Faculty Bibliography

Deficits in forepaw adjusting steps in rats have been proposed as a non-drug-induced model of the akinesia associated with Parkinson's disease. The present study examined the relationship between contralateral forepaw adjusting steps and dopamine depletion after medial forebrain bundle lesions with 6-hydroxydopamine. Depletion of striatal dopamine by >80% resulted in dramatic reductions in the ability of rats to make adjusting steps, but rats with < 80% dopamine depletion had no detectable deficit. The deficit in forepaw adjusting steps was evident by three days after lesions and did not recover for up to 13 weeks. Compared to apomorphine-induced rotation, the deficit in adjusting steps was evident at milder dopamine depletion. Discrete striatal lesions were also utilized to localize the striatal subregions that mediate forepaw adjusting steps. Forepaw adjusting steps were reduced after lesions of dorsolateral, ventrolateral or ventrocentral striatum, but not after lesions of dorsomedial, dorsocentral or ventromedial striatum. The reductions in adjusting steps after the discrete striatal lesions were not as severe as after medial forebrain bundle lesions. Furthermore, none of the discrete striatal lesions resulted in rotation after apomorphine administration, although a few resulted in increase in amphetamine-induced rotation. Administration of L-3,4-dihydroxyphenylalanine partially reversed the reductions of forepaw adjusting steps in both sets of lesion experiments. Together, these results suggest that forepaw adjusting step deficits in the rat provide a good model for the akinesia of Parkinson's disease both in medial forebrain bundle and striatal lesions, and would be a useful tool for investigating the efficacy of various therapeutic strategies.

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.

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.