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.     

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.     

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.     

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.     

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.     

Quantitative analyses of GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the nigrostriatal system reveal bilateral compensatory changes following unilateral 6-OHDA lesions in the rat

Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.     

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.     

Recombinant adeno-associated viral vector (rAAV) delivery of GDNF provides protection against 6-OHDA lesion in the common marmoset monkey (Callithrix jacchus)

Glial cell line-derived neurotrophic factor (GDNF) has shown potential as a treatment for Parkinson's disease. Recombinant adeno-associated viral vectors expressing the GDNF protein (rAAV-GDNF) have been used in rodent models of Parkinson's disease to promote functional regeneration after 6-OHDA lesions of the nigrostriatal system. The goal of the present study was to assess the anatomical and functional efficacy of rAAV-GDNF in the common marmoset monkey (Callithrix jacchus). rAAV-GDNF was injected into the striatum and substantia nigra 4 weeks prior to a unilateral 6-OHDA lesion of the nigrostriatal bundle. Forty percent of the dopamine cells in the lesioned substantia nigra of the rAAV-GDNF-treated monkeys survived, compared with 21% in the untreated monkeys. Fine dopaminergic fibres were observed microscopically in the injected striatum of some rAAV-GDNF-treated monkeys, suggesting that rAAV-GDNF treatment may have prevented, at least in part, the loss of dopaminergic innervation of the striatum. Protection of dopamine cells and striatal fibre innervation was associated with amelioration of the lesion-induced behavioural deficits. rAAV-GDNF-treated monkeys showed partial or complete protection not only in the amphetamine and apomorphine rotation but also in head position and the parkinsonian disability rating scale. Therefore, our study provides evidence for the behavioural and anatomical efficacy of GDNF delivered via an rAAV vector as a possible treatment for Parkinson's disease.     

Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons

Glial cell-lined derived neurotrophic factor (GDNF) has been shown to promote survival of developing mesencephalic dopaminergic neurons in vitro. In order to determine if there is a positive effect of GDNF on injured adult midbrain dopaminergic neurons in situ, we have carried out experiments in which a single dose of GDNF was injected into the substantia nigra following a unilateral lesion of the nigrostriatal system. Rats were unilaterally lesioned by a single stereotaxic injection of 6-hydroxydopamine (6-OHDA; 9 μg/4 μl normal saline with 0.02% ascorbate) into the medial forebrain bundle and tested weekly for apomorphine-induced (0.05 mg/kg s. c. ) contralateral rotation behavior, Rats that manifested >300 turns/hour received a nigral injection of 100 μg GDNF, or cytochrome C as a control, 4 weeks following the 6-OHDA lesion, Rotation behavior was quantified weekly for 5 weeks after GDNF. Rats were subsequently anesthetized, transcardially perfused, and processed for tyrosine hydroxylase immunohistochemistry. It was found that 100 μg GDNF decreased apomorphine-induced rotational behavior by more than 85%. Immunohistochemical studies revealed that tyrosine hydroxylase immunoreactivity was equally reduced in the striatum ipsilateral to the lesion in both cytochrome C and GDNF-injected animals. In contrast, large increments in tyrosine hydroxylase immunoreactivity were observed in the substantia nigra of animals treated with 100 μg of GDNF, with a significant increase in numbers of tyrosine hydroxylase-immunoreactive cell bodies and neurites as well as a small increase in the cell body area of these neurons. The results suggest that GDNF can maintain the dopaminergic neuronal phenotype in a number of nigral neurons following a unilateral nigrostriatal lesion in the rat.     

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinson's disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.     

Effect of glial cell line-derived neurotrophic factor (GDNF) co-transplantation with fetal ventral mesencephalic cells (VMC) on functional restoration in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson’s disease: neurobehavioral, neurochemical and immunohistochemical studies

Among trophic factors already known, glial cell line-derived neurotrophic factor (GDNF) and other members of its family have potent and specific action on dopaminergic neurons. In the present investigation an attempt has been made to validate the role of GDNF co-transplantation with fetal ventral mesencephalic cells (VMC) on functional viability and restoration using neurobehavioral, neurochemical and immunohistochemical parameters at 6 weeks post-transplantation in 6-hydroxydopamine (6-OHDA) lesioned rat model of Parkinson's disease (PD). A significant restoration (P<0.01) in D-amphetamine induced rotations, spontaneous and apomorphine induced locomotor activity in rats co-transplanted with VMC and GDNF was observed as compared to VMC alone transplanted rats. Level of dopamine (DA), 3,4-dihydroxy-phenyl acetic acid (DOPAC) and dopamine D2 (DA-D2) receptors in the caudate putamen (CPu) were significantly (P<0.001) restored in co-transplanted group as compared to VMC transplanted or GDNF administered animals. The functional viability of transplanted VMC was confirmed by tyrosine hydroxylase (TH) expression and quantification of TH-positive cells by image analysis revealed a significant restoration in TH-IR fibers density as well as TH-IR neurons counts in co-transplanted animals over VMC transplanted animals. Results suggest that co-transplantation of VMC and GDNF may be a better approach towards functional restoration in 6-OHDA lesioned rat model of Parkinson's disease.     

