Alpha galactosidase A activity in Parkinson's disease.

Glucocerebrosidase (GCase, deficient in Gaucher disease) enzymatic activity measured in dried blood spots of Parkinson's Disease (PD) cases is within healthy range but reduced compared to controls. It is not known whether activities of additional lysosomal enzymes are reduced in dried blood spots in PD. To test whether reduction in lysosomal enzymatic activity in PD is specific to GCase, we measured GCase, acid sphingomyelinase (deficient in Niemann-Pick disease types A and B), alpha galactosidase A (deficient in Fabry), acid alpha-glucosidase (deficient in Pompe) and galactosylceramidase (deficient in Krabbe) enzymatic activities in dried blood spots of PD patients (n = 648) and controls (n = 317) recruited from Columbia University. Full sequencing of glucocerebrosidase (GBA) and the LRRK2 G2019S mutation was performed. Enzymatic activities were compared between PD cases and controls using t-test and regression models adjusted for age, gender, and GBA and LRRK2 G2019S mutation status. Alpha galactosidase A activity was lower in PD cases compared to controls both when only non-carriers were included (excluding all GBA and LRRK2 G2019S carriers and PD cases with age-at-onset below 40) [2.85 µmol/l/h versus 3.12 µmol/l/h, p = 0.018; after controlling for batch effect, p = 0.006 (468 PD cases and 296 controls)], and when including the entire cohort (2.89 µmol/l/h versus 3.10 µmol/l/h, p = 0.040; after controlling for batch effect, p = 0.011). Because the alpha galactosidase A gene is X-linked, we stratified the analyses by sex. Among women who were non-carriers of GBA and LRRK2 G2019S mutations (PD, n = 155; control, n = 194), alpha galactosidase A activity was lower in PD compared to controls (2.77 µmol/l/h versus 3.10 µmol/l/h, p = 0.044; after controlling for a batch effect, p = 0.001). The enzymatic activity of acid sphingomyelinase, acid alpha-glucosidase and galactosylceramidase was not significantly different between PD and controls. In non-carriers, most lysosomal enzyme activities were correlated, with the strongest association in GCase, acid alpha-glucosidase, and alpha galactosidase A (Pearson correlation coefficient between 0.382 and 0.532). In a regression model with all five enzymes among non-carriers (adjusted for sex and age), higher alpha galactosidase A activity was associated with lower odds of PD status (OR = 0.54; 95% CI:0.31-0.95; p = 0.032). When LRRK2 G2019S PD carriers (n = 37) were compared to non-carriers with PD, carriers had higher GCase, acid sphingomyelinase and alpha galactosidase A activity. We conclude that alpha galactosidase A may have a potential independent role in PD, in addition to GCase.

Intracerebral grafting in the dopaminergic system: issues and controversy.

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.

Brain-derived neurotrophic factor-transduced fibroblasts: production of BDNF and effects of grafting to the adult rat brain.

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.

Dopamine beta-hydroxylase activity in cerebrospinal fluid of idiopathic torsion dystonia.

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.

Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions.

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.

Deduced amino acid sequence of bovine aromatic L-amino acid decarboxylase: homology to other decarboxylases.

Nucleotide sequence of cDNA for bovine aromatic L-amino acid decarboxylase (AADC) was analyzed. The deduced amino acid sequence of bovine AADC shows 57% identity to drosophila AADC and 37% to plant Catharanthus roseus AADC. The 7-amino acid sequence of the pyridoxal phosphate binding site is completely conserved among drosophila, pig and bovine AADC. AADC primary structure also shows high homology to that of feline glutamic acid decarboxylase.

The localization and functional contribution of striatal aromatic L-amino acid decarboxylase to L-3,4-dihydroxyphenylalanine decarboxylation in rodent parkinsonian models.

