Cultured human foetal cerebral cortex, transfected with tyrosine hydroxylase cDNA, as a source of neural transplant material.

Obtaining an adequate supply of foetal dopaminergic tissue to treat Parkinson's disease by neural transplantation can be difficult. In this study primary cultures of human foetal cerebral cortex cells were transfected, using cationic lipids, with a eukaryotic expression vector (pCIneo-THI) containing the cDNA for human tyrosine hydroxylase isoform I (TH). Cortical cells from human (10-14 week) foetuses were cultured for 11 days in vitro and transfected twice with pCIneo-THI during this time. The double transfection process resulted in 3-4% of the cells becoming TH positive. When grafted into the striatum of 6-OHDA lesioned rats the transfected foetal cerebral cortex cells reduced amphetamine-induced circling behaviour by 75%, while grafts of untransfected cells had no significant effect on turning. TH transfected foetal cerebral cortex cells may therefore be a useful alternative supply of tissue for use in neural transplants to treat Parkinson's disease.

Parallel induction of the formation of dopamine and its metabolites with induction of tyrosine hydroxylase expression in foetal rat and human cerebral cortical cells by brain-derived neurotrophic factor and glial-cell derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF; 50 ng/ml), dopamine (DA; 10 microM) and forskolin (Fsk; 10 microM) have previously been shown by this and other laboratories to induce the tyrosine hydroxylase (TH) enzyme in foetal human and rat cerebral cortex during specified sensitive developmental periods. In the present study, these findings were extended for human and rat cells by showing that the induced TH+ cells also produce dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). In addition to this, TH induction and DA plus DOPAC production was observed in foetal human and rat cerebral cortex by using glial-cell derived neurotrophic factor (GDNF) in place of BDNF. The degree of induction by GDNF (1-10 ng/ml) was similar to that produced by BDNF and did not increase further when the two neurotrophic factors were used together. The time-course of induction in human cultures was followed: GDNF was found to cause a more rapid induction process than BDNF during the first 2 weeks. However the degree of induction after 3 weeks was the same for both neurotrophic factors. Inhibitors of transcription (actinomycin D) or of translation (cycloheximide) eliminated all the increase in DA+DOPAC contents elicited by these compounds, indicating that de novo transcription and translation were required for increased expression of the TH and other related enzymes. The intracellular pathways by which these molecules exert this dopaminergic phenotype induction effect are discussed. This study indicates a new source of dopaminergic brain tissue for use as transplants to neurosurgically treat Parkinson's disease patients.

The neuronal survival effects of rasagiline and deprenyl on fetal human and rat ventral mesencephalic neurones in culture.

The neuronal survival properties of rasagiline (R(+)-N-propargyl-1-aminoindane mesylate or TVP-1012), a novel monoamine oxidase B inhibitor, have been investigated using neuronal cell cultures from fetal rat and human ventral mesencephalon. The ability of rasagiline to reduce the rate of neuronal cell loss in vitro was tested using primary neuronal cell lines and immunohistochemistry to quantify the reduction in cell death. Direct comparison was made with deprenyl, a widely used and long established monoamine oxidase B inhibitor. Rasagiline was shown to act 15-20% more effectively as a neuronal survival agent than deprenyl, increasing both the survival of the total number of neurones and selectively increasing the survival of dopaminergic neurones with no statistically significant increase in survival of GABAergic neurones.

Forskolin-induced expression of tyrosine hydroxylase in human foetal brain cortex.

Brain-derived neurotrophic factor (BDNF) has previously been shown by this and other laboratories to work in concert with dopamine (DA) to induce the dopaminergic phenotype in foetal rat and human cerebral cortex during specified sensitive developmental stages. In the present study this induction by BDNF/DA was found to be greatly amplified by adding forskolin (fsk: 10 microM) to the rat and human cerebral cortex cultures together with DA (10 microM) and BDNF (50 ng/ml). This amplification was 14-fold for human tissue and 2-fold for rat tissue treated over an 80% shorter period. Compared to treatment with BDNF alone, the additional fsk increased tyrosine hydroxylase-positive (TH+) cell numbers by 220-fold in the human and 26-fold in the rat tissue. Parallel reverse transcription-polymerase chain reaction (RT-PCR) measurement of TH mRNA showed substantial increases above control levels when BDNF/DA or BDNF/DA/fsk treatments were applied. Since fsk boosts intracellular levels of cyclic AMP (cAMP), its amplifying action when added together with BDNF/DA is likely to be due to interactions via the cAMP response element/cAMP response element binding protein (CRE/CREB) systems. This is discussed.

