humans with PD.     

Fetal intra-nigral ventral mesencephalon and kidney tissue bridge transplantation restores the nigrostriatal dopamine pathway in hemi-parkinsonian rats

We have previously demonstrated that intranigral transplantation of fetal ventral mesencephalic (VM) tissue and nigrostriatal administration of glial cell line-derived neurotrophic factor (GDNF) restores striatal dopamine input in hemiparkinsonian rats. Since it has been found that GDNF is highly expressed in fetal kidney, we examined the possibility that fetal kidney tissue may provide trophic support, similar to GDNF, to an intranigral dopamine (DA) transplant and restore the nigrostriatal pathway. Adult Sprague-Dawley rats were anesthetized and unilaterally injected with 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. Completeness of the lesion was evaluated by measuring amphetamine-induced rotation. One month after 6-OHDA lesioning, fetal VM cells were grafted into the lesioned nigral area followed by transplantation of fetal kidney tissue or vehicle along a pathway from nigra to striatum. Animals receiving these transplants showed a significant decrease both in amphetamine-induced rotation and in postural asymmetry 1 to 3 months after grafting. Immunocytochemical studies demonstrated tyrosine hydroxylase (TH) positive fiber tracts in the lesioned striatum. Control animals that received vehicle injection after the intranigral graft or no transplantation showed no alterations in amphetamine-induced turning and no TH-positive fibers in the lesioned striatum. These results indicate that combinations of fetal nigral and kidney transplants may restore the nigrostriatal DA pathway in Parkinsonian rats. As fetal kidney contains a variety of trophic proteins, it may provide a synergistic admixture to optimally promote DA fiber outgrowth.     

Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration

Previous studies have demonstrated that either the neurotrophin glial-derived neurotrophic factor (GDNF) or the antiapoptotic peptide Bcl-2 delivered into striatum by a viral vector protects dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In this study we used recombinant, replication-incompetent, genomic herpes simplex virus-based vectors to deliver the genes coding for Bcl-2 and GDNF into rat substantia nigra (SN) 1 week prior to 6-OHDA injection into the striatum. Vector-mediated expression of either Bcl-2 or GDNF alone each resulted in a doubling in cell survival as measured by retrograde labeling with fluorogold (FG) and a 50% increase in tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the lesioned SN compared to the unlesioned side. Gene transfer of Bcl-2 and GDNF were equivalent in this effect. Coadministration of the Bcl-2-expressing vector with the GDNF-expressing vector improved the survival of lesioned SN neurons as measured by FG labeling by 33% and by the expression of TH-IR by 15%. These results suggest that the two factors delivered together act in an additive fashion to improve DA cell survival in the face of 6-OHDA toxicity.     

Differential expression of GDNF, BDNF, and NT-3 in the aging nigrostriatal system following a neurotoxic lesion

Protein levels for brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell line-derived neurotrophic factor (GDNF) were measured in the striatum and ventral midbrain of young and aged Brown Norway/F344 F1 (F344BNF(1)) hybrid rats following a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway. At 2 weeks post-lesion, protein levels of BDNF and GDNF were higher in the denervated striatum when compared to the intact striatum for young (4-5 months old) but not old (31-33 months old) rats. Interestingly, in old rats BDNF protein in the denervated striatum was significantly lower than that measured in the intact striatum. At the same time point BDNF protein levels in the ventral midbrain were higher on the lesioned versus intact side for both young and old rats while no significant side differences were detected for GDNF protein in the ventral midbrain of young or old rats. No significant differences in NT-3 protein levels were detected between the lesioned and intact sides for striatal or ventral midbrain regions in either young or old brain. While no significant age effects were detected for BDNF or NT-3 protein, young rats showed higher GDNF protein levels in both the striatum (lesioned or intact) and ventral midbrain (lesioned or intact) than old rats. These data show that two endogenous neurotrophic factors, BDNF and GDNF, are differentially affected by a 6-OHDA lesion in the aging nigrostriatal system with young brain showing a significant compensatory increase of these two factors in the denervated striatum while no compensatory increase is observed in aged brain.     

Neuroprotection in the rat Parkinson model by intrastriatal GDNF gene transfer using a lentiviral vector

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.     

