Bruce D. Howard

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β‐Bungarotoxin, a presynaptically‐acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ‐aminobutyric acid and 2‐deoxy‐d‐glucose. The toxin‐induced efflux of γ‐aminobutyric acid occurred by a Na+ ‐dependent process while that of 2‐deoxyglucose was Na+ ‐independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β‐bungarotoxin, synaptosomes exhibited increased production of 14CO2 from [U‐14C]glucose and decreased ATP levels. β‐Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin‐treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β‐bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β‐bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.

A dopaminergic cell line variant resistant to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is known to cause parkinsonism by killing dopaminergic neurons; the toxic substance is a metabolite, 1‐methyl‐4‐phenylpyridinium ion (MPP+). PC12 cells, which are dopaminergic, are killed in culture by MPTP and MPP+ but at concentrations much higher than that required to kill affected neurons in vivo. However, at low concentrations (10–100 μM), MPP+ caused an increased production of lactate by PC12 cells. MPP+‐treated PC12 cells exhibited decreased mitochondrial respiration. Mitochondria from the treated cells respired normally in the presence of added succinate but not β‐hydroxybutyrate, a finding indicating that MPP+ inhibits the oxidation of some substrates selectively. MPP+ was more effective in killing the cells when glycolysis was reduced with 2‐deoxyglucose or by lowering the glucose content of the culture medium. Under these conditions, MPP+ inhibited ATP synthesis and depleted cellular stores of ATP. A PC12 variant that is even more resistant to MPTP and MPP+ than are wild‐type cells has been isolated. The MPTP‐resistant variant is also more resistant to the lethal effects of oligomycin, antimycin A, and rotenone. This variant exhibited altered lactate production and mitochondrial respiration. It is suggested that some brain neurons that accumulate MPP+ without being killed by it may also have an energy metabolism somewhat different from that of more sensitive neurons.

Rat C6 and human astrocytic tumor cells express a neuronal type of glutamate transporter

C6 glioma cells take up aspartate and glutamate by a Na(+)-dependent transporter. Using the polymerase chain reaction and degenerate oligonucleotide primers corresponding to conserved regions of previously cloned glutamate transporters, we isolated from these cells a partial cDNA clone with a sequence of the neuronal type EAAC1 glutamate transporter. The cells express a 4.4 kb message that hybridizes to this cDNA, and they do not express either of the previously described glial type glutamate transporters, GLT-1 or GLAST. The cells were sensitive to the toxic aspartate analog alanosine, which enters the cells by a glutamate transporter. Several human brain tumors examined, including some astrocytic tumors, expressed the EAAT3 glutamate transporter, which is the human homolog of the rodent EAAC1 transporter. Some of the tumors also expressed the other types of glutamate transporter.

Phage Lambda Mutants Deficient in rII Exclusion

The lambda gene responsible for rII exclusion is distinct from other lambda genes and lies between the N and CI genes on the genetic map.

β-NEUROTOXIN REDUCES NEUROTRANSMITTER STORAGE IN BRAIN SYNAPSES

β‐neurotoxin, a component of Bungarus multicinctus venom, is known to cause neuromuscular blockade by first increasing the rate of spontaneous ACh release and then inhibiting the nerve impulse‐induced release of ACh. We report that the toxin also affects the storage of several neurotransmitters in rat brain and it is active on synaptosomes and brain minces. Synaptosomes prepared from brain tissue that had been treated with β‐toxin have a reduced ability to accumulate radioactive NE, GABA, serotonin and the ACh precursor, choline. The toxin also causes a release of previously accumulated NE and GABA from synaptosomes, suggesting that the storage process rather than the uptake transport process is affected. The toxin does not contain phospholipase A, phospholipase C, protease or hyaluronidase activity.

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K+ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K+ media. When labeled choline was present during the release incubation, a‘newly‐synthesized’pool of acetylcholine was formed in the tissue. In toxin‐treated slices exposed to high K+, both the production and the release of this‘newly‐synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [3H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxins interaction with the mechanisms regulating acetylcholine turnover.

