Neuroprotective potential of pleiotrophin overexpression in the striatonigral pathway compared with overexpression in both the striatonigral and nigrostriatal pathways.

Intrastriatal injection of recombinant adeno-associated viral vector serotype 2/1 (rAAV2/1) to overexpress the neurotrophic factor pleiotrophin (PTN) provides neuroprotection for tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc), increases THir neurite density in the striatum (ST) and reverses functional deficits in forepaw use following 6-hydroxydopamine (6-OHDA) toxic insult. Glial cell line-derived neurotrophic factor (GDNF) gene transfer studies suggest that optimal neuroprotection is dependent on the site of nigrostriatal overexpression. The present study was conducted to determine whether enhanced neuroprotection could be accomplished via simultaneous rAAV2/1 PTN injections into the ST and SN compared with ST injections alone. Rats were unilaterally injected in the ST alone or injected in both the ST and SN with rAAV2/1 expressing either PTN or control vector. Four weeks later, all rats received intrastriatal injections of 6-OHDA. Rats were euthanized 6 or 16 weeks relative to 6-OHDA injection. A novel selective total enumeration method to estimate nigral THir neuron survival was validated to maintain the accuracy of stereological assessment. Long-term nigrostriatal neuroprotection and functional benefits were only observed in rats in which rAAV2/1 PTN was injected into the ST alone. Results suggest that superior preservation of the nigrostriatal system is provided by PTN overexpression delivered to the ST and restricted to the ST and SN pars reticulata and is not improved with overexpression of PTN within SNpc neurons.

LRRK2 mutations in a clinic-based cohort of Parkinson's disease.

In the last decade, major breakthroughs in the understanding of genetic contributions to Parkinson's disease (PD) have been achieved. Recently, mutations in LRRK2, encoding dardarin, have been found to be responsible for an autosomal dominant parkinsonism (OMIM 607060). We screened 311 subjects (cases: n = 202, controls: n = 109) for the three previously reported LRRK2 mutations. Our investigation revealed a sporadic case of PD with a heterozygous mutation G2019S (c.6055G>A). Here, we present the clinical phenotype of this patient and discuss the implications of genetic testing for the G2019S mutation in patients with sporadic PD.

RNA knockdown as a potential therapeutic strategy in Parkinson's disease.

Parkinson's disease is a prevalent progressive degenerative disorder of the elderly. There is a current need for novel therapeutic strategies because the standard levodopa pharmacotherapy is only temporarily efficacious. Recently, there have been some high-profile successful preclinical results obtained in animal models of neurological disorders using small interfering RNAs delivered by viral vectors. RNA interference can theoretically be applied to Parkinson's disease since over-expression of various proteins is known to kill the dopamine neurons of the substantia nigra in animal models and in familial forms of Parkinson's disease. Potential RNA interfering strategies and caveats are discussed in this review.

Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse.

The melanocortin system is involved in hypothalamic regulation of energy homeostasis. The melanocortin-4 receptor (MC4R) has been linked to both obesity and reproductive dysfunction. Deletion of the MC4R from the mouse genome has resulted in phenotypes including adult onset obesity, hyperphagia, and difficulty in reproducing when homozygote parents are bred. Additionally, polymorphisms of the human MC4R have been identified in morbidly obese children and adults. Herein, we have identified that voluntary exercise, provided via the presence of a running wheel, impedes the monogenetic obesity (at 20 weeks of age running wheel housed body weight=31+/-1.8 g versus conventionally housed body weight=41+/-2.3 g, a 25% decrease in body weight p<0.01), hyperphagia (average cumulative food intake is not statistically different than wild type mice housed in running wheel cages), and reproductive dysfunction phenotypes associated with the MC4R knockout mice housed by conventional means. These data demonstrate the novel finding that voluntary exercise at a young age may hinder genetically induced obesity.

Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model.

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.

Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra.

Guanosine triphosphate cyclohydrolase I (GTPCHI) is a critical enzyme in catecholamine function and is rate limiting for the synthesis of the catecholamine co-factor tetrahydrobiopterin. The present study assessed the distribution of GTPCHI immunoreactivity (-ir) within the monkey and human ventral midbrain and determined whether its expression is altered as a function of age. Light and confocal microscopic analyses revealed that young monkeys and humans displayed GTPCHI-ir within melanin-containing and tyrosine-hydroxylase-ir neurons in primate substantia nigra. Stereological counts revealed that there was a 67.4% reduction in GTPCHI-ir neuronal number, a 63.5% reduction in GTPCHI-ir neuronal density, and a 37.6% reduction in neuronal volume in aged monkeys relative to young cohorts. Similar age-related changes were seen in humans, in whom there were significant reductions in the number of GTPCHI-ir nigral neurons in middle age (58.4%) and aged (81.5%) cases relative to young cohorts. The density of GTPCHI-ir neurons within the nigra was similarly reduced in middle-aged (63.0%) and aged (81.8%) cases. In contrast to monkeys, aged humans did not display shrinkage in the volume of GTPCHI-ir nigral neurons. The presence of numerous melanin-positive, but GTPCHI-ir immunonegative, neurons in the aged monkey and human nigra indicates that these decreases represent an age-related phenotypic downregulation of this enzyme and not a loss of neurons per se. These data indicate that there is a dramatic decrease in GTPCHI-ir in nonhuman primates and humans as a function of age and that loss of this enzyme may be partly responsible for the age-related decrease in dopaminergic tone within nigrostriatal systems.