Calretinin-containing axons and neurons are resistant to an intrastriatal 6-hydroxydopamine lesion

Relative preservation of dopaminergic axons in patches and a subcallosal layer was observed in the dorsal, lateral and caudal striatum 4 weeks after intrastriatal injection of 6-hydroxydopamine (6-OHDA), a neurotoxin selective for catecholaminergic neurons. Since calcium binding proteins are reported to provide neuroprotective influence in neurons, differences in the distribution of the calcium binding proteins might be related to the different vulnerabilities of dopaminergic neurons and axons to neurotoxins. To address this possibility, we characterized patches of relatively dense tyrosine hydroxylase-immunoreactive (TH-IR) axons in intrastriatal 6-OHDA lesioned rats, focusing on two calcium binding proteins, calbindin (CB) and calretinin (CR). The patches and subcallosal layer of preserved dopaminergic axons in the striatum of rats lesioned with 6-OHDA contained CR, a 31-kDa calcium-binding protein, but interestingly not CB. Dopaminergic neurons containing CR in the substantia nigra pars compacta (SNpc) were relatively spared compared to those that did not contain CR. Taken together, our data indicate that dopaminergic axons and neurons containing CR in the nigrostriatal pathway are more resistant to 6-OHDA lesion than those that do not contain CR.     

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.     

PD模型中GDNF与星形胶质细胞对黑质DA能神经元的影响

目的探讨星形胶质细胞和胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)在帕金森病(Parkinson's disease,PD)中对多巴胺(dopamine neurons,DA)能神经元损伤的影响。方法成年大鼠右侧前脑侧束注射6羟多巴胺(6-OHDA)制备PD模型。PD模型右侧黑质内注射GDNF,于注射后第6周采用免疫组织化学方法观察星形胶质细胞神经纤维酸性蛋白(glial fibrillary acidic protein,GFAP)以及多巴胺能神经元酪氨酸羟化酶(tyrosine hydroxylasa,TH)的变化。结果模型组、PBS和GDNF组注射侧与非注射侧星形胶质细胞相比,均发现GFAP阳性细胞明显增多,DA能神经元数量明显减少(P<0.05)。GDNF组与模型组相比,发现GFAP阳性细胞明显增多,同时残存的DA能神经元数量有所增加(P<0.05)。结论黑质内注射GDNF可能通过激活的星形胶质细胞保护PD大鼠模型黑质DA能神经元。     

Neuroprotection in a rat Parkinson model by GDNF gene therapy using EIAV vector

Vectors based on lentiviruses are opening up new approaches for the treatment of neurodegenerative diseases. Currently, the equine infectious anaemia virus (EIAV) vector is one of the most attractive gene delivery systems with respect to neuronal tropism. The aim was to validate EIAV-lentiviral vectors as a gene delivery system for neurotrophic factor genes in an animal model of Parkinson's disease. EIAV carrying the glial cell line-derived neurotrophic factor (GDNF) gene was unilaterally injected into rat striatum and above the substantia nigra (SN). One week later, the rats received a 6-OHDA lesion into the ipsilateral striatum. GDNF delivery led to extensive expression of GDNF protein within the striatum. In addition, near complete protection against dopaminergic cell death was observed in the GDNF-treated group.     

