Factors involved in the determination of the neurotransmitter phenotype of developing neurons of the CNS: applications in cell replacement treatment for Parkinson's disease.

The developmental stages involved in the conversion of stem cells to fully functional neurons of specific neurotransmitter phenotype are complex and not fully understood. Over the past decade many studies have been published that demonstrate that in vitro manipulation of the epigenetic environment of the stem cells allows experimental control of final neuronal phenotypic choice. This review presents the evidence for the involvement of a number of endogenous neurobiochemicals, which have been reported to potently influence DAergic (and other neurotransmitter) phenotype expression in vitro. They act at different stages on the pathway to neurotransmitter phenotype determination, and in different ways. Many are better known for their involvement in other aspects of development, and in other biochemical roles. Their proper place, and precise roles, in neurotransmitter phenotype determination in vivo will no doubt be determined in the future. Meanwhile, considerable medical benefits are offered from producing large, long-term, viable cryostores of self-regenerating multipotential neural precursor cells (i.e., brain stem cells), which can be used for cell replacement therapies in the treatment of degenerative brain diseases, such as Parkinson's disease.

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.

Striatal extracts promote the survival and phenotypic expression of rat fetal dopaminergic neurons in vitro.

To begin to identify novel protein(s) that acts on nigral dopaminergic (DA) neurons, we characterized trophic effects of DA-depleted striatum on survival of fetal DA neurons in the present study. Treatment of ventral mesencephalic cultures with the striatal extracts delayed DA cell death in a dose-dependent manner. This effect was partially dependent on brain-derived neurotrophic factor (BDNF), but not glial cell line-derived neurotrophic factor (GDNF), present in the extracts. Furthermore, we addressed the hypothesis that the striatum-derived substances can elicit DA phenotypic expression of striatal cells in cultures. The striatal extract was found to be able to induce expression of tyrosine hydroxylase in cultured striatal cells in the presence of dopamine. These data suggest that denervation of the striatum resulted in production of neurotrophic factors, including BDNF and as-yet-unidentified trophic substances, which may be responsible for the increased survival and DA phenotype expression in DA neurons.

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.

Anticonvulsant and glutamate release-inhibiting properties of the highly potent metabotropic glutamate receptor agonist (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV).

The anticonvulsant effects of intracerebral administration of the highly potent group II metabotropic glutamate receptor agonist, DCG-IV, were tested in fully kindled rats following daily electrical stimulation of the basolateral amygdala. The agonist caused a dose-dependent increase in the generalized seizure threshold (GST) of these seizure susceptible animals within the dose range tested (0. 01-1.0 nmol). The estimated GST100 value (dose causing a 100% increase in GST) for this effect was 0.22 nmol. The anti-seizure activity of DCG-IV was fully inhibited in the presence of the group II metabotropic glutamate receptor antagonist (2S,1'S, 2'S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG; 40 nmol), while MCCG alone showed no significant inhibitory effect on seizure activity. DCG-IV also powerfully inhibited depolarization-induced release of [3H]D-aspartate from rat cerebrocortical synaptosomes, with an IC50 value of 0.39 microM. In this respect, DCG-IV was approximately 70-fold more potent than the clinically effective anticonvulsant drug lamotrigine (IC50=27.7 microM), a proposed neurotransmitter release inhibitor known to inhibit glutamate release, also tested in this assay. These findings demonstrate the high potency of DCG-IV as an anticonvulsant agent and confirm a key role for group II metabotropic glutamate receptors in the control of seizure activity via their modulatory action on neuronal glutamate release.

Specific group II metabotropic glutamate receptor activation inhibits the development of kindled epilepsy in rats.

The effects of intracerebral administration of the group II metabotropic glutamate receptor agonist, 2R,4R-APDC, were tested on both the development of amygdaloid kindling and on fully developed stage 5 amygdala kindled seizures. The development of amygdaloid kindling was significantly retarded in 2R,4R-APDC (10 nmol in 0.5 microl) treated animals compared to control animals over a period of 8 days. At a low dose, 2R,4R-APDC (0.1 nmol) caused a 42.5+/-26.6% increase of the generalised seizure threshold in fully kindled animals. As higher doses were administered, however, the changes in generalised seizure threshold were less marked, and even a small decrease in the threshold was seen (-19.6+/-5.36% at 10 nmol). The agonist 2R,4R-APDC inhibited depolarization-induced release of [3H]d-aspartate from cortical synaptosomes with an IC50 value of 0. 29 microM. This effect was maximal at 1 microM, and decreased with dose thereafter. These findings suggest that the selective activation of the group II metabotropic glutamate receptors by agonists such as 2R,4R-APDC may be of therapeutic potential in the treatment of seizure disorders.

The effect of 2-amino-3-arsonopropionate and 2-amino-4-arsonobutyrate on the development and maintenance of amygdala kindled seizures.