L-3,4-Dihydroxyphenylalanine (L-dopa) is the mainstay of therapy for patients with Parkinson's disease (PD), and mediates its primary effects through conversion into dopamine by aromatic L-amino acid decarboxylase (AADC). Given the loss of AADC-containing nigrostriatal dopaminergic neurons in PD, however, the location of residual AADC that converts L-dopa into dopamine remains controversial. The first objective of this study was to establish the presence of AADC expression in striatal neurons and glia using reverse transcriptase and PCR. Transcripts for the neuronal but not nonneuronal forms of AADC were detected in striatal tissue, cultured striatal neurons, and glia. We then examined whether this striatal AADC expression represents a physiologically significant source of dopaine production. No dopamine release was detected following incubation of striatal cultures with L-dopa or transduction with adenovirus expressing tyrosine hydoxylase. Our data establish the presence of AADC expression in the striatum both in vivo and in vitro, but suggest that striatal components do not represent a primary source of L-dopa decarboxylation following nigrostriatal denervation in rats. Understanding the source and localization of AADC is important in understanding the complications of L-dopa therapy and in designing rational therapeutic strategies for PD, including cellular transplantation and gene therapy.

Preferential resistance of dopaminergic neurons to glutathione depletion in a reconstituted nigrostriatal system.

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD), and could initiate dopaminergic neuronal degeneration. Nevertheless, we have previously demonstrated that mesencephalic dopaminergic neurons in primary monolayer cultures are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. To extend this finding to a system that more closely resembles the in vivo situation, we characterized the effects of glutathione depletion on reaggregate cultures derived from ventral mesencephalic and their striatal target neurons, as well as supporting elements including glia. Dopaminergic neurons were found to be more resistant to the toxicity of buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis, than other nigrostriatal neurons, while striatal target cells exhibited an intermediate susceptibility when examined after 48 h. Glutathione depletion, however, decreased the intracellular content of catecholamines after 48 h and eventually led to the loss of dopaminergic neurons after 7 days. Our data indicate that the intrinsic resistance of dopaminergic neurons to the toxicity of glutathione depletion occurs in a variety of experimental paradigms, and suggest that global glutathione depletion alone is unlikely to account for the selective loss of dopaminergic neurons in PD. Rather, it is more likely that either the selective loss of glutathione from dopaminergic neurons, or the combination of glutathione loss with other insults contributes to the preferential death of dopaminergic neurons in PD.

Dopa-decarboxylation in the striata of rats with unilateral substantia nigra lesions.

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.

Tardive dystonia.

We retrospectively reviewed the clinical course and response to treatment of 67 patients with tardive dystonia. The age at onset ranged from 13 to 72 years without predilection to any particular age group or sex. Patients developed tardive dystonia even after relatively short duration of exposure to dopamine antagonists (21% within 1 year). Five of 42 patients withdrawn from these drugs remitted. Overall clinical improvement occurred in 52% of patients. Tetrabenazine and reserpine were most effective (greater than 50% response rate) in controlling dystonia. Anticholinergic drugs diminished dystonia in 46% of patients. In conclusion, this review supports our original concept of tardive dystonia as a subtype of tardive dyskinesia, which is quite disabling, usually persistent, and difficult to treat. Although anticholinergics and dopamine-depleting drugs frequently improved symptoms, treatment with them only rarely led to a remission or satisfactory control of symptoms. The difficulty of treating this condition necessitates reemphasis of one important observation of this study, that this condition may develop early in the course of dopamine antagonist treatment; there is no minimum period of exposure which can be considered safe. These drugs must therefore be used only for correct medical indications, and every attempt should be made to withdraw them at the first sign of dyskinesia, particularly of the dystonic type.

Gene therapy for Parkinson's disease: determining the genes necessary for optimal dopamine replacement in rat models.

This article reviews the mechanism of dopamine delivery in the CNS in order to determine the optimal set of genes for effective gene therapy in Parkinson's disease (PD). Systematic neurobiological investigation of the biochemical steps has revealed that tyrosine hydroxylase (TH), which has been used in earlier studies, functions only when the essential cofactor, tetrahydrobiopterin (BH1) is present. Transduction of the gene for GTP cyclohydrolase I, the first and rate-limiting step in BH1 synthesis, along with the TH gene, generated cells that are capable of producing L-DOPA spontaneously both in vitro and in vivo. When the aromatic L-amino acid decarboxylase (AADC) gene was added as a third gene, in an attempt to increase the conversion of L-DOPA to dopamine, feedback inhibition by the end product, dopamine, on TH activity resulted. To circumvent this problem, we employed a complementary strategy. Gene transfer of the vesicular monoamine transporter was combined with AADC and produced genetically modified cells that can convert L-DOPA to dopamine and store it for gradual release. This approach provided a means to regulate final dopamine delivery by controlling precursor doses and to achieve more sustained delivery of dopamine. Our investigation into determining the genes necessary for optimal dopamine delivery has been facilitated by in vivo biochemical assays using microdialysis. This technique has provided us with a clear and quantitative tool to compare the effects of various genes involved in dopamine synthesis and processing.