Vitamin D(3) attenuates 6-hydroxydopamine-induced neurotoxicity in rats

Previous reports have demonstrated that exogeneous administration of glial cell line-derived neurotrophic factor (GDNF) reduces ventral mesencephalic (VM) dopaminergic (DA) neuron damage induced by 6-hydroxydopamine (6-OHDA) lesioning in rats. Recent studies have shown that 1,25-dihydroxyvitamin D(3) (D3) enhances endogenous GDNF expression in vitro and in vivo. The purpose of present study was to investigate if administration of D3 in vivo and in vitro would protect against 6-OHDA-induced DA neuron injury. Adult male Sprague-Dawley rats were injected daily with D3 or with saline for 8 days and then lesioned unilaterally with 6-OHDA into the medial forebrain bundle. Locomotor activity was measured using automated activity chambers. We found that unilateral 6-OHDA lesioning reduced locomotor activity in saline-pretreated animals. Pretreatment with D3 for 8 days significantly restored locomotor activity in the lesioned animals. All animals were sacrificed for neurochemical analysis 6 weeks after lesioning. We found that 6-OHDA administration significantly reduced dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanilic acid (HVA) levels in the substantia nigra (SN) on the lesioned side in the saline-treated rats. D3 pretreatment protected against 6-OHDA-mediated depletion of DA and its metabolites in SN. Using primary cultures obtained from the VM of rat embryos, we found that 6-OHDA or H(2)O(2) alone caused significant cell death. Pretreatment with D3 (10(-10) M) protected VM neurons against 6-OHDA- or H(2)O(2)-induced cell death in vitro. Taken together, our data indicate that D3 pretreatment attenuates the hypokinesia and DA neuronal toxicity induced by 6-OHDA. Since both H(2)O(2) and 6-OHDA may injure cells via free radical and reactive oxygen species, the neuroprotection seen here may operate via a reversal of such a toxic mechanism.     

A tetracycline-regulated adenovirus encoding dominant-negative caspase-9 is regulated in rat brain and protects against neurotoxin-induced cell death in vitro, but not in vivo

Caspase-9 is a critical downstream effector molecule involved in apoptosis, a cell death process thought to be involved in the demise of dopamine (DA) neurons in the substantia nigra (SN) affected by Parkinson's disease (PD). In this study, we determined that a tetracycline-regulated adenovirus harboring a dominant-negative form of caspase-9 (Casp9DN) and the marker gene, enhanced green fluorescent protein (EGFP), under the control of a bidirectional promoter could each be regulated in vitro and in vivo by doxycycline. We next observed that Casp9DN gene delivery significantly protected against TNFalpha and cycloheximide-induced chromatin condensation in HeLa cells and prevented chromatin condensation and the appearance of the early apoptotic marker annexin V in 6-hydroxydopamine (6-OHDA) treated MN9D cells, a dopaminergic cell line. Effects of Casp9DN on DA neurons in vivo were also assessed. DA neurons were retrogradely labeled with fluorogold (FG) and transduced with Casp9DN and EGFP or EGFP alone. A progressive lesion of DA neurons was induced by striatal injection of 6-OHDA 1 week later. At 2 weeks post-lesion, a morphometric analysis of FG+ neurons in the SN revealed that the mean cell diameter of FG labeled neurons in the Casp9DN group was 8% and 21% larger than the EGFP and PBS groups, respectively (P <0.05). However, there was no difference among the treatment groups in the number of neurons remaining in the lesioned SN. These results suggest that while inhibiting apoptosis at the level of caspase-9 is protective in vitro, it is not protective against 6-OHDA-induced cell death in vivo.     

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.     

Preservation of a functional nigrostriatal dopamine pathway by GDNF in the intrastriatal 6-OHDA lesion model depends on the site of administration of the trophic factor

Here we studied whether glial cell line-derived neurotrophic factor (GDNF), given as a single bolus injection before an intrastriatal 6-hydroxydopamine (6-OHDA) lesion, can protect the nigrostriatal dopamine neurons against the toxin-induced damage and preserve normal motor functions in the lesioned animals. GDNF or vehicle was injected in the striatum (25 microg), substantia nigra (25 microg) or lateral ventricle (50 microg) 6 h before the 6-OHDA lesion (20 microg/3 microL). Motor function was evaluated by the stepping and drug-induced motor asymmetry tests. Lesioned animals given vehicle alone showed a clear ipsilateral-side bias in response to amphetamine (13 turns/min), a moderate contralateral-side bias to apomorphine (4.5 turns/min) and a moderate to severe stepping deficit on the contralateral forepaw (three to four steps, as compared with 11-13 steps on the unimpaired side). Injection of GDNF into the striatum had a significant protective effect both on nigrostriatal function (1-2 turns/min in the rotation tests and seven to eight steps in the stepping test), and the integrity of the nigrostriatal pathway, seen as a protection of both the cell bodies in the substantia nigra and the dopamine innervation in the striatum. Injection of GDNF in the nigra had a protective effect on the nigral cell bodies, but not the striatal innervation, and failed to provide any functional benefit. In contrast, intranigral GDNF had deleterious effects on both the striatal TH-positive fibre density and on drug-induced rotation tests. Intraventricular injection had no effect. We conclude that preservation of normal motor functions in the intrastriatal 6-OHDA lesion model requires protection of striatal terminal innervation, and that this can be achieved by intrastriatal, but not nigral or intraventricular, administration of GDNF.