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Neuromodulation by spinal cord stimulation has been proposed as a symptomatic treatment for Parkinson's disease. We tested the chronic effects of spinal cord stimulation in a progressive model of Parkinson's based on overexpression of alpha-synuclein in the substantia nigra. Adult Sprague Dawley rats received unilateral injections of adeno-associated virus serotype 6 (AAV6) in the substantia nigra to express alpha-synuclein. Locomotion and forepaw use of the rats were evaluated during the next 10 weeks. Starting on week 6, a group of AAV6-injected rats received spinal cord stimulation once a week. At the end of the experiment, tyrosine hydroxylase and alpha-synuclein immunostaining were performed. Rats with unilateral alpha-synuclein expression showed a significant decrease in the use of the contralateral forepaw, which was mildly but significantly reverted by spinal cord stimulation applied once a week from the 6th to the 10th week after the AAV6 injection. Long-term spinal cord stimulation proved to be effective to suppress or delay motor symptoms in a sustained and progressive model of Parkinson's and might become an alternative, less invasive neuromodulation option to treat this disease.

Accumulation and misfolding of the alpha-synuclein protein are core mechanisms in the pathogenesis of Parkinson's disease. While the normal function of alpha-synuclein is mainly related to the control of vesicular neurotransmission, its pathogenic effects are linked to various cellular functions, which include mitochondrial activity, as well as proteasome and autophagic degradation of proteins. Remarkably, these functions are also affected when the renewal of macromolecules and organelles becomes impaired during the normal aging process. As aging is considered a major risk factor for Parkinson's disease, it is critical to explore its molecular and cellular implications in the context of the alpha-synuclein pathology. Here, we discuss similarities and differences between normal brain aging and Parkinson's disease, with a particular emphasis on the nigral dopaminergic neurons, which appear to be selectively vulnerable to the combined effects of alpha-synuclein and aging.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

An increase in α-synuclein levels due to gene duplications/triplications or impaired degradation is sufficient to trigger its aggregation and cause familial Parkinson disease (PD). Therefore, lowering α-synuclein levels represents a viable therapeutic strategy for the treatment of PD and related synucleinopathies. Here, we report that Polo-like kinase 2 (PLK2), an enzyme up-regulated in synucleinopathy-diseased brains, interacts with, phosphorylates and enhances α-synuclein autophagic degradation in a kinase activity-dependent manner. PLK2-mediated degradation of α-synuclein requires both phosphorylation at S129 and PLK2/α-synuclein complex formation. In a rat genetic model of PD, PLK2 overexpression reduces intraneuronal human α-synuclein accumulation, suppresses dopaminergic neurodegeneration, and reverses hemiparkinsonian motor impairments induced by α-synuclein overexpression. This PLK2-mediated neuroprotective effect is also dependent on PLK2 activity and α-synuclein phosphorylation. Collectively, our findings demonstrate that PLK2 is a previously undescribed regulator of α-synuclein turnover and that modulating its kinase activity could be a viable target for the treatment of synucleinopathies.

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. While sporadic in the majority of cases, PD-linked dominant mutations in the α-synuclein and LRRK-2 genes, and recessive mutations in the parkin, DJ-1 and PINK-1 genes have been identified in PD families in recent years. In this review we describe viral animal models for PD, i.e. models that are based on PD-associated mutations, and have been generated by viral delivery of the respective disease genes to the substantia nigra of rodents and non-human primates. To date, viral PD models comprise α-synuclein and LRRK-2-based overexpression models, as well as models that mimic parkin loss of function by overexpression of the parkin substrates Pael-R, CDCrel-1, p38/JTV or synphilin-1. These viral models provide valuable insights into Parkinson disease mechanisms, help to identify therapeutic targets and may contribute to the development of therapeutic approaches., (Copyright © 2011 Elsevier Inc. All rights reserved.)

With the recent development of effective gene delivery systems, gene therapy for the central nervous system is finding novel applications. Here, we review existing viral vectors and discuss gene therapy strategies that have been proposed for Parkinson's disease. To date, most of the clinical trials were based on viral vectors to deliver therapeutic transgenes to neurons within the basal ganglia. Initial trials used genes to relieve the major motor symptoms caused by nigrostriatal degeneration. Although these new genetic approaches still need to prove more effective than existing symptomatic treatments, there is a need for disease-modifying strategies. The investigation of the genetic factors implicated in Parkinson's disease is providing precious insights in disease pathology that, combined with innovative gene delivery systems, will hopefully offer novel opportunities for gene therapy interventions to slow down, or even halt disease progression.

