Disruption of ephrin-A/EphA binding alters synaptogenesis and neural connectivity in the hippocampus.

Ephrins are guidance cues that modulate axonal growth and the subsequent axonal topographic maps in many regions of the CNS. Here we studied the functional roles of ephrin-A/EphA interactions in the layer-specific pattern of axonal projections in the hippocampus by disrupting the ephrin-A signaling by over-expression of a soluble EphA receptor. Tracing experiments in EphA5-Fc over-expressing mice revealed that reduction of ephrin-A/EphA interactions did not affect the proper distribution of the main hippocampal afferents, i.e. entorhinal and commissural projections. However, further ultrastructural analyses showed a reduction in the density of synaptic terminals in the entorhinal and commissural termination layers in these mice. In addition, using anti-calbindin antibodies, we analyzed the dentate mossy fiber projections following disruption of ephrin-A/EphA interactions throughout developing hippocampus. While the main mossy fiber bundle appeared normal, the infrapyramidal bundle formed longer projections that established ectopic contacts in these transgenic mice. Later, the expected specific pruning of the infrapyramidal bundle was not observed at adult stages. Ultrastructural analyses confirmed a higher number of mossy fiber terminals in the infrapyramidal bundle in adult EphA5-Fc transgenic mice and showed that these terminals were larger and established a greater number of contacts than in controls. Our results demonstrate that ephrin-A/EphA interactions regulate the synaptogenesis of hippocampal afferents and the proper development and refinement of granule cell projections.

Functional receptor for GDNF encoded by the c-ret proto-oncogene.

Glial-cell-line-derived neutrophic factor (GDNF) promotes the survival and phenotype of central dopaminergic noradrenergic and motor neurons, as well as various subpopulations of peripheral sensory and sympathetic neurons. GDNF is structurally related to members of the transforming growth factor (TGF)-beta superfamily, several members of which have well-characterized receptor systems; however, GDNF receptors still remain undefined. Here we show that GDNF binds to, and induces tyrosine phosphorylation of, the product of the c-ret proto-oncogene, an orphan receptor tyrosine kinase, in a GDNF-responsive motor-neuron cell line. Ret protein could also bind GDNF and mediate survival and growth responses to GDNF upon transfection into naive fibroblasts. Moreover, high levels of c-ret mRNA expression were found in dopaminergic neurons of the adult substantia nigra, where exogenous GDNF protected Ret-positive neurons from 6-hydroxydopamine-induced cell death. Thus the product of the c-ret proto-oncogene encodes a functional receptor for GDNF that may mediate its neurotrophic effects on motor and dopaminergic neurons.

Multiple astrocyte transcripts encode nigral trophic factors in rat and human.

The recent discovery of glial cell line-derived neurotrophic factor (GDNF) identified a novel trophin that selectively increases survival of substantia nigra dopaminergic neurons, which degenerate in Parkinson's disease. Our previous studies indicated that GDNF RNA can be amplified from cultured rat nigral type 1 astrocytes and from rat striatum in vivo, implying local as well as target trophic support. The current study establishes the regional pattern of GDNF RNA expression in adult human brain. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the highest expression of GDNF mRNA in the human caudate, with low levels in the putamen and no detectable message in the nigra, suggesting that GDNF is a target-derived factor in humans. We also report the isolation of two additional GDNF-related cDNAs, termed astrocyte-derived trophic factors (ATF), which apparently result from differential RNA processing. Sequence analysis of rat ATF-1 revealed a 78-bp deletion corresponding to a loss of 26 amino acids within the prepro region of the predicted GDNF protein. The RNA processing events responsible for ATF-1 formation in rat brain are conserved in humans; we report the isolation of a full-length human ATF-1 homologue. We identified a second alternative transcript, human ATF-2; the transcript encodes a protein which differs in its first 18 amino acids from the predicted mature GDNF and ATF-1 proteins and shares the terminal 115 residues with the other two forms. To begin assessing the biologic significance of multiple transcript expression we characterized the actions of COS-expressed GDNF and ATF-1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on species sensitivity to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Part 1: Systemic administration.