Bilateral effects of unilateral GDNF administration on dopamine- and GABA-regulating proteins in the rat nigrostriatal system

Dopamine (DA) affects GABA neuronal function in the striatum and together these neurotransmitters play a large role in locomotor function. We recently reported that unilateral striatal administration of GDNF, a growth factor that has neurotrophic effects on DA neurons and enhances DA release, bilaterally increased striatal neuron activity related to locomotion in aged rats. We hypothesized that the GDNF enhancement of DA function and resulting bilateral enhancement of striatal neuronal activity was due to prolonged bilateral changes in DA- and GABA-regulating proteins. Therefore in these studies we assessed dopamine- and GABA-regulating proteins in the striatum and substantia nigra (SN) of 24 month old Fischer 344 rats, 30 days after a single unilateral striatal delivery of GDNF. The nigrostriatal proteins investigated were the DA transporter (DAT), tyrosine hydroxylase (TH), and TH phosphorylation and were examined by blot-immunolabeling. The striatal GABA neuron-related proteins were examined by assay of the DA D₁ receptor, DARPP-32, DARPP-32 Thr³⁴ phosphorylation, and glutamic acid decarboxylase (GAD). Bilateral effects of GDNF on TH and DAT occurred only in the SN, as 30 μg GDNF increased ser19 phosphorylation, and 100 μg GDNF decreased DAT and TH protein levels. GDNF also produced bilateral changes in GAD protein in the striatum. A decrease in DARPP-32 occurred in the ipsilateral striatum, while increased D₁ receptor and DARPP-32 phosphorylation occurred in the contralateral striatum. The 30 μg GDNF infusion into the lateral striatum was confined to the ipsilateral striatum and substantia nigra. Thus, long-lasting bilateral effects of GDNF on proteins regulating DA and GABA neuronal function likely alter physiological properties in neurons, some with bilateral projections, associated with locomotion. Enhanced nigrostriatal excitability and DA release by GDNF may trigger these bilateral effects.     

Aberrant sprouting and downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer

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.     

Adenoviral vector-mediated GDNF gene therapy in a rodent lesion model of late stage Parkinson's disease

A recombinant adenoviral vector encoding the human glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was used to express the neurotrophic factor GDNF in the unilaterally 6-hydroxydopamine (6-OHDA) denervated substantia nigra (SN) of adult rats ten weeks following the 6-OHDA injection. 6-OHDA lesions significantly increased apomorphine-induced (contralateral) rotations and reduced striatal and nigral dopamine (DA) levels by 99% and 70%, respectively. Ad-GDNF significantly (P<0.01) decreased (by 30–40%) apomorphine-induced rotations in lesioned rats for up to two weeks following a single injection. Locomotor activity, assessed 7 days following the Ad-GDNF injection, was also significantly (P<0.05) increased (by 300–400%). Two weeks after the Ad-GDNF injection, locomotor activity was still significantly increased compared to the Ad-β-gal-injected 6-OHDA lesioned (control) group. Additionally, in Ad-GDNF-injected rats, there was a significant decrease (10–13%) in weight gain which persisted for approximately two weeks following the injection. Consisitent with the behavioral changes, levels of DA and the metabolite dihydroxyphenylacetic acid (DOPAC) were elevated (by 98% and 65%, respectively) in the SN, but not the striatum of Ad-GDNF-injected rats. Overall, a single Ad-GDNF injection had significant effects for 2–3 weeks following administration. These results suggest that virally delivered GDNF promotes the recovery of nigral dopaminergic tone (i.e.: increased DA and DOPAC levels) and improves behavioral performance (i.e.: decreased rotations, increased locomotion) in rodents with extensive nigrostriatal dopaminergic denervation. Moreover, our results suggest that viral delivery of trophic factors may be used eventually to treat neurodegenerative diseases such as Parkinson's disease.     

Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson's disease after administration into the striatum or the lateral ventricle

Both glial cell line-derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to exert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6-hydroxydopamine (6-OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6-OHDA injection. GDNF provided almost complete (90-92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion-induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6-OHDA-induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine-induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF-treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth-stimulating agent in the IS 6-OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.     