Induction of tyrosine hydroxylase gene expression in human foetal cerebral cortex.

Human foetal cerebral cortex (9-14 weeks gestational age) was dissected out and cultured in microwell plates. It was then treated with brain-derived neurotrophic factor (BDNF, 50 ng/ml), dopamine (10 mM) or their combination. After 5 weeks of this treatment tyrosine hydroxylase (TH)-immunopositive neurones were detected at a level of 0.73% of total neurones present. This represented 300-500 TH + neurones per microwell. None were seen in untreated cultures. This correlates with induction of the entire dopaminergic phenotype in foetal rat cerebral cortex (E1214) by the same co-treatment applied for a much shorter time period (7 days), which implies that the complete dopaminergic phenotype is also induced in cultured human foetal tissue over a longer period, reflecting the 5-fold longer neuronal gestational period.

Influence of BDNF on the expression of the dopaminergic phenotype of tissue used for brain transplants.

Brain-derived neurotrophic factor (BDNF) has previously been shown by this laboratory among others to promote survival and differentiation of central dopaminergic neurons and to stimulate expression of the dopaminergic phenotype in fetal cerebrocortex in vitro. We have examined the effect of BDNF antibody on nigral dopaminergic neurons in vivo and in vitro. It reduced the survival of rat fetal dopaminergic neurons in culture (up to 40% died). The BDNF antibody also caused ipsilateral rotation after a single in vivo intranigral injection in the adult rats. Pre-treatment of fetal nigral neurons with BDNF improved the performance of dopaminergic cells in fetal nigral transplants based on surviving TH+ cells numbers. Thus, parkinsonian rats receiving fetal nigral cells treated with BDNF showed a significantly greater reduction of turning over the 3 weeks following transplantation, compared with the rats receiving untreated nigral transplants. However, the average number of tyrosine hydroxylase (TH)-positive neurons in the grafts of rats receiving fetal nigral cells treated with BDNF was 211 +/- 35 which was only about 20% of the cell number (1012 +/- 223, mean +/- S.E.M.) found in those receiving untreated nigral transplants. These results suggest that pretreatment of nigral dopaminergic neurons with BDNF may improve their functional performance, but not their survival in transplants. The ability of artificially induced cerebrocortical 'dopaminergic' cells to ameliorate behavioral asymmetry of Parkinsonian rats was assessed. A proportion (1.0% maximum) of the TH+ neurons in these transplants survived in the host brain and were likely to be responsible for the prominent reduction in rotation scores observed to occur 6 weeks after implantation. Thus, the combined treatment of fetal cerebral cortex with BDNF and dopamine created long-lived TH-expressing neuronal populations which were very effective in alleviating the rat parkinsonian model, and thus may be suitable for use in transplantation in treating human Parkinson's disease.

The BDNF content of postnatal and adult rat brain: the effects of 6-hydroxydopamine lesions in adult brain.

Brain-derived neurotrophic factor (BDNF) has been well studied for its effects in improving survival and differentiation of the central and peripheral nervous system. In order to understand the developing CNS and the pathogenesis of brain injury, an enzyme immunoassay was employed to detect BDNF protein in the various tissues of developing and adult animals. Increased levels of the BDNF were found in the hippocampus, cerebrocortex, striatum, cerebellum and ventral mesencephalon in 2-week-old rats, compared with that in postnatal day 0 pups. In the adult rat, the highest level of BDNF was detected in the hippocampus (14.5 +/- 0.8 ng/g wet tissue), with a relatively high level also observed in the cerebrocortex and striatum. In peripheral tissues, a substantial amount of BDNF protein was observed in various organs. The changes in BDNF levels in the striatum and the ventral mesencephalon of unilaterally 6-hydroxydopamine-lesioned young adult rats were also examined. Significant increases of BDNF levels were detected during 2 weeks after lesion.

