The effects of perturbed energy metabolism on the processing of amyloid precursor protein in PC12 cells.

The mismetabolism of amyloid precursor protein (APP), favouring the production of A beta, is considered to be central to the pathogenesis of Alzheimer's disease (AD). However it remains to be established whether the causative factor is the reported toxicity of A beta or reduced production of secretory derivatives of APP which may have trophic or neuroprotective properties. One possible contributory factor to an imbalance in APP metabolism is the impaired cellular energy availability described in AD. The aim of this study was to investigate processing of APP-like proteins following inhibition of oxidative energy metabolism in PC12 cells. Under these conditions, intracellular and secreted APP-like proteins were significantly reduced. Treatment of energy perturbed cells with the lysosomotropic agent chloroquine restored intracellular concentrations of APP-like proteins to the control range, while the secretion was completely restored by activation of protein kinase C. These findings raise the possibility that energy related metabolic stress may lead to altered metabolism of APP-like proteins favouring a potentially amyloidogenic pathway. Furthermore, the observation that activation of PKC is able to overcome this potentially pathogenic process has important implications for treatment of AD with the current generation of cholinomimetic drugs, suggesting that such drugs may slow disease progression as well as improve cognitive dysfunction.

A novel protein, amyloid precursor-like protein 2, is present in human brain, cerebrospinal fluid and conditioned media.

A monoclonal antibody, 3B11, was raised to a novel protein, amyloid precursor-like protein 2, which did not recognize amyloid precursor protein. Multiple bands were detected in human brain fractions and cell lysate by Western blotting, indicating the presence of isoforms, 3B11 immunoreactivity was also detected in cerebrospinal fluid and conditioned medium, indicating that the protein is secreted. Immunocytochemistry revealed 3B11 immunoreactivity in sections of human brain.

Effect of isonicotinic acid hydrazide on extracellular amino acids and convulsions in the rat: reversal of neurochemical and behavioural deficits by sodium valproate.

We have studied the effect of isonicotinic acid hydrazide (INH), a convulsant agent, on the extracellular levels of amino acids in the hippocampus, and the effect of sodium valproate (VPA) administration in INH-treated rats. INH (250 mg/kg) caused a rapid and sustained decrease in basal levels of GABA, and during this period convulsions of increasing severity were observed. Basal levels of glutamine, taurine, aspartate, and glutamate were unchanged by INH. When VPA was coadministered with INH, basal GABA levels were increased and no convulsions were observed. When transmitter release was evoked using 100 mM K+, the increase in dialysate GABA observed in INH-treated animals was less than that seen in controls and convulsions increased in frequency. K(+)-evoked release of glutamate and aspartate tended to be higher following INH treatment, and in the case of aspartate, this increase was significant. VPA reversed the changes in evoked release of glutamate and aspartate, and release of GABA was considerably greater than that seen in control or INH-treated rats. No drug effect on evoked changes in taurine or glutamine level was seen. These are the first data to show decreased extracellular GABA in conjunction with convulsions in freely moving animals in vivo.

Regional effects of sodium valproate on extracellular concentrations of 5-hydroxytryptamine, dopamine, and their metabolites in the rat brain: an in vivo microdialysis study.

The effects of sodium valproate (VPA; 100, 200, and 400 mg/kg, i.p.) on ventral hippocampal and anterior caudate putamen extracellular levels of dopamine (DA) and 5-hydroxytryptamine (5-HT) were examined using in vivo microdialysis. VPA induced dose-related increases in dialysate DA, 3,4-dihydroxyphenylacetic acid, and 5-HT in the ventral hippocampus. Anterior caudate putamen dialysate 5-HT was also dose dependently elevated by the drug, whereas DA levels tended to decrease with increasing VPA dose. In contrast, VPA (200, 400, and 800 mg/kg, i.p.) produced no significant elevation of DA in posterior caudate putamen dialysates, although 5-HT levels were significantly elevated at the 400- and 800-mg/kg doses. In all three regions studied, dialysate concentrations of 5-hydroxyindoleacetic acid and homovanillic acid remained at basal levels following VPA treatments. The results are discussed with regard to the possible anticonvulsant mode of action of VPA.

The effect of sodium valproate on extracellular GABA and other amino acids in the rat ventral hippocampus: an in vivo microdialysis study.

We report the effects of i.p. administration of sodium valproate (VPA) on extracellular concentrations of various amino acids in the rat ventral hippocampus studied using in vivo microdialysis, followed by HPLC with fluorometric detection. At the doses used (100, 200 and 400 mg/kg), VPA had no effect on extracellular aspartate, glutamine and taurine, whilst inducing a small, but not statistically significant increase in glutamate at 200 and 400 mg/kg. In contrast, VPA administration produced a biphasic effect on extracellular GABA levels which was dependent on the dose used. At 100 mg/kg, VPA reduced GABA concentrations by 50% when compared to basal. 200 mg/kg VPA had virtually no effect, whilst 400 mg/kg VPA raised extracellular GABA levels to 200% of basal. The results are discussed in relation to the known pharmacological and anticonvulsant actions of VPA.

