Faculty Bibliography

Primary skin fibroblasts were genetically modified with catecholamine-synthesizing enzyme genes and studied as potential syngeneic donor cells to supply catecholamines in animal models of Parkinson's disease. Primary skin fibroblasts obtained from inbred Fischer 344 rats were transduced with tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) cDNAs using retroviral vector system. The transduced cells were characterized in vitro by enzymatic assay, immunocytochemistry, and HPLC analysis of catecholamine production and release. Accumulation of high levels of dopamine was detected in the media in a time-dependent manner. Secretion of dopamine and its metabolites appeared to be constitutive without significant storage capacity in vesicles or regulation at the level of secretion. The feasibility of regulating the final dopamine production by the AADC-transduced cells was explored in two ways. First, administration of various doses of the precursor, L-dopa, resulted in a controlled production of dopamine by these cells. Second, coculturing AADC-transduced cells with TH-transduced cells in various proportions allowed control of dopamine production. TH-transduced cells served as an endogenous source of precursor. We propose the use of these cells to study the role of AADC in restoring the dopamine-deficient behavior and to compare the effect of dopamine-producing cells with L-dopa-producing cells either by cografting TH-transduced cells with AADC-transduced cells or by grafting TH-transduced cells alone. The role of AADC in vivo will be assessed in future experiments involving animal models of Parkinson's disease.

Aromatic L-amino acid decarboxylase catalyzes the biosynthesis of the neurotransmitters dopamine and serotonin. This enzyme is also expressed in nonneuronal tissues. Two reported cDNA sequences show that the pheochromocytoma message differs from the liver message only at the 5' untranslated region. We present the complete exonal organization and promoter sequences of the rat gene encoding this enzyme. The rat aromatic L-amino acid decarboxylase gene is composed of two promoters and 16 exons spanning more than 80 kb in the genome. The first exon carries the majority of the 5' untranslated sequence of the liver cDNA, and the second exon carries that of the pheochromocytoma cDNA. In the third exon, there are two alternatively utilized splicing acceptors specific to the first and second exons. Therefore, both alternative promoter usage and alternative splicing are operative for the differential expression of this gene. The sequence of each promoter region shows putative binding sites for octamer factors and AP-2.

The source and site of the DOPA decarboxylation to dopamine in Parkinson's disease (PD) and animal models of PD are controversial. Since most of aromatic L-amino acid decarboxylase (AADC) are lost along with the degenerating dopaminergic neurons, we addressed the possibility that other decarboxylases or a novel protein that is structurally different from AADC decarboxylate L-DOPA in the denervated striatum. Immunotitration of the extracts from the denervated striatum with AADC antibody showed that all activity can be attributed to AADC-immunoreactive protein. We then investigated if there are non-dopaminergic intrinsic striatal neurons that express AADC. No evidence of such neurons was noted by immunocytochemistry and in situ hybridization.

A review of work in the dopaminergic system reveals both progress and controversy. More than 100 papers on intracerebral grafting were published last year. Several groups have published the clinical outcome of fetal substantia nigra implants in Parkinsonian patients and studies suggest that sprouting of dopaminergic fibers in response to grafting procedures ameliorates the behavioral deficits of dopamine-depleted animals. Trophic factors for dopamine neurons have also been identified. In addition, genetically modified cells continue to be developed as an alternative method for delivering molecules to the brain. The speed with which neural grafting has become a therapeutic procedure in Parkinsonian patients continues to stimulate debate.

We used single fiber electromyography (SFEMG) to study 42 patients who had enrolled in a double-blind, placebo-controlled trial undertaken to assess the efficacy of botulinum toxin (BTX) injection of neck muscles to treat torticollis. SFEMG in a limb muscle was performed before treatment, 2, and 12 weeks after injection of placebo or BTX. Before treatment, the mean jitter was 26.8 microsec in patients who were to receive BTX, and 25.7 microsec in the placebo group. Two weeks after injection, mean jitter in the group receiving BTX was 43.6 microsec. In the placebo group, it was 26.5 microsec (P = less than .05). Twelve weeks after injection, mean jitter in the BTX group was 35.5; for the placebo group it was 24.5. Fiber density did not change in any patient during the study. There were no remote clinical effects of BTX. Injection of BTX into muscles affected with focal dystonia is a promising and safe treatment, but there are subclinical effects on uninjected muscles.

Neural transplantation, a mode of cellular replacement, has been used as a therapeutic trial for Parkinson's disease. Studies indicate that tonic release of the metabolites from the graft that can be utilized by the host brain, is likely to be the major mechanism responsible for the therapeutic effect. The use of fetal tissue is complicated by ethical controversy and immunological incompatibility. Autografting adult tissue has not been successful mainly due to poor survival. Genetically engineered cells are promising alternative sources of donor cells. We have investigated the potential of primary skin fibroblasts as donor cells for intracerebral grafting. Primary skin fibroblasts survive in the brain and remain in situ. A number of genes (nerve growth factor, tyrosine hydroxylase, glutamic acid decarboxylase, and choline acetyltransferase) have been successfully introduced and expressed in the primary fibroblasts. The L-dopa-secreting primary fibroblasts exhibited a behavioral effect in a rat model of Parkinson's disease up to 8 weeks after being grafted into denervated striatum. Factors that can maximize gene transfer, transgene expression, and fibroblast survival in the brain make up the future direction of investigation.

Since a postmortem biochemical study and a genetic linkage study of idiopathic torsion dystonia suggested possible involvement of dopamine beta-hydroxylase (DBH), we determined CSF DBH activities of Jewish and non-Jewish patients with childhood-onset idiopathic torsion dystonia and found no differences from a control population.

We enrolled 55 patients in a double-blind, placebo-controlled, parallel design study of the effectiveness of botulinum toxin (Botox) injections for the treatment of spasmodic torticollis. Patients received a standard series of injections, either placebo or Botox. We determined the sites of injection and dose per muscle by the nature of head deviation. Compared with placebo, Botox produced statistically significant improvement in the severity of torticollis, disability, pain, and degree of head turning. There were no serious side effects. During the double-blind phase, 61% of patients injected with Botox improved; 74% of patients subsequently improved during a later open phase at a higher dose of Botox. Direction of head turning, severity of torticollis, and presence or absence of jerky movements did not significantly influence the response rate. We conclude that Botox is a valuable treatment for spasmodic torticollis.