Several parameters necessary for the expression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity to dopaminergic neurons were examined in both mice and rats in order to determine if differences in these processes might underlie the marked differences in the sensitivity of the two species to the neurotoxic effects of MPTP. Monoamine oxidase-B activity was greater in brain tissues from rats than from mice. The kinetics of 1-methyl-4-phenylpyridinium (MPP+) uptake into neostriatal synaptosomal preparations from the two species were similar. Brain and neostriatal levels of MPP+ were 2-fold higher in rats after the administration of MPTP at 60 mg/kg and were 10 to 20 times higher in rats than in mice after MPTP treatment which produced similar decrements in the content of neostriatal dopamine. MPP+ concentrations in the extracellular fluid of the neostriatum of the two species were similar after the administration of the same dose of MPTP (40 mg/kg). However, this dose induced a 40-fold increase in neostriatal dopamine efflux in mice, whereas in rats only a 3-fold increase was observed. In addition, pretreatment of rats with guanethidine, a ganglionic blocking agent, permitted the use of high doses of MPTP which resulted in substantial damage to the striatal dopaminergic nerve terminals. It is concluded that nigrostriatal dopaminergic neurons in the rat require exposure to a much higher concentration of MPP+ than do those in mice for the induction of toxicity.

Nigral type I astrocytes release a soluble factor that increases dopaminergic neuron survival through mechanisms distinct from basic fibroblast growth factor.

Our studies have been directed to the identification of local, naturally-occurring molecules that support substantia nigra (SN) dopaminergic (DA) neuron survival. We have previously demonstrated that local Type I astrocytes selectively increase the dopaminergic population [30,31]. However, the mechanism of action remains to be defined. To determine whether survival is elicited through diffusible agents, Type I astrocyte conditioned medium (CM) was tested on SN dissociates. After 7 days of exposure to CM, DA neuronal integrity was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme, or by TH catalytic assay. CM increased TH+ cell number greater than 2-fold, suggesting that a soluble factor(s) promoted neuron survival. Neurons cultured in serum free medium (SFM) are known to contain few, but detectable numbers of glia [34]. To examine whether CM affected neurons directly, or indirectly through glia, glial populations were stained with antibody against the glial marker, glial fibrillary acidic protein (GFAP). We employed several approaches to define the potential role of glia. Initially, CM was compared to basic fibroblast growth factor (bFGF), a glial mitogen that reportedly enhances nigral DA neuron survival. bFGF enhanced TH activity in our system, as well, but the effect was blocked by the mitotic inhibitor 5-fluorodeoxyuridine (FDUR), which kills dividing glia. In parallel studies CM increased enzyme activity and TH cell number in cultures exhibiting GFAP+ cells. To define the role of these glial cells in the CM effect, we completely eliminated astrocytes in CM-treated cultures employing alpha-aminoadipic acid (AA; 10-30 microM), a specific gliotoxin. At a concentration of AA that eliminated detectable GFAP+ cells, CM continued to elicit a significant increase in TH cell number. These data suggest that, in contrast to effects of bFGF, the DA neurotrophic activity in CM acts directly on nigral neurons to enhance survival.

Regional and cell-specific expression of GDNF in rat brain.

The survival of ventral mesencephalic substantia nigra (SN) dopamine neurons, which degenerate in Parkinson's disease, is enhanced by glial cells in vitro. The recent isolation of glial cell line-derived growth factor (GDNF), a molecule with apparently selective effects on dopamine (DA) neurons in vitro, raises the question of whether this factor is found in normal brain cells. In this study, the polymerase chain reaction (PCR) was employed to determine the regional distribution and cellular localization of GDNF in the rat central nervous system. GDNF was expressed by SN and basal forebrain Type 1 (T1) astrocytes, with trace transcript levels present in cortical T1 astrocytes. Neuronal cultures of embryonic SN also expressed GDNF. Regionally, postnatal striatum contained the highest GDNF mRNA levels in vivo under the PCR conditions employed. Our data suggest a role for GDNF in both local and target-derived support of DA neurons, as well as potential involvement in the support of other neuronal populations in vivo.

