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.
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 ϳ85% 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.
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 (Ϸ70%) 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 (Ϸ70%); 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.
Multiple system atrophy (MSA) is a rare and fatal α-synucleinopathy characterized by a distinctive oligodendrogliopathy with glial cytoplasmic inclusions and associated neuronal multisystem degeneration. The majority of patients presents with a rapidly progressive parkinsonian disorder and atypical features such as early autonomic failure and cerebellar ataxia. We have previously reported that complete MSA pathology can be modeled in transgenic mice overexpressing oligodendroglial α-synuclein under conditions of oxidative stress induced by 3-nitropropionic acid (3-NP) including striatonigral degeneration, olivopontocerebellar atrophy, astrogliosis, and microglial activation. Here, we show that myeloperoxidase (MPO), a key enzyme involved in the production of reactive oxygen species by phagocytic cells, is expressed in both human and mouse MSA brains. We also demonstrate that in the MSA mouse model, MPO inhibition reduces motor impairment and rescues vulnerable neurons in striatum, substantia nigra pars compacta, cerebellar cortex, pontine nuclei, and inferior olives. MPO inhibition is associated with suppression of microglial activation but does not affect 3-NP induced astrogliosis in the same regions. Finally, MPO inhibition results in reduced intracellular aggregates of α-synuclein. This study suggests that MPO inhibition may represent a novel candidate treatment strategy against MSA-like neurodegeneration acting through its anti-inflammatory and anti-oxidative properties.
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 pmol͞mg 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. R estoration of striatal dopamine by peripheral administration of L-dopa can provide efficient symptomatic relief in patients with Parkinson's disease (PD). However, the therapeutic efficacy of L-dopa treatment diminishes over time, concomitant with the appearance of on-off fluctuations and drug-induced dyskinesia. These complications may be caused, at least in part, by the intermittent, pulsatile supply of L-dopa provided by the standard oral L-dopa medication. Indeed, there is considerable evidence that these side effects can be reduced greatly if L-dopa is administered at a constant level, and continuous L-dopa delivery may provide a more physiological stimulation of denervated striatal dopamine receptors (1, 2). One promising delivery method is via ex vivo or in vivo gene transfer of the L-dopasynthesizing enzyme, tyrosine hydroxylase (TH). This approach has the additional advantage of local site-specific L-dopa delivery in striatum where the dopamine deficiency is most prominent.Studies in rodent PD models have shown that this approach is feasible by either implantation of cells engineered to produce L-dopa (3-9) or direct injection of TH-expressing viral vectors into the denervated striatum (10-14). However, expression of TH alone may not be sufficient. L-Dopa synthesis by the transduced TH enzyme critically depends on the availability of its cofactor, tetrahydrobiopterin (BH 4 ), and it has bee...
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