Several lines of evidence suggest that phosphorylation of α-synuclein (α-syn) at S87 or S129 may play an important role in regulating its aggregation, fibrillogenesis, Lewy body formation, and neurotoxicity in vivo. However, whether phosphorylation at these residues enhances or protects against α-syn toxicity in vivo remains unknown. In this study, we investigated the cellular and behavioral effect of overexpression of wild-type (WT), S87A, and S87E α-syn to block or to mimic S87 phosphorylation, respectively, in the substantia nigra of Wistar rats using recombinant adeno-associated vectors. Our results revealed that WT and S87A overexpression induced α-syn aggregation, loss of dopaminergic neurons, and fiber pathology. These neuropathological effects correlated well with the induction of hemi-parkinsonian motor symptoms. Strikingly, overexpression of the phosphomimic mutant S87E did not show any toxic effect on dopaminergic neurons and resulted in significantly less α-syn aggregates, dystrophic fibers, and motor impairment. Together, our data demonstrate, for the first time, that mimicking phosphorylation at S87 inhibits α-syn aggregation and protects against α-syn-induced toxicity in vivo, suggesting that phosphorylation at this residue would play an important role in controlling α-syn neuropathology. In addition, our results provide strong evidence for a direct correlation between α-syn-induced neurotoxicity, fiber pathology, and motor impairment and the extent of α-syn aggregation in vivo, suggesting that lowering α-syn levels and/or blocking its aggregation are viable therapeutic strategies for the treatment of Parkinson's disease and related synucleinopathies.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common genetic cause of Parkinson's disease (PD), accounting for a significant proportion of both autosomal dominant familial and sporadic PD cases. Our aim in the present study is to generate a mammalian model of mutant G2019S LRRK2 pathogenesis, which reproduces the robust nigral neurodegeneration characteristic of PD. We developed adenoviral vectors to drive neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in the nigrostriatal system of adult rats. Wild-type LRRK2 did not induce any significant neuronal loss. In contrast, under the same conditions and levels of expression, G2019S mutant LRRK2 causes a progressive degeneration of nigral dopaminergic neurons. Our data provide a novel rat model of PD, based on a prevalent genetic cause, that reproduces a cardinal feature of the disease within a rapid time frame suitable for testing of neuroprotective strategies.

Although the overabundance of human alpha-synuclein in nigral dopaminergic neurons is considered to play a pathogenic role in Parkinson's disease (PD), it remains unclear how alpha-synuclein leads to neuronal degeneration and motor symptoms. Here, we explored the effect of human alpha-synuclein in the rat substantia nigra following AAV-mediated gene delivery inducing a moderate loss of dopaminergic neurons together with motor impairments. A significant fraction of the surviving nigral neurons were found to express human αSyn and displayed a pathological fragmentation of the Golgi apparatus. This observation prompted further investigation on the role of the secretory pathway, in particular at the ER/Golgi level, in alpha-synuclein toxicity. To address this question, we co-expressed human alpha-synuclein with Rab1A, a regulator of ER-to-Golgi vesicular trafficking, and found a significant reduction of Golgi fragmentation. Rab1A did not protect the dopaminergic neurons from the alpha-synuclein-induced degeneration that occurred within several months following vector injection. However, we observed in animals co-expressing Rab1A an improvement of motor behavior that correlates with the rescue of normal Golgi morphology in alpha-synuclein-expressing dopaminergic neurons. The non-prenylable mutant Rab1A-DeltaCC did not produce any of the effects observed with the wild-type form of Rab1A, linking the protective role of Rab1A with its activity in ER-to-Golgi vesicular trafficking. In conclusion, Rab1A can rescue the Golgi fragmentation caused by the overabundance of alpha-synuclein in nigral dopaminergic neurons, improving the ability of the surviving neurons to control motor function in hemiparkinsonian animals.