The depolarisation-induced release of [125I]BDNF from brain tissue.

The pattern of release of radioactive brain-derived neurotrophic factor ([125I]BDNF) from brain tissue was studied. Rat brain slices from cerebral cortex and synaptosomes from cerebral cortex and hippocampus were preloaded with [125I]BDNF. Depolarising stimulation by veratridine (final conc. 50 microM) and high KCl (final conc. 45 mM) caused a short-term, greatly enhanced depolarisation-induced release of [125I]BDNF during superfusion and batch protocol experiments. The results suggested that the evoked release was independent of the presence of extracellular calcium ions, but dependent on intracellular calcium ion stores, since the intracellular calcium ion chelator BAPTA-AM, but not the extracellular chelator EGTA abolished the high-potassium-induced [125I]BDNF release from synaptosomes. The release was blocked by tetrodotoxin (1 microM) when synaptosomes were stimulated by veratridine or potassium chloride. Short time-fraction (30 s) superfusion experiments showed that the [125I]BDNF release from synaptosomes appeared in two temporal phases.

Neural toxicity of retroviruses.

Recombinant retroviruses containing the cDNA for human tyrosine hydroxylase-1 and Escherichia coli lacZ gene were used to infect primary foetal ventral mesencephalon and cortical cultures from rat brain. Severe neuronal toxicity resulted 3-4 days after infection, glial cells seemed to be much more resistant. The toxicity was likely to have resulted from an agent present within the virus-containing medium itself, rather than from the retrovirus itself. The results of this study indicate that retroviruses are not suitable vectors for the introduction of tyrosine hydroxylase into primary neuronal cultures.

Induction of dopaminergic neurotransmitter phenotype in rat embryonic cerebrocortex by the synergistic action of neurotrophins and dopamine.

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), have been shown to enhance survival and differentiation of a variety of central neuronal populations, such as those with the dopaminergic, cholinergic, GABAergic phenotype during development. In this paper we present evidence that BDNF, NT-3 and NT-4/5 acting synergistically with dopamine (DA) can artificially induce the full dopaminergic phenotype in rat fetal cerebral cortex which normally has very few dopaminergic neurons in adulthood. Thus, BDNF/DA, NT-3/DA, NT-4/DA elicited a great increase in the number of tyrosine hydroxylase (TH)-immunoreactive cells, which was up to 5-7% of total neuronal population in cultures of fetal rat cortical cells. This stimulatory effect was not dependent on glial proliferation, or on addition of serum to the culture. Pharmacological studies showed that dopamine receptors D1 and D2 were involved in this effect. The TH+ cortical cells possessed other biochemical phenotypic features of dopaminergic neurons. Thus, high-affinity DA uptake was elevated in cortical cultures treated with neurotrophin/DA. Also DA and 3,4-dihydroxyphenlacetic acid production was detected (5.42 +/- 1.24 and 13.72 +/- 2.84 pmol/dish respectively, zero in controls). This show the presence of functionally active TH, aromatic acid decarboxylase and monoamine oxidase. Neurotrophins/DA had no effect on noradrenergic phenotype expression by cortical fetal cells. Taken together, these results support the long-standing view that development of the central nervous system is determined not only by intrinsic genetic programmes, but also involves environmental influences such as the action of growth factors and extracellular neurotransmitter. In this case we report the effect of specific DA phenotype-inducing agents.

The therapeutic potential of moclobemide, a reversible selective monoamine oxidase A inhibitor in Parkinson's disease.