Increased dopamine release from striata of rats after unilateral nigrostriatal bundle damage

Dopaminergic control of striatal neurons is retained in rats sustaining lesions of the nigrostriatal bundle (NSB) as long as 10% of the projection remains, suggesting that enhanced efficiency of dopamine (DA) transmission may compensate for the denervation of the striatum. To examine this hypothesis we have studied the extracellular concentration of striatal DA using brain dialysis. In control rats, haloperidol (1 mg/kg, i.p.) or depolarization of striatal tissue with 25 mM KCl increased, and gamma-butyrolactone (500 mg/kg, i.p.) decreased DA and homovanillic acid (HVA) levels in striatal dialysates. Three weeks after unilateral injection of 6-hydroxydopamine (6-OHDA) to substantia nigra, DA content in the ipsilateral striatum was decreased by 60-98%. Nevertheless, extracellular DA concentration in the lesioned striata remained unchanged in rats with 60-90% DA depletions. More extensive lesions (96% DA depletion) were accompanied by 60% reduction in DA release. In contrast, extracellular HVA levels in the lesioned striata decreased proportionally to the depletion of tissue DA, indicating decreased inactivation of extracellular DA. We propose that the capacity of the residual DA terminals to maintain normal levels of extracellular DA after 60-90% NSB lesions may serve to compensate for the partial denervation of the striatal tissue. Disruption of striatal DA functions and postsynaptic supersensitivity after more extensive lesions may be associated with the failure of the NSB to fully compensate for loss of DA terminals. In striata contralateral to the 6-OHDA lesions, increased DA release was also observed. In addition, 60-90% ipsilateral DA depletions were accompanied by 32% and 42% increases in DA and HVA content in contralateral tissue, respectively. The possibility of the contralateral sprouting of DA terminals is discussed.     

The CRF-like peptide urocortin produces a long-lasting recovery in rats made hemiparkinsonian by 6-hydroxydopamine or lipopolysaccharide

We have recently observed that the corticotropin releasing factor related peptide urocortin (UCN) reverses key features of nigrostriatal neurodegeneration following intracerebral injection of either 6-hydroxydopamine (6-OHDA) or lipopolysaccharide (LPS). To determine the potential therapeutic utility of UCN here we have studied whether these effects are sustained for several weeks following peptide injection. In addition we have studied whether UCN still shows efficacy in rats with more pronounced nigrostriatal lesions. Rats were lesioned using 6-OHDA or LPS and injected with UCN either 7 or 14 days later. At different time points animals were tested for rotational behaviour (apomorphine, 0.5 mg/kg) and subsequently implanted with bilateral dialysis probes into the striata. The following day rats were dialysed to estimate extracellular striatal dopamine (DA) and then sacrificed for estimation of striatal tissue DA and subsequent immunohistochemistry of TH(+) cells in the substantia nigra (SN). Toxin treated rats given UCN 7 days later showed clear evidence of reduced nigrostriatal damage both 28 and 84 days following UCN compared with saline injection. In rats given UCN 14 days after toxin injection, by which time deficits were maximal, a restoration of nigrostriatal damage was observed. This suggests that UCN is able to elicit a sustained restoration of functional nigrostriatal integrity and has the ability to produce a recovery in severely lesioned rats. These findings suggest that stimulation of CRF (probably CRF(1)) receptors could have therapeutic utility in PD.     

Single administration of GDNF into the striatum induced protection and repair of the nigrostriatal dopaminergic system in the intrastriatal 6-hydroxydopamine injection model of hemiparkinsonism

Purpose: Neurotrophic factor delivery into the brain is a promising approach in the treatment of Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent neurotrophic factors for dopaminergic neurons. Although multiple injections of GDNF into the brain are commonly performed in experimental studies, the present study investigates the efficacy of using a single injection of GDNF, which may be useful in elinically applying this treatment. Methods: Unilateral 6-hydroxydoparnine (6-OHDA) administration into the striatum was perforrned in Sprague-Dawley rats to create a partial lesion of the nigrostriatal DA system. These parkinsonian model rats received a single injection of human recombinant GDNF into the same portion of the striatum either 24 h before or 4 weeks after 6-OHDA treatrnent. Results: GDNF injected into the striatum before 6-OHDA administration potently protected the dopaminergic system, as shown by the numbers of mesencephalic dopaminergie neuron cell bodies and dopaminergic nerve terminal densities in the striatum. Dopaminergic neuron cell bodies and fiber densities were also significantly restored when GDNF was given after 6-OHDA administration, although the degree of restoration was lower than in the protective experiment. ODNF administration ameliorated apomorphine-induced rotational behavior in animals receiving it either before or after 6-OHDA treatment. However, the degree of improvement was less prominent when GDNF was iniected after 6-OHDA. Conclusion: Intracerebral GDNF adininistration exerts both protective and regenerative effects on the nigrostriatal dopaminergic system, a finding which may have implications for the development of new treatment strategies for Parkinson's disease.     