We explored the relationship between ubiquitin proteasome system (UPS) and lysosomal markers and the formation of alpha-synuclein (alpha-syn) inclusions in nigral neurons in Parkinson disease (PD). Lysosome Associated Membrane Protein 1(LAMP1), Cathepsin D (CatD), and Heat Shock Protein73 (HSP73) immunoreactivity were significantly decreased within PD nigral neurons when compared to age-matched controls. This decrease was significantly greater in nigral neurons that contained alpha-syn inclusions. Immunoreactivity for 20S proteasome was similarly reduced in PD nigral neurons, but only in cells that contained inclusions. In aged control brains, there is staining for alpha-syn protein, but it is non-aggregated and there is no difference in LAMP1, CatD, HSP73 or 20S proteasome immunoreactivity between alpha-syn positive or negative neuromelanin-laden nigral neurons. Targeting over-expression of mutant human alpha-syn in the rat substantia nigra using viral vectors revealed that lysosomal and proteasomal markers were significantly decreased in the neurons that displayed alpha-syn-ir inclusions. These findings suggest that alpha-syn aggregation is a key feature associated with decline of proteasome and lysosome and support the hypothesis that cell degeneration in PD involves proteosomal and lysosomal dysfunction, impaired protein clearance, and protein accumulation and aggregation leading to cell death.

Loss of function of parkin, an ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). Parkin-associated endothelin receptor-like receptor (Pael-R) was identified as an authentic substrate of parkin and is thought to accumulate abnormally following loss of parkin activity, causing neurodegeneration of nigral dopaminergic neurons in AR-JP patients. Our aim is therefore to generate a model of AR-JP through overexpression of Pael-R in the nigrostriatal system of adult rats. Using recombinant adeno-associated virus pseudotyped with the serotype 6 capsid (rAAV2/6) as a gene delivery tool, we achieved targeted and robust overexpression of rat Pael-R in nigral neurons and their striatal terminals. Overexpressed Pael-R was shown to accumulate very rapidly in an insoluble form. Accumulation of the receptor triggered a rapid and severe loss of nigral neurons and nigrostriatal fibers and terminals. No cell recovery was observed for up to 6 months post-injection. GABAergic neurons of the globus pallidus were unaffected by Pael-R overexpression, highlighting the selective vulnerability of nigral dopaminergic neurons to abnormal levels of Pael-R. Pael-R-induced degeneration also led to a depletion of striatal dopamine resulting in spontaneous motor impairments, as measured in the cylinder and stepping tests for forelimb akinesia. Interestingly, behavioral deficits of individual animals were correlated with the extent of the nigrostriatal lesion. Insoluble accumulation of Pael-R in the nigrostriatal system of adult rats represents, therefore, a chronic, rapidly progressing and specific model of AR-JP, which recapitulates major pathological hallmarks of the disease.

Phosphorylation is involved in numerous neurodegenerative diseases. In particular, alpha-synuclein is extensively phosphorylated in aggregates in patients suffering from synucleinopathies. However, the share of this modification in the events that lead to the conversion of alpha-synuclein to aggregated toxic species needed to be clarified. The rat model that we developed through rAAV2/6-mediated expression of alpha-synuclein demonstrates a correlation between neurodegeneration and formation of small filamentous alpha-synuclein aggregates. A mutation preventing phosphorylation (S129A) significantly increases alpha-synuclein toxicity and leads to enhanced formation of beta-sheet-rich, proteinase K-resistant aggregates, increased affinity for intracellular membranes, a disarrayed network of neurofilaments and enhanced alpha-synuclein nuclear localization. The expression of a mutation mimicking phosphorylation (S129D) does not lead to dopaminergic cell loss. Nevertheless, fewer but larger aggregates are formed, and signals of apoptosis are also activated in rats expressing the phosphorylation-mimicking form of alpha-synuclein. These observations strongly suggest that phosphorylation does not play an active role in the accumulation of cytotoxic pre-inclusion aggregates. Unexpectedly, the study also demonstrates that constitutive expression of phosphorylation-mimicking forms of alpha-synuclein does not protect from neurodegeneration. The role of phosphorylation at Serine 129 in the early phase of Parkinson's disease is examined, which brings new perspective to therapeutic approaches focusing on the modulation of kinases/phosphatases activity to control alpha-synuclein toxicity.