Dopamine is equally well deaminated oxidatively by monoamine oxidase (MAO) A and B types. Selegiline (L-deprenyl), a selective inhibitor of MAO-B, ameliorates the "wearing off" akinesia and delays the need for levodopa in mild, previously untreated Parkinson's disease. The therapeutic potential of selective inhibition of MAO-A in Parkinson's disease has not been examined in detail. MAO-A accounts for only about 20% of total MAO activity in the human basal ganglia, and it differs from MAO-B in distribution. In contrast to MAO-B, which is confined to the extraneuronal compartment, MAO-A is found both extraneuronally and within the presynaptic dopaminergic terminals. The inhibition of MAO-A might alter the intraneuronal handling of dopamine reuptaken from synaptic clefts and thereby prolong oral levodopa benefit. We have given moclobemide, a selective, reversible inhibitor of MAO-A, to nondepressed patients with Parkinson's disease receiving standard levodopa/peripheral decarboxylase inhibitor or levodopa with dopaminergic agonist (bromocriptine, pergolide). Selegiline was discontinued at least 8 weeks earlier. A standard oral levodopa challenge was performed at the patient's entry to the study and repeated on the 22nd day of moclobemide treatment (150 mg thrice daily). The overall time spent "on" and "off" before the onset of treatment and during the last week on the drug was estimated from the patients' diaries. Neuropsychological assessments were also made before and after 3 weeks of moclobemide to measure possible effects on cognitive performance and mood. In acute levodopa challenge, the latency of motor response was significantly shortened and its duration was prolonged during moclobemide treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The stimulatory effect of brain-derived neurotrophic factor on dopaminergic phenotype expression of embryonic rat cortical neurons in vitro.

Cells of embryonic (E12-16) rat cerebral cortex were cultured for 7 days in vitro (7DIV) in the presence of either brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), with or without dopamine (DA). Chronic treatment of cells with DA or BDNF alone increased (300% and 600%, respectively) the number of the cells that expressed tyrosine hydroxylase (TH). However, the combination of BDNF and DA treatment greatly increased the expression of TH in E14 cortical cells in a dose-dependent manner, to a much greater extent than DA or BDNF alone. This marked response due to treatment with both BDNF and DA was greater in cortical tissue from E12 embryos than that from E14 embryos. The combination of CNTF and DA also increased expression of the dopaminergic phenotype whilst CNTF alone was ineffective, but this effect was largely due to DA. No effect of DA, or of neurotrophic factors, was observed on cortical cells from E16 embryos under any of the treatment conditions. The present study reveals how chemical environment plays an important role in determining the final phenotype of cortical neurons during early periods in brain development. BDNF, but not CNTF, may influence the differentiation of fetal cortical cells towards a dopaminergic phenotype via a unique mechanism, different from that due to DA. This combination of nerve growth factor and neurotransmitter may be of general importance in phenotype determination in the early developmental stages of the nervous system.

The response of human and rat fetal ventral mesencephalon in culture to the brain-derived neurotrophic factor treatment.

Brain-derived neurotrophic factor (BDNF) has been shown to increase the survival of dopaminergic neurons in rodent mesencephalic cultures. The mRNAs of BDNF and trkB receptor have been found to be expressed in the substantia nigra of rat. In this study, the action of BDNF was studied on the survival and transmitter-specific differentiation of dopaminergic neurons of fetal human CNS aged 9-10-week in vitro. Dopaminergic neuron viability and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunohistochemistry and measurement of dopamine (DA) content with HPLC, respectively. After seven days of treatment with BDNF there were 2.2-fold greater number of TH+ neurons surviving than in untreated cultures. Although very low levels of DA were detectable in human tissue, considerable amounts of DA was found in the culture medium from around 13 days in vitro (DIV), indicating that DA in human fetal tissue tended to be synthesised and released into the incubation medium more readily than from cultured rat fetal tissue during the same period. The content of DA in the BDNF-treated cultures was approximately double that of untreated cultures after 7 days. In rat fetal tissue, the capacity of each TH+ neuron to produce DA was not changed in the BDNF-treated cultures (7 DIV) compared with control cultures, suggesting that BDNF does not up-regulate the production of DA but rather acts to reduce cell death rates. Ciliary neurotrophic factor (CNTF) treatment of rat mesencephalic culture failed to improve the period of survival of fetal dopaminergic neurons and had no effect on the production of DA in cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Motor response to levodopa in patients with parkinsonian motor fluctuations: a follow-up study over three years.