Expansion of the first PolyA tract of ARX causes infantile spasms and status dystonicus.

BACKGROUND: ARX is a paired-type homeobox gene located on the X chromosome that contains five exons with four polyalanine (PolyA) tracts, a homeodomain, and a conserved C-terminal aristaless domain. Studies in humans have demonstrated remarkable pleiotropy: malformation phenotypes are associated with protein truncation mutations and missense mutations in the homeobox; nonmalformation phenotypes, including X-linked infantile spasms (ISS), are associated with missense mutations outside of the homeobox and expansion of the PolyA tracts. OBJECTIVE: To investigate the role of ARX, we performed mutation analysis in 115 boys with cryptogenic ISS. This included two pairs of brothers. RESULTS: We found an expansion of the trinucleotide repeat that codes for the first PolyA tract from 10 to 17 GCG repeats (c.333_334ins[GCG]7) in six boys (5.2%) ages 2 to 14, from four families, including the two pairs of brothers. In addition to ISS, all six boys had severe mental retardation and generalized dystonia that appeared around the age of 6 months and worsened, eventually leading to stable severe quadriplegic dyskinesia within age 2 years. Three children experienced recurrent, life-threatening status dystonicus. In four children brain MRI showed multiple small foci of abnormal cavitation on T1 and increased signal intensity on T2 in the putamina, possibly reflecting progressive multifocal loss of tissue. CONCLUSION: The phenotype of infantile spasms with severe dyskinetic quadriparesis increases the number of human disorders that result from the pathologic expansion of single alanine repeats. ARX gene testing should be considered in boys with infantile spasms and dyskinetic cerebral palsy in the absence of a consistent perinatal history.

Potential of gene therapy for Parkinson's disease: neurobiologic issues and new developments in gene transfer methodologies.

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.

Spread of symptoms in idiopathic torsion dystonia.

We reviewed the histories of 115 patients who had idiopathic dystonia and who were 21 years of age and younger at onset of symptoms and 472 patients with onset older than age 21. Patients with onset in the lower extremities tended to be younger at onset (mean age 8.4 years), have rapid spread of symptoms to other body parts, and to develop generalized dystonia. Patients with onset in the upper extremities tended to be older at onset (mean age 11.2 years) and were less likely to develop generalized dystonia, but were more likely to experience spread of symptoms many years after the disease began. Almost 20% of patients younger than age 22 began with torticollis, and 67% of these remained focal after a mean 14.9 years. In adults, dystonic symptoms remained focal in the majority, but approximately 15-30% of patients presenting with blepharospasm, torticollis, laryngeal or brachial dystonia eventually experienced dystonia outside the initially involved segment. Almost 50% of children and adolescents with dystonia reported a family history of dystonia, compared with approximately 10-13% of patients with torticollis, laryngeal or brachial dystonia. Less than 3% of patients with blepharospasm reported a family history of dystonia. There were only minor differences in the pattern of spread of dystonic symptoms between Jewish and non-Jewish patients. Although younger patients were more likely to report a family history of dystonia, patterns of spread were the same for familial and sporadic patients in the same age range.

The role of glutathione in dopaminergic neuronal survival.

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.

Role of aromatic L-amino acid decarboxylase for dopamine replacement by genetically modified fibroblasts in a rat model of Parkinson's disease.

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.

Natural history and treatment of tardive dystonia.

We retrospectively reviewed the clinical course and response to treatment of 67 patients with tardive dystonia. The age at onset ranged from 13 to 72 years without predilection to any particular age group or sex. Patients developed tardive dystonia even after relatively short duration of exposure to dopamine antagonists (21% within one year). Five of 42 patients withdrawn from these drugs remitted. Overall clinical improvement occurred in 52% of patients. Tetrabenazine and reserpine were most effective (greater than 50% response rate) in controlling dystonia. Anticholinergic drugs diminished dystonia in 46% of patients.

Intrastriatal implantation of fibroblasts genetically engineered to produce brain-derived neurotrophic factor prevents degeneration of dopaminergic neurons in a rat model of Parkinson's disease.

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.