Parkinson disease (PD) is characterized by dopaminergic neurodegeneration and intracellular inclusions of alpha-synuclein amyloid fibers, which are stable and difficult to dissolve. Whether inclusions are neuroprotective or pathological remains controversial, because prefibrillar oligomers may be more toxic than amyloid inclusions. Thus, whether therapies should target inclusions, preamyloid oligomers, or both is a critically important issue. In yeast, the protein-remodeling factor Hsp104 cooperates with Hsp70 and Hsp40 to dissolve and reactivate aggregated proteins. Metazoans, however, have no Hsp104 ortholog. Here we introduced Hsp104 into a rat PD model. Remarkably, Hsp104 reduced formation of phosphorylated alpha-synuclein inclusions and prevented nigrostriatal dopaminergic neurodegeneration induced by PD-linked alpha-synuclein (A30P). An in vitro assay employing pure proteins revealed that Hsp104 prevented fibrillization of alpha-synuclein and PD-linked variants (A30P, A53T, E46K). Hsp104 coupled ATP hydrolysis to the disassembly of preamyloid oligomers and amyloid fibers composed of alpha-synuclein. Furthermore, the mammalian Hsp70 and Hsp40 chaperones, Hsc70 and Hdj2, enhanced alpha-synuclein fiber disassembly by Hsp104. Hsp104 likely protects dopaminergic neurons by antagonizing toxic alpha-synuclein assemblies and might have therapeutic potential for PD and other neurodegenerative amyloidoses.

The involvement of alpha-synuclein in familial forms of Parkinson's disease suggests a potential causative role in the pathogenesis. We have explored the possibility of generating animal models of Parkinson's disease by overexpressing alpha-synuclein in the nigrostriatal pathway using viral vectors. Both lentiviral and adeno-associated vectors efficiently transduce dopaminergic neurons in the substantia nigra, and transgenic expression of alpha-synuclein leads to the progressive loss of neurons positive for dopaminergic markers, with the formation of intraneuronal alpha-synuclein aggregates. With a high tropism for nigral dopaminergic neurons, adeno-associated vectors allow for the monitoring of dopaminergic function using spontaneous and drug-induced behaviour. We propose that virus-based rodent alpha-synuclein models provide a valuable approach for the preclinical testing of therapeutics.

Reactive oxygen species are considered to contribute to the pathogenesis of Parkinson's disease (PD). In order to study viral vector-mediated overexpression of the antioxidant enzyme glutathione peroxidase (GPX) as a potential neuroprotective approach in both an in vitro and in vivo model of PD, we have developed a lentiviral vector carrying the human GPX1 gene. Neuroblastoma cells infected with this vector showed a 2-fold increase in GPX activity compared to cells infected with a control vector. In addition, overexpression of GPX protected 83.0 +/- 14.2% of these cells against 6-hydroxydopamine (6-OHDA)-induced toxicity, while only 22.9 +/- 4.6% of the cells infected with a control vector survived. Furthermore, lentivirus-mediated expression of GPX1 in nigral dopaminergic neurons in vivo prior to intrastriatal injection of 6-OHDA led to a small, but significant protection of these cells against drug-induced toxicity. These results indicate that antioxidative gene therapy strategies may be relevant for PD.

Parkinson's disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic system. Brain delivery of glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore the dopaminergic pathway in various animal models of PD. However, GDNF overexpression in the dopaminergic pathway leads to a time-dependent down-regulation of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis. In order to elucidate GDNF-mediated biochemical effects on dopaminergic neurons, we overexpressed GDNF in the intact rat striatum using a lentiviral vector-mediated gene transfer technique. Long-term GDNF overexpression led to increased GTP cyclohydrolase I (GTPCH I) activity and tetrahydrobiopterin (BH4) levels. Further, we observed a down-regulation of TH enzyme activity in morphologically intact striatal dopaminergic nerve terminals, as well as a significant decrease of dopamine levels in striatal tissue samples. These results indicate that long-term GDNF delivery is a major factor affecting dopamine biosynthesis via a direct or indirect modulation of TH and GTPCH I and further underscore the importance of assessing both GDNF dose and delivery duration prior to clinical application in order to circumvent potentially adverse pharmacological effects on the biosynthesis of dopamine.