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.

Effect of acute L-Dopa pretreatment on apomorphine-induced rotational behavior in a rat model of Parkinson's disease.

Currently, reduction of apomorphine-induced rotational behavior in the 6-hydroxydopamine (6-OHDA) lesioned rat is the most utilized drug-induced paradigm for assessing functional efficacy in a rat model of Parkinson's disease (PD). Any clinically predictive animal model of PD should include a positive response to l-dopa, the standard pharmacotherapy for PD. However, the acute interaction between L-dopa and apomorphine has never been studied to determine if L-dopa pretreatment could reduce apomorphine-induced rotational behavior in a 6-OHDA lesioned rat. The present study was designed to explore whether, indeed, pretreatment with subrotational doses of L-dopa could inhibit apomorphine-induced rotations. The data indicate that L-dopa significantly reduced apomorphine-induced rotational behavior only at one dose (5.0 mg/kg) for 12 min. Based on these and other data, it is concluded that although the apomorphine-induced rotational paradigm may continue to be utilized as one additional indicator of efficacy in the 6-OHDA rat model of PD, it is not in itself a completely valid functional assay.

The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS.

A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Progress in direct striatal delivery of L-dopa via gene therapy for treatment of Parkinson's disease using recombinant adeno-associated viral vectors.

Viral vectors have recently been used successfully to transfer genes and express different proteins in the brain. This review discusses the requirements to consider human clinical trials in which recombinant adeno-associated virus vectors are used to transfer the genes necessary to produce l-dihydroxyphenylalanine (l-dopa) directly into the striatum of Parkinson's patients. Preclinical data that apply to the criteria defined as prerequisite for clinical trials are discussed. Thus, in animal models using recombinant adeno-associated virus vectors it has been demonstrated that l-dopa can be synthesized in the striatum after in vivo transduction. In addition, these l-dopa levels are sufficient to affect behavior in a dopamine-deficient animal model, the expression is extremely long-lasting, and the ability to transcriptionally regulate tyrosine hydroxylase has been demonstrated but not fully characterized. However, while immune responses to recombinant adeno-associated virus infection in the periphery have been studied, direct assessment of the potential immune response in the brain has not been sufficiently defined. Therefore, the rationale for delivering l-dopa directly to the striatum to treat Parkinson's disease is sound and the preclinical data are promising but all the issues surrounding this strategy are not resolved.

Long-term restoration of striatal L-aromatic amino acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a rodent model of Parkinson's disease.

As a potential treatment for Parkinson's disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinson's disease patients may be an attractive gene therapy strategy.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Nerve growth factor expressed in the medial septum following in vivo gene delivery using a recombinant adeno-associated viral vector protects cholinergic neurons from fimbria-fornix lesion-induced degeneration.

Nerve growth factor (NGF) has been shown to support the survival of axotomized medial septal cholinergic neurons after aspirative lesions of the fimbria-fornix (FF). This survival effect has been achieved utilizing intraventricular and intraparenchymal delivery of the NGF protein. While the use of NGF for the treatment of the cholinergic deficits present in Alzheimer's disease shows promise based on its efficacy in animal models, concerns about side-effects of intraventricular NGF delivery in humans have been raised. In the present study, NGF was delivered directly to the medial septum via a recombinant adeno-associated viral vector (rAAV) encoding the cDNA for human NGF prior to a FF lesion in rats. This rAAV-mediated NGF delivery was shown to significantly attenuate the medial septal cholinergic cell loss observed in animals receiving an equivalent injection of a control rAAV vector.

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.

Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

A third-generation lentivirus vector with a conditional packaging system.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

Regulation of gene expression in vivo following transduction by two separate rAAV vectors.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

In vivo L-DOPA production by genetically modified primary rat fibroblast or 9L gliosarcoma cell grafts via coexpression of GTPcyclohydrolase I with tyrosine hydroxylase.

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.