To clarify the way in which the clinical response to levodopa changes with the progression of Parkinson's disease, a longitudinal study was performed to quantify motor response characteristics to single doses of levodopa by mouth over three years in 23 patients with fluctuating motor function. A significant increase in motor disability in "on" (time of peak motor improvement) and "off" (before levodopa dose) phases occurred and "on" phase dyskinesia increased by 24%, though the amplitude of motor response was conserved. There was no evidence of progressive loss of response of certain motor deficits affecting axial muscles and gait. The mean duration of motor response decreased by 17%. Both shortening of response duration and increase in "off" phase disability contribute to the development of motor fluctuations. A short response time to the levodopa test dose was not an invariable finding in patients with severe fluctuations, whereas all had large response amplitudes and high "off" phase disability scores. Patients who have developed motor fluctuations may continue to respond to dopaminergic treatment until late in the disease course, despite the unstable nature of their responses.

Subcutaneous apomorphine in Parkinson's disease: response to chronic administration for up to five years.

Subcutaneous apomorphine, administered by continuous waking-day infusion with boluses, or by repeated intermittent injection, was given to 71 parkinsonian patients with severe refractory levodopa related on-off fluctuations for 1-5 years. A mean reduction in daily off period time of approximately 50% was maintained, and the incidence of neuropsychiatric toxicity remained low on long-term follow-up. No clinically significant tolerance or loss of therapeutic effect was seen, although increasingly severe on-phase dyskinesias and postural instability marred the long-term therapeutic response in many patients. Despite these drawbacks, apomorphine, when combined with the peripheral dopamine receptor agonist domperidone, represents a significant therapeutic advance in the management of late-stage Parkinson's disease and should certainly be considered before experimental implantation procedures.

Increased prevalence of undernutrition in Parkinson's disease and its relationship to clinical disease parameters.

An anthropometric study was performed in 95 subjects (53 male, 42 female) with Parkinson's disease. Weight, height, triceps and biceps skin-fold thicknesses, and mid-arm circumference were recorded. A high incidence of undernutrition was found (23.6% of males and 22.5% of females, as defined by recent British guidelines). A subgroup of severely disabled patients with Parkinson's disease had a significantly lower mean body mass index than a similarly disabled control group with chronic pyramidal upper motor neuron lesions (males 20.6 v 23.2 kg/m2 p < 0.05; females 20.6 v 26.6 kg/m2 p < 0.01), suggesting that the undernutrition is not due to chronic illness or immobility alone. Correlation between anthropometric indices and clinical features of disease demonstrated that the presence of moderate or severe dyskinetic movements was the clinical parameter most strongly related to undernutrition. The reduction in anthropometric indices was most marked for skin fold thickness (related to percentage body fat) and least for arm muscle circumference (related to lean body mass); therefore the weight loss seen in Parkinson's disease is primarily due to fat loss rather than muscle loss.

The properties of cultured fetal human and rat brain tissue and its use as grafts for the relief of the parkinsonian syndrome.

Primary cultures were derived from human fetal ventral mesencephalon and cerebral cortex at 7-11 weeks gestation, and from fetal rat mesencephalon and cortex at embryonic day 14-15. Immunohistochemical analysis of the mesencephalic cultures using antibodies to tyrosine hydroxylase (TH) showed between 0.1-0.5% of human cells to be TH positive and 0.1-1% of rat cells to be TH positive. HPLC analysis of extracts from the cultures showed that they had the ability to synthesise and store dopamine. Implantation of the cultured human and rat mesencephalic tissue into a 6-hydroxydopamine rat model of Parkinson's disease produced marked recovery from amphetamine induced rotational asymmetry in the recipient rats, but no such recovery was observed following implantation of cortical cultures. Histological examination demonstrated the presence of surviving human mesencephalic and cortical grafts at least 6 months after implantation. Implants of cultured fetal rat tissue were less obviously but still significantly effective in these experiments. These rat tissue grafts were detectable for periods of at least 6-8 weeks by histological staining.