Parkinson's disease (PD) is characterized by a progressive loss of midbrain dopamine neurons and the presence of cytoplasmic inclusions called Lewy bodies. Mutations in several genes including alpha-synuclein and parkin have been linked to familial PD. The loss of parkin's E3-ligase activity leads to dopaminergic neuronal degeneration in early-onset autosomal recessive juvenile parkinsonism, suggesting a key role of parkin for dopamine neuron survival. To evaluate the potential neuroprotective role of parkin in the pathogenesis of PD, we tested whether overexpression of wild-type rat parkin could protect against the toxicity of mutated human A30P alpha-synuclein in a rat lentiviral model of PD. Animals overexpressing parkin showed significant reductions in alpha-synuclein-induced neuropathology, including preservation of tyrosine hydroxylase-positive cell bodies in the substantia nigra and sparing of tyrosine hydroxylase-positive nerve terminals in the striatum. The parkin-mediated neuroprotection was associated with an increase in hyperphosphorylated alpha-synuclein inclusions, suggesting a key role for parkin in the genesis of Lewy bodies. These results indicate that parkin gene therapy may represent a promising candidate treatment for PD.

Viral delivery of glial cell line-derived neurotrophic factor (GDNF) currently represents one of the most promising neuroprotective strategies for Parkinson's Disease (PD). However, the effect of this neurotrophic factor has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. In this study, we evaluated the ability of lentiviral vectors coding for GDNF (lenti-GDNF) to prevent nigral dopaminergic degeneration associated with the lentiviral mediated expression of the A30P mutant human alpha-synuclein (lenti-A30P). This virally based rat model develops a progressive and selective loss of dopamine neurons associated with the appearance of alpha-synuclein containing inclusions, thus recapitulating the major hallmarks of PD. Lenti-GDNF was injected in the substantia nigra 2 weeks before nigral administration of lenti-A30P. Although a robust expression of GDNF was observed in the whole nigrostriatal pathway due to retrograde and/or anterograde transport, lenti-GDNF did not prevent the alpha-synuclein-induced dopaminergic neurodegeneration in the lentiviral-based genetic rat model of PD. These results suggest that sustained GDNF treatment cannot modulate the cellular toxicity related to abnormal folded protein accumulation as mutated human alpha-synuclein.

Parkinson disease (PD) is characterized by the progressive degeneration of substantia nigra dopaminergic neurons projecting to the striatum. Since the deficit in striatal dopamine is the main cause of PD symptoms, it appears critical to preserve axon terminals. Significant axon protection from peripheral nerve Wallerian degeneration is observed in Wlds mice, a phenotype conferred by a spontaneous dominant mutation. To assess any Wlds-mediated rescue of dopamine fibers in a PD model, the nigrostriatal pathway of Wlds mice was lesioned with 6-hydroxydopamine (6-OHDA), a catecholaminergic neurotoxin. Following 6-OHDA injection in the medial forebrain bundle, Wlds mice showed remarkable dopamine fiber protection in the striatum. Drug-induced rotational behavior confirmed the nigrostriatal fiber ability to release dopamine, although revealing an abnormal neurotransmitter control presumably due to disrupted axonal transport. Following 6-OHDA injection in the midstriatum, only a protection trend was observed. Strikingly, no protection of Wlds nigral dopaminergic cell bodies was obtained following either nigrostriatal lesion. Besides showing subtle differences in the degeneration process between subcellular compartments, the reported Wlds-mediated protection of the dopamine axon terminals in an animal model of PD may lead to the understanding of mechanisms underlying axon loss and to the development of new therapeutic approaches.

Parkinson's disease (PD) is characterized by the progressive loss of substantia nigra dopaminergic neurons and the presence of cytoplasmic inclusions named Lewy bodies. Two missense mutations of the alpha-synuclein (alpha-syn; A30P and A53T) have been described in several families with an autosomal dominant form of PD. alpha-Syn also constitutes one of the main components of Lewy bodies in sporadic cases of PD. To develop an animal model of PD, lentiviral vectors expressing different human or rat forms of alpha-syn were injected into the substantia nigra of rats. In contrast to transgenic mice models, a selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum was observed in animals expressing either wild-type or mutant forms of human alpha-syn. This neuronal degeneration correlates with the appearance of abundant alpha-syn-positive inclusions and extensive neuritic pathology detected with both alpha-syn and silver staining. Lentiviral-mediated expression of wild-type or mutated forms of human alpha-syn recapitulates the essential neuropathological features of PD. Rat alpha-syn similarly leads to protein aggregation but without cell loss, suggesting that inclusions are not the primary cause of cell degeneration in PD. Viral-mediated genetic models may contribute to elucidate the mechanism of alpha-syn-induced cell death and allow the screening of candidate therapeutic molecules.