IMPAIRED DIFFERENTIATION OF HPRT-DEFICIENT DOPAMINERGIC NEURONS : A POSSIBLE MECHANISM UNDERLYING NEURONAL DYSFUNCTION IN LESCH-NYHAN SYNDROME

Lesch‐Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self‐mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine‐guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT‐deficient PC12 mutants that have a normal or near normal dopamine content (55–97% of that of wild‐type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor‐induced differentiation is near normal under these conditions in PC12 HPRT‐deficient mutants containing much lower dopamine levels (<8% of that of wild type cells), indicating a neurotoxic effect of the endogenous dopamine in the mutants. The degree of inhibition of the de novo pathway of purine synthesis was the same in both classes of HPRT‐deficient mutants. Expression of BCl‐2 in a PC12 mutant that has a normal dopamine content allowed partial NGF‐induced differentiation suggesting that the apoptotic pathway might be involved in the failure of differentiation when the de novo pathway of purine synthesis is partially inhibited. J. Neurosci. Res. 53:78–85, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Inhibition of dopamine uptake by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a cause of parkinsonism

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism in man and monkeys, producing behavioral effects within 5 min of administration. The compound reversibly and competively inhibited (IC50 = 2 microM) dopamine uptake into PC12, a clonal line of rat pheochromocytoma cells that store and secrete dopamine and acetylcholine. Uptake of choline and 2-deoxyglucose was not affected. Prolonged exposure to the compound was lethal to PC12; survivors of this treatment lost the ability to store dopamine and acetylcholine and to extend neurites upon incubation with nerve growth factor.

PC12 Variants Deficient in Catecholamine Transport

Abstract We have isolated PC12 cell variants deficient in transporter‐mediated uptake of 3,4‐dihydroxyphenylethylamine (dopamine). The variants either were obtained nonselectively, or they were selected by resistance to guanethidine or N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). Dopamine uptake into guanethidine‐resistant cells occurred with a decreased Vmax; the Km for dopamine and inhibition by guanethidine were normal. MPTP‐resistant cells lacked the capacity to take up dopamine. Most of the variants resembled wild‐type PC12 in their response to nerve growth factor and the storage and secretion of dopamine. MPTP‐resistant cells exhibited several deficiencies in addition to dopamine transport, i.e., no measurable storage of dopamine or acetylcholine and no observable response to nerve growth factor. Wild‐type and variant cells were compared with respect to the labeling of cell proteins with [3H]xylamine, which binds covalently to certain proteins apparently only after entering PC12 via the catecholamine transporter. When intact variant cells were used, there was markedly reduced labeling of the proteins by [3H]xylamine. Almost all of these proteins were readily labeled when cell homogenates were exposed to [3H]xylamine. However, MPTP‐resistant cells were missing three of these proteins. Northern blot analysis with cDNA clones revealed that the MPTP‐resistant cells had markedly reduced levels of several specific mRNA species.

Wnt Signaling Induces GLT‐1 Expression in Rat C6 Glioma Cells

Abstract : The regulation of glial and neuronal Na+ ‐dependent glutamate/aspartate transporters is of interest because abnormal glutamate transport may be responsible for certain neurological diseases. Because expression of the Wnt‐1 protooncogene results in induction of the glial‐type glutamate transporter GLAST in PC12 neuron‐like cells, we have evaluated the effect of Wnt‐1‐induced signaling on glutamate transporter expression in rat C6 glioma cells. C6 cells are known normally to express EAAC1, a neuronal glutamate transporter, but not the GLAST or the GLT‐1 glutamate transporter. C6 cells that ectopically expressed Wnt‐1 contained a GLT‐1 RNA species similar in size (> 10 kb) to the GLT‐1 transcript present in rat brain, and they also contained a previously unreported 3.3‐kb GLT‐1 RNA species. Both GLT‐1 RNAs contain large parts of the coding region. However, the 3.3‐kb GLT‐1 species contains at least one small deletion within the coding region. The Wnt‐1‐expressing C6 cells contained little, if any, GLT‐1 protein as determined by immunological techniques. We suggest that one or both of the GLT‐1 RNA species induced by Wnt‐1 either fail to be translated or yield abnormal translation products that are quickly degraded. Wnt‐1‐expressing C6 cells may thus represent a novel in vitro system for studying GLT‐1 transporter expression at the transcriptional and/or posttranscriptional levels.