The effects of 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate, the arsono analogues of aspartate and glutamate respectively, on the development of electrically-induced kindling in the amygdala, and on seizures induced in fully kindled rats, were compared to the effects of 3-amino-propylarsonate the arsono analogue of GABA. Intra-amygdaloid micro-injection of 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate (10 nmol in 0.5 microl buffer phosphate) reduced the rate of epileptogenesis without preventing the development of generalized seizure responses, after 14 daily stimulations. In fully electrically kindled animals with stage 5 amygdala-kindled seizures, 3-aminopropy-larsonate (10 nmol/0.5 microl) increased after-discharge threshold (ADT) by 82% (P< or =0.005) without having any effect on mean seizure score or after-discharge duration. Chemical reduction of 3-aminopropylarsonate with glutathione diminished the anti-seizure activity of the drug. 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate the arsono analogues of aspartate and glutamate were not effective when they were micro-injected into the amygdala of fully kindled animals at equivalent doses i.e. (10 nmol/0.5 microl). Higher doses (100 nmol/0.5 microl) of 2-a-3-arsonopropionate the analogue of aspartate increased the generalized seizure threshold by 40% (P < or = 0.025), while 2-a-4-arsonobutyrate was not effective even at high doses.

Nerve growth factors and the control of neurotransmitter phenotype selection in the mammalian central nervous system.

Determination of neurotransmitter phenotype in the peripheral nervous system (PNS) has been intensively characterized. However, relatively little is known about the underlying molecular and biochemical events involved in determination of transmitter phenotype in the central nervous system (CNS). It has been well established that nerve growth factors regulate cell growth and differentiation. They are increasingly recognized as playing an important role in many decision-making steps during development. Published data suggest that neurotransmitter phenotype is determined largely by exogenous stimuli, such as nerve growth factors--acidic/basic fibroblast growth factor, epidermal growth factor, neurotrophins, etc., working in concert with the genetic programmes. They exert potent effects independently or synergistically with other molecules by acting either on neural precursor cells or differentiated neuronal cells. However, the process of transmitter phenotype determination in the CNS is only beginning to be understood, with more uncharacterized substances, with considerable potency in this respect being reported and in need of isolation and further study. These studies will bring great advances in our existing knowledge of brain development and have potential value for the development of new treatments for neurodegenerative diseases.

Brain-derived neurotrophic factor in human platelets.

A sensitive, capture enzyme-linked immunosorbent assay (CELISA) has been applied to the accurate and reproducible measurement of brain-derived neurotrophic factor (BDNF) protein in normal human blood platelets, a mean concentration of 1.03 +/- 0.04 ng (SEM)/mg of platelet protein being observed. The method, which requires only 10 ml blood, is now suitable for the investigation of a variety of clinical disorders.

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 protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures.

The influence of 2-chloroadenosine, a non-metabolizable adenosine A1 receptor agonist, was tested on the development of electrically kindled amygdala and on the seizure responses of fully kindled rats. Focal intra-amygdaloid injection of 2-chloroadenosine (1-10 nmol/0.5 microl) 20 min before applying the daily kindling stimulus prevented the development of the kindling process. The behavioural seizure score and the afterdischarge duration were reduced below their initial values. The antiepileptogenic effects of 1 and 10 nmol of 2-chloroadenosine were reversible 8-10 days after withdrawal of the drug. When 2-chloroadenosine was tested on fully developed stage 5 amygdala-kindled seizures, it increased the generalised seizure threshold in a dose-dependent manner. A maximum efficiency of 125% (P < 0.001) was achieved with 5 nmol and the median effective dose was 0.55 nmol. Higher doses resulted in the reduced anticonvulsant effect (P < 0.05). With the same daily stimulation, 2-chloroadenosine 5 nmol in 0.5 microl vehicle, significantly reduced the maximum seizure score by 90%, the afterdischarge duration by 88% and completely blocked the generalised seizure duration. The antiseizure activity of the drug lasted for 3 days. In conclusion, 2-chloroadenosine not only acts as an anticonvulsant against electrically induced kindled seizures as described here, and against audiogenic seizures, electroshock and a variety of chemical convulsants as described by others, it prevents the development of the epileptic state by kindling-stimulation, i.e., it is antiepileptogenic. We theorise here that this is due to its blockade of presynaptic glutamate release.

Anti-epileptogenic and anticonvulsant activity of L-2-amino-4-phosphonobutyrate, a presynaptic glutamate receptor agonist.