The primate striatum contains tyrosine hydroxylase (TH)-immunoreactive (ir) neurons, the numbers of which are augmented after dopamine depletion. Glial cell line-derived neurotrophic factor (GDNF) strongly modulates the viability and phenotypic expression of dopamine ventral mesencephalic neurons. The effect of GDNF on TH-ir neurons intrinsic to the striatum has yet to be investigated. In the present study, stereological counts of TH-ir striatal neurons in aged and parkinsonian nonhuman primates revealed that GDNF delivered via a lentiviral vector (lenti-) further increased the number of these cells. Aged monkeys treated with lenti-GDNF displayed an eightfold increase in TH-ir neurons relative to lenti-beta-galactosidase-treated monkeys. Unilateral 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine treatment alone in young monkeys resulted in a bilateral eightfold increase in TH-ir striatal cells. This effect was further magnified sevenfold on the side of lenti-GDNF treatment. These cells colocalized with the neuronal marker neuronal-specific nuclear protein. Some of these cells colocalized with GDNF-ir, indicating that an alteration in phenotype may occur by the direct actions of this trophic factor. Thus, GDNF may mediate plasticity in the dopamine-depleted primate brain, which may serve to compensate for cell loss by converting striatal neurons to a dopaminergic phenotype.

Lentiviral delivery of glial cell line-derived neurotrophic factor (lenti-GDNF) was tested for its trophic effects upon degenerating nigrostriatal neurons in nonhuman primate models of Parkinson's disease (PD). We injected lenti-GDNF into the striatum and substantia nigra of nonlesioned aged rhesus monkeys or young adult rhesus monkeys treated 1 week prior with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Extensive GDNF expression with anterograde and retrograde transport was seen in all animals. In aged monkeys, lenti-GDNF augmented dopaminergic function. In MPTP-treated monkeys, lenti-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Additionally, lenti-GDNF injections to intact rhesus monkeys revealed long-term gene expression (8 months). In MPTP-treated monkeys, lenti-GDNF treatment reversed motor deficits in a hand-reach task. These data indicate that GDNF delivery using a lentiviral vector system can prevent nigrostriatal degeneration and induce regeneration in primate models of PD and might be a viable therapeutic strategy for PD patients.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor disturbances caused by an alteration of the dopaminergic nigrostriatal system. Current symptomatic treatments for PD include dopaminergic drug administration, deep brain stimulation, ablative surgery and fetal cell transplantation. Though these approaches have significant beneficial effects, they are hampered by limiting side-effects, but more importantly they do not change the disease progression. Alternative restorative and neuroprotective strategies have therefore to be considered. Neuroprotective effects of neurotrophic factors, anti-apoptotic and antioxidant molecules are currently being investigated for this purpose. Among neurotrophic molecules, the potential of the glial cell line-derived neurotrophic factor (GDNF) to protect the nigral dopaminergic neurons and/or rescue striatal dopamine levels has been extensively documented. For GDNF to become a clinical reality, appropriate delivery techniques will have to be developed. This chapter focuses on the potential of encapsulated cells and viral vectors to locally release neurotrophic factors in experimental models of PD.

Parkinson's disease, a neurologic disorder characterized by a dopamine deficit within the striatum, may be improved by transplantation of polymer encapsulated neurosecretory cells. Surrounding cells with a selectively permeable barrier offers several advantages, including preventing immune rejection and tumor formation while allowing functional efficacy. Bovine adrenal medullary chromaffin cells, or PC12 cells, a rat-derived pheochromocytoma cell line, were encapsulated within polyelectrolyte-based microcapsules or thermoplastic-based macrocapsules. Histologic and biochemical analyses revealed that both types of cells survived and that neurotransmitter release from capsules was sustained for several months in vitro, irrespective of the encapsulation method employed. Both of the encapsulation systems protected the enclosed cells from an immunologic challenge in vitro, and prevented immune rejection when cell containing capsules were implanted in an immunologically incompatible host. Chromaffin or PC12 cell containing capsules implanted into the dopamine (DA) depleted striatum of rats reduced the lesion associated functional deficit. These results suggest that encapsulated neurosecretory cell implants may be useful in treating various central nervous system (CNS) deficits, particularly in cases involving a specific neurochemical lesion.

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)