Mesencephalic type I astrocytes mediate the survival of substantia nigra dopaminergic neurons in culture.

We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between the neostriatal content of the 1-methyl-4-phenylpyridinium species and dopaminergic neurotoxicity following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration to several strains of mice.

In the present study we observed pronounced differences in the capacity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce dopaminergic neurotoxicity in several strains of mice. For example, there was no MPTP-induced decrement in neostriatal dopamine content in Ace Swiss-Webster mice and a 92% decrement in Taconic Farms C57 bl mice. Several parameters which could possibly explain this differential sensitivity to MPTP were studied. These include: 1) neostriatal monoamine oxidase-B (MAO-B) activity; 2) the capacity of neostriatal synaptosomes prepared from the mouse strains to accumulate 1-methyl-4-phenylpyridinium (MPP+), the major metabolite of MPTP formed via oxidation by MAO-B; and 3) the neostriatal MPP+ content after MPTP administration to the mice. There were no significant differences in the Km values for MAO-B in the neostriatum among the strains of mice examined. Neostriatal Vmax values for MAO-B differed somewhat among the strains, with a low of 2915 +/- 172 nmol/g of tissue per hr (CD-1 mice from Charles River) and a high of 3884 +/- 203 nmol/g of tissue per hr (C57 bl mice from Taconic Farms). However, Vmax values for MAO-B in the mouse strains did not correlate significantly with the relative sensitivity of the strains to MPTP. There were no significant differences in the capacity of neostriatal synaptosomes prepared from the mouse strains to accumulate MPP+. Studies on the metabolism of MPTP after peripheral administration revealed that there was a significant (P less than .01) positive correlation between the relative sensitivity of the mouse strains to MPTP and their neostriatal MPP+ content after MPTP administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoamine oxidase and the bioactivation of MPTP and related neurotoxins: relevance to DATATOP.

The DATATOP study is a clinical trial in which deprenyl, a selective inhibitor of monoamine oxidase-B (MAO-B), is being given to newly diagnosed Parkinsonian patients in an attempt to halt the progression of their disorder. In part, this is being done because of the working hypothesis than an MPTP-like molecule may be the cause of Parkinsonism, and deprenyl is known to protect against MPTP-induced dopaminergic neurotoxicity in experimental animals. In the present study we point out that several analogs of MPTP are good substrates not only for MAO-B but also for MAO-A. In addition, we point out that with long-term administration to rodents, deprenyl loses its selectivity as an inhibitor of MAO-B and also inhibits MAO-A. We believe that these observations have relevance for the DATATOP study.

Structure-activity study of the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. I. Evaluation of the biological activity of MPTP analogs.

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and compared to MPTP for their ability to be oxidized by monoamine oxidase (MAO) and for their ability to cause nigrostriatal dopaminergic neurotoxicity in mice. Most of the compounds were oxidized by mouse brain MAO, either predominantly by the B-form or by both the A- and B-forms. The MAO-catalyzed oxidation of all of the MAO substrates resulted in the formation of dihydropyridinium intermediates which, in turn, except for the dihydropyridinium of 1-methyl-4-benzyl-1,2,3,6-tetrahydropyridine, formed pyridinium species as the final oxidation product. Nine analogs were found to be neurotoxic; all were oxidized by MAO to pyridinium compounds. However, some non-neurotoxic MPTP analogs were also oxidized by MAO. Neither 1-methyl-4-benzyl-1,2,3,6-tetrahydropyridine nor the compounds which were not substrates for MAO were neurotoxic. Also, the neurotoxicity of all of the compounds tested was blocked by inhibiting either MAO-B, MAO-A or both MAO-B and MAO-A together, indicating that MAO activity was necessary for the neurotoxicity of the compounds to be manifested. The capacity of an MPTP analog to be oxidized by MAO to a pyridinium appears to be a necessary, but not sufficient, parameter in determining the neurotoxic potential of the compound.