Regional effects of MK-801 on dopamine and its metabolites studied by in vivo microdialysis.

The non-competitive N-methyl-D-aspartate receptor antagonist MK-801 was observed to have regionally specific effects on the extracellular concentration of dopamine and its metabolites. In rat anterior striatum, MK-801 transiently decreased extracellular dopamine, in spite of inducing intense circling behaviour which is generally associated with an increase in this neurotransmitter. In contrast, hippocampal extracellular dopamine was increased in a dose-related manner by MK-801. The possible significance of these data is discussed in relation to some of the known behavioural actions of MK-801.

MK-801 increases extracellular 5-hydroxytryptamine in rat hippocampus and striatum in vivo.

The effect of MK-801 (0.25 or 0.5 mg/kg) on the extracellular concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus and striatum was studied using intracerebral dialysis. The dialysate 5-HT concentration was dose-dependently increased by MK-801 in both regions. In the hippocampus, at the higher drug dose a slow increase in the 5-HIAA level was observed, and this became significant 3 h after treatment. In contrast to this, the extracellular 5-HIAA content in the striatum was significantly decreased 150 min after administration of both doses of MK-801. The data are discussed in the light of the known behavioural effects of MK-801 and possible N-methyl-D-aspartic acid receptor regulation of 5-HT release.

Growth cones isolated from developing rat forebrain: uptake and release of GABA and noradrenaline.

A growth cone-enriched fraction isolated from neonatal rat forebrain was shown to accumulate gamma-amino [3H]butyric acid ([3H]-GABA) and [3H]noradrenaline ([3H]NA). Uptake of both neurotransmitters was sodium- and temperature-dependent and exhibited saturation kinetics with Km values of 17.7 microM and 4.5 microM respectively and Vmax values of 114 pmol/min/mg protein and 59 pmol/min/mg protein respectively. Electron microscopic autoradiography showed that about 50% of isolated growth cones can accumulate [3H]GABA. Inhibitor studies showed that beta-alanine was a relatively weak inhibitor of [3H]GABA uptake compared to nipecotic acid and diamino-butyric acid. Growth cone fractions preloaded with [3H]GABA and [3H]NA demonstrated a K+ (25 mM) -induced release of both neurotransmitters. Of the K+-stimulated release of [3H]GABA 50% was Ca2+-dependent, whereas the release of [3H]NA was entirely Ca2+-independent.

Uptake and release of [3H]GABA by growth cones isolated from neonatal rat brain.

A subcellular fraction highly enriched in neuronal growth cones was isolated from 5-day-old rat forebrain by a recently described method. The growth cone fraction was shown to have a sodium- and temperature-dependent, high-affinity (Km = 4.4 microM) uptake system for [3H]GABA. Electron microscopic autoradiography confirmed that this uptake was into growth cones since only these structures were heavily labelled with silver grains. High potassium induced the release of newly accumulated [3H]GABA from the growth cone fraction, about half of which was Ca2+-dependent. The presence of uptake and release systems for GABA in growth cones may simply reflect the development of growth cones into nerve terminals. Alternatively, these observations may indicate a role for neurotransmitter release in synaptogenesis.

[3H]Kainic acid binding and choline acetyltransferase activity in Alzheimer's dementia.

The binding of [3H]kainic acid to caudate nucleus membranes prepared from brains of examples of Alzheimer's dementia and controls has been determined. No changes were detected in either the affinity or the number of kainate binding sites in the Alzheimer samples compared to control, although there was a large decrease in choline acetyltransferase activity.

Neurotransmitter dysfunction and atrophy of the caudate nucleus in Alzheimer's disease.

The caudate nucleus from examples of Alzheimer's disease (mean age 68, range 51-77 yr) had a mean wet weight and total protein content that were significantly lower than control. Biochemical markers of various specific nerve cells were determined. These are thought to reflect intrinsic cholinergic neurones (choline acetyltransferase activity) and corticostriatal (L-[3H]glutamate binding), nigrostriatal (dopamine and homovanillate concentrations), and ascending brain stem (serotonin, 5-hydroxyindoleacetate, and noradrenaline concentrations) tracts. There is evidence of selective vulnerability, with cholinergic, dopaminergic, and possibly glutamergic neurons being affected, but not serotonergic and noradrenergic cells. Dopaminergic neurons are probably not markedly reduced in number, but may not be fully operating. Some observations made for the monoamines, as well as the alteration in L-[3H]glutamate binding, seem to lay emphasis on the importance of cortical pathology in Alzheimer's disease.

Induction of a stress protein in developing cell cultures of the rat cerebellum.

We examined the ability of developing cerebellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd2+ treatment. The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used. Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd2+ was