1,25-Dihydroxyvitamin D3 administration to 6-hydroxydopamine-lesioned rats increases glial cell line-derived neurotrophic factor and partially restores tyrosine hydroxylase expression in substantia nigra and striatum

It has previously been demonstrated that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] administration, whether in cell cultures or in vivo to rats, increases glial cell line-derived neurotrophic factor (GDNF) expression levels, suggesting that this hormone may have beneficial effects in neurodegenerative disorders. This study was carried out to explore the effects of 1,25(OH)(2)D(3) administration in a 6-OHDA-lesioned rat model of Parkinson's disease on GDNF and tyrosine hydroxylase (TH) expression in substantia nigra (SN) and striatum. Two groups of animals received 1,25(OH)(2)D(3) intraperitoneally, the first group 7 days before the unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) and the second group 21 days (days 21-28) after the unilateral injection of 6-OHDA. Animals of both groups were sacrificed on day 28. In addition, two other groups received a unilateral injection of either saline or 6-OHDA into the MFB. Rats were killed, and the SN and striatum were then removed for GDNF and TH determination. Striatal GDNF protein expression was increased on the ipsilateral with respect to the contralateral side after 6-OHDA injection alone as well as in 1,25(OH)(2)D(3)-treated rats before or after 6-OHDA administration. As expected, 6-OHDA injection induced an ipsilateral decrease in TH-immunopositive neuronal cell bodies and axonal terminals in the SN and striatum. However, treatment with 1,25(OH)(2)D(3) before and after 6-OHDA injection partially restored TH expression in SN. These data suggest that 1,25(OH)(2)D(3) may help to prevent dopaminergic neuron damage.     

Single administration of GDNF into the striatum induced protection and repair of the nigrostriatal dopaminergic system in the intrastriatal 6-hydroxydopamine injection model of hemiparkinsonism

Purpose: Neurotrophic factor delivery into the brain is a promising approach in the treatment of Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent neurotrophic factors for dopaminergic neurons. Although multiple injections of GDNF into the brain are commonly performed in experimental studies, the present study investigates the efficacy of using a single injection of GDNF, which may be useful in elinically applying this treatment. Methods: Unilateral 6-hydroxydoparnine (6-OHDA) administration into the striatum was perforrned in Sprague-Dawley rats to create a partial lesion of the nigrostriatal DA system. These parkinsonian model rats received a single injection of human recombinant GDNF into the same portion of the striatum either 24 h before or 4 weeks after 6-OHDA treatrnent. Results: GDNF injected into the striatum before 6-OHDA administration potently protected the dopaminergic system, as shown by the numbers of mesencephalic dopaminergie neuron cell bodies and dopaminergic nerve terminal densities in the striatum. Dopaminergic neuron cell bodies and fiber densities were also significantly restored when GDNF was given after 6-OHDA administration, although the degree of restoration was lower than in the protective experiment. ODNF administration ameliorated apomorphine-induced rotational behavior in animals receiving it either before or after 6-OHDA treatment. However, the degree of improvement was less prominent when GDNF was iniected after 6-OHDA. Conclusion: Intracerebral GDNF adininistration exerts both protective and regenerative effects on the nigrostriatal dopaminergic system, a finding which may have implications for the development of new treatment strategies for Parkinson's disease.     

Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system.

Previous studies have used recombinant adeno-associated viral (rAAV) vectors to deliver glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra to protect the nigral dopamine (DA) neurons from 6-hydroxydopamine-induced damage. However, no regeneration or functional recovery was observed in these experiments. Here, we have used an rAAV-GDNF vector to express GDNF long-term (6 months) in either the nigral DA neurons themselves, in the striatal target cells, or in both of these structures. The results demonstrate that both nigral and striatal transduction provide significant protection of nigral DA neurons against the toxin-induced degeneration. However, only the rats receiving rAAV-GDNF in the striatum displayed behavioral recovery, accompanied by significant reinnervation of the lesioned striatum, which developed gradually over the first 4-5 months after the lesion. GDNF transgene expression was maintained at high levels throughout this period. These results provide evidence that rAAV is a highly efficient vector system for long-term expression of therapeutic proteins in the nigrostriatal system.     

