Faculty Bibliography

Gene transfer techniques have been explored as therapeutic modalities and neurobiologic tools to understand the role of various genes in animal models of Parkinson's disease. The gene for tyrosine hydroxylase, the rate-limiting step of dopamine synthesis, has been transferred into animal models by viral vectors or by implantable cells that have been modified by retrovirus vectors. The role of additional genes such as GTP cyclohydrolase 1 and aromatic L-amino acid decarboxylase in optimal delivery of dopamine in animal models is reviewed. Gene therapy also allows goals beyond replacement of dopamine. Neurotrophic factors such as brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor can be introduced to promote sprouting of neurites and protect the dopaminergic neurons from degeneration. Genes involved in apoptosis, free radical scavenger pathway, or other cell death mechanism could also be used to prevent the degeneration of the neurons. Current technology of gene therapy is limited in its long-term expression and ability to regulate the gene expression. However, recent developments provide better understanding of these limitations and suggest potential solutions to these technical hurdles.

Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic L-amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic L-amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic L-amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. L-DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic L-amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic L-amino acid decarboxylase into the 6-hydroxydopamine-denervated rat striatum. Grafts containing aromatic L-amino acid decarboxylase produced less L-DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic L-amino acid decarboxylase near striatal grafts producing L-DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.

An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1-methyl-4-phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1-methyl-4-phenylpyridinium toxicity, although 1-methyl-4-phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1-methyl-4-phenylpyridinium to cause the selective death of dopaminergic neurons.

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of the dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although various treatments are successfully used to alleviate the symptoms of PD, none of them prevents or halts the neurodegenerative process of the disease. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family of proteins, supports the survival and the differentiation of dopaminergic neurons. BDNF also prevents the death of dopaminergic neurons in vitro, which suggests that it may be of possible use in the development of neuroprotective therapies for PD. To determine whether BDNF is neuroprotective for SNpc dopaminergic neurons in the adult brain, we used a rat model of PD in which degeneration of 60-70% of these neurons was induced by an intrastriatal injection of 6-hydroxydopamine (6-OHDA). We report here that intrastriatal grafts of fibroblasts genetically engineered to produce BDNF partially prevent the loss of nerve terminals and completely prevent the loss of cell bodies of the nigrostriatal dopaminergic pathway that is induced by the intrastriatal injection of 6-OHDA. In contrast, the implantation of control fibroblasts that did not produce BDNF failed to protect nerve terminals and cell bodies against 6-OHDA-induced damage. Our observation that grafts of BDNF-producing fibroblasts protect against 6-OHDA-induced degeneration of SNpc dopaminergic neurons in the adult rat brain opens new perspectives for treatments aimed at the prevention of neurodegeneration in PD, using gene therapy and neurotrophic factors such as BDNF.

Local delivery of brain-derived neurotrophic factor (BDNF) by genetically modified cells provides the unique opportunity to examine the effects of BDNF on adult dopaminergic and cholinergic neurons in vivo. Primary rat fibroblasts were genetically engineered to produce BDNF. Conditioned media from BDNF-transduced fibroblasts supported embryonic chick dorsal root ganglion neurons as well as rat fetal mesencephalic neurons. BDNF-transduced fibroblasts grafted to the rat brain survived and showed continued mRNA production for at least 2 weeks. The effects of BDNF-transduced fibroblast grafts on the dopaminergic and cholinergic systems were then assessed. BDNF-transduced fibroblasts grafted into the normal intact substantia nigra induced sprouting of tyrosine hydroxylase- and neurofilament-immunoreactive fibers into the graft. Fibroblast grafts implanted into the normal intact striatum and midbrain as well as the 6-hydroxydopamine-lesioned brain did not induce sprouting of dopaminergic fibers; neither did they affect drug-induced rotational behavior. BDNF-transduced fibroblasts did, however, significantly increase the homovanillic acid/dopamine ratio when grafted into the normal midbrain. Following transection of the fimbriafornix, BDNF-transduced fibroblasts grafted into the septum were unable to rescue the septal cholinergic population, as did nerve growth factor-producing fibroblast grafts. Genetically modified fibroblast grafts may provide an effective, localized method of BDNF delivery in vivo to test biological effects of this factor on the central nervous system.