The protective effect of amygdaloid (focally administered) doses of the presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate (L-AP4) was tested on the development of electrical kindling and in fully kindled animals. L-AP4 inhibited epileptogenesis at 10 nmol in 0.5 microl buffer, by preventing the increase in both seizure score and afterdischarge duration. The effects were reversible after withdrawal of the drug, with all treated animals subsequently progressing to the fully kindled state at the same rate as control animals. The same concentration of the drug was also effective when injected into fully kindled animals. It significantly decreased the mean seizure score by 88% (P < 0.005) and increased the mean generalized seizure threshold (GST) by 85% (P < 0.005). The increase in GST was accompanied by a significant delay before the onset of generalized seizure and by a 37% reduction in generalized seizure duration. MPPG ((RS)-alpha-methyl-4-phosphonophenyl glycine) a selective antagonist of L-AP4 at glutamate pre-synaptic receptors inhibited the depressant effect of L-AP4 in a dose-dependent manner. MPPG (10 nmol) inhibited the antiseizure activity of L-AP4, whilst MPPG (40 nmol) reduced both the anti-epileptogenic and antiseizure activities of L-AP4. MPPG (40 nmol) by itself had no effect on generalized seizure activity, and it had no detectable influence on the normal rate of kindled epileptogenesis. During in vitro studies using a microsuperfusion method, L-AP4 inhibited depolarization-induced release of [3H]D-aspartate from rat cortical synaptosomes (IC50 125.1 microM) and decreased the depolarization-evoked uptake of 45Ca2+ in a dose-dependent manner. Both actions of L-AP4 were reduced by the selective antagonist MPPG. When applied alone MPPG (200 microM) had no detectable action on veratridine-evoked 45Ca2+ uptake by the synaptosomes. These results suggest the mechanisms by which presynaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kindling, and indicate that their anticonvulsive activity is due to inhibition of presynaptic glutamate and/or aspartate release following blockade of presynaptic Ca2+ entry.

The effects of focal N-methyl-D-aspartate pretreatment on the parameters of amygdaloid electrical kindling.

Evidence is accumulating for a role of glutamate in both the development (epileptogenesis) and spread of epileptic neuronal hyperactivity in the brain. In the present investigation we examined the influence of daily focal pretreatment with the selective glutamate receptor agonist N-methyl-D-aspartate (NMDA) on the parameters of amygdaloid electrical kindling, an animal model of human complex partial and secondary generalised focal seizures. Pretreatment with NMDA significantly increased the electrical afterdischarge threshold in this model. With subsequent daily suprathreshold electrical stimulation, however, NMDA pretreatment enhanced the kindling process as shown by both electroencephalographic and motor seizure responses. Marked reductions in the number of stimulations required to reach each distinct stage of kindling development were evident. The number of stimulations required to achieve the fully kindled state was approximately halved by pretreatment with NMDA (6.8 +/- 1.6 stimulations) compared with control, buffer-pretreated animals (11.6 +/- 1.4 stimulations; mean +/- S.E.M.; P < 0.05). Consistent with this, the mean durations of the electrically-evoked afterdischarges on most NMDA pretreatment days were significantly increased compared to those recorded in control animals. Importantly, fully kindled animals showed a markedly enhanced sensitivity to focally applied NMDA. The results of the present experiments provide strong in vivo evidence to support the concept that ion fluxes through NMDA receptor-linked cation channels play a major role in the mechanisms of kindling epileptogenesis. Extracellular glutamate at abnormally raised levels, acting at least in part via NMDA receptors, may be the principal agent triggering many forms of epilepsy.

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.

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 anti-epileptic effect of 3-aminopropylarsonate on electrically-kindled and N-methyl-D-aspartate-kindled amygdala.

The effects of 3-aminopropylarsonate, an arsono analogue of GABA, was tested on the development of electrically-kindled amygdala and on the expression of generalized seizure activity in electrically and NMDA fully amygdala-kindled rats. Intra-amygdaloid microinjection of 3-aminopropylarsonate (10 nmol in 0.5 microl injection vehicle) inhibited electrical epileptogenesis by keeping the seizure score at or below stage 1 on the Racine scale, and the afterdischarge duration (ADD) at or below 19.70 +/- 4.59 s. The effect was reversible after withdrawal of the drug, since the animals developed a generalized seizure activity when kindling stimuli continued in the absence of drug. In fully electrically kindled animals with stage 5 amygdala-kindled seizures, the drug increased afterdischarge threshold (ADT) by 30-70%, without any effect on mean seizure score or ADD. The changes were reversible after 7 days. In fully NMDA-kindled rats, intra-amygdala administration of 3-aminopropylarsonate (10 nmol/0.5 microl) 20 min before injection of NMDA (4 nmol/0.5 microl) reduced the seizure score from 3.80 +/- 0.37(5) on the Racine scale to 0.83 +/- 0.40(6) (P < 0.01). The effect was partially reversible after washing with phosphate buffer. 2-Amino-4-arsonobutyrate, the analogue of glutamate, had no effect on seizure score following treatment with the same concentration of the drug and the same route of injection. The inhibitory effect of 3-aminopropylarsonate on NMDA kindled activity was dose-dependent, since higher doses of NMDA reduced the effect of the drug. The effect of 3-aminopropylarsonate was also selective to NMDA receptors since it had no effect on kainate-induced seizures. With both models of kindling, no gross behavioural abnormalities were observed 3-6 months after treatment with the drug. These findings show the potent antiepileptogenic and anti-convulsant activity of the arsonoanalogue of GABA which appears to be non-toxic and therefore potentially useful as the basis for developing a new family of clinically useful anticonvulsants for treating epilepsy.