Delayed gene therapy of glial cell line-derived neurotrophic factor is efficacious in a rat model of Parkinson's disease

Gene transfer of glial cell line-derived neurotrophic factor (GDNF) in rodent models of Parkinson's disease (PD) has been shown to protect against neurodegeneration either prior to or immediately after neurotoxin-induced lesions; however, the nigrostriatal pathway was largely intact when gene delivery was completed in these models, which may not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, replication-incompetent adenoviral vectors encoding the rat GDNF gene were administered into the striatum 4 weeks following 6-hydroxydopamine (6-OHDA) injection in the unilateral striatum, more closely resembling fully developed PD. Apomorphine-induced rotational behavior testing was performed every week following 6-OHDA injection. At the 10th week after gene transfer, the striatal dopamine concentrations were measured by HPLC with an electrochemical detector and the number of tyrosine hydroxylase (TH)-positive dopamine neurons in the substantia nigra (SN) was determined by immunohistochemistry. Injection of 6-OHDA into the striatum produced stable increases in rotation, which reached a plateau between 4 and 5 weeks post-injection. The number of TH-positive neuron in the SN and dopamine levels in the striatum was significantly lower in the 6-OHDA group compared to the normal group. Gene transfer of GDNF, but not beta-galactosidase, significantly increased the number of TH-positive neurons and dopamine levels, with a subsequent behavioral recovery between 5 and 10 weeks following GDNF transduction. These findings demonstrate that adenovirus-mediated gene transfer of GDNF is efficacious even in the late stages of 6-OHDA-induced PD rats. They also provide further evidence on the effectiveness of GDNF-based gene therapy for experimental Parkinson's disease.     

Sequential administration of GDNF into the substantia nigra and striatum promotes dopamine neuron survival and axonal sprouting but not striatal reinnervation or functional recovery in the partial 6-OHDA lesion model

Glial cell line-derived neurotrophic factor (GDNF) has prominent survival-promoting effects on lesioned nigrostriatal dopamine neurons, but understanding of the conditions under which functional recovery can be obtained remains to be acquired. We report here the time course of nigrostriatal axon degeneration in the partial lesion model of Parkinson's disease and the morphological and functional effects of sequential administration of GDNF in the substantia nigra (SN) and striatum during the first 5 weeks postlesion. By 1 day postlesion, the nigrostriatal axons had retracted back to the level of the caudal globus pallidus. Over the next 6 days axonal retraction progressed down to the SN, and during the following 7 weeks 74% of tyrosine hydroxylase-positive (TH(+)) and 84% of retrogradely labeled nigral neurons were lost, with a more pronounced loss in the rostral part of the SN. GDNF administration protected 70 and 72% of the nigral TH(+) and retrogradely labeled cell bodies, respectively, but did not prevent the die-back of the lesioned nigrostriatal axons. Although clear signs of sprouting were observed close to the injection site in the striatum as well as in the globus pallidus, the overall DA innervation of the striatum [as measured by [(3)H]-N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine-binding autoradiography] was not improved by the GDNF treatment. Moreover, the lesion-induced deficits in forelimb akinesia and drug-induced rotation were not attenuated. We conclude that functional recovery in the partial lesion model depends not only on preservation of the nigral cell bodies, but more critically on the ability of GDNF to promote significant reinnervation of the denervated striatum.     

Biochemical and behavioral effects of serotonin neurotoxins on the nigrostriatal dopamine system: Comparison of injection sites

Unilateral injections of the serotonin neurotoxin, 5,7-dihydroxytryptamine (DHT), at various points along the 5-HT pathway to the forebrain produce a turning syndrome associated with alterations of dopamine synthesis in the ipsilateral striatum. Unilateral injections of DHT into the SN produced an ipsilateral increase in striatal dopamine (DA) turnover and contralateral rotation in response to amphetamine or apomorphine. Injection of DHT into the MFB produced an ipsilateral decrease in striatal DA turnover and tyrosine hydroxylase (TOH) activity, and ipsilateral rotation in response to amphetamine or apomorphine. After the injection of DHT into the SN or MFB, there was a significant correlation between the rates of drug-induced rotation, the decrease in cortical 5-HT turnover, and the change in striatal DA turnover, suggesting that the unilateral change in DA turnover (and, presumably, the increased stimulation of DA receptors) is causally linked to turning. Injection of DHT into the zones of the striatum and GP richest in 5-HT terminals produced the same responses as the MFB-lesioned rats: ipsilateral rotation and a decrease in striatal TOH activity. Injection of DHT into the area of the striatum richest in DA terminals failed to produce rotation or a significant change in TOH activity. We suggest that 5-HT neurons from the raphe nuclei exert a tonic inhibition on the nigrostriatal pathway at the level of the SN through direct synapses on DA neurons, whereas their neostriatal terminals have an indirect effect on DA terminals, perhaps via interaction with cholinergic and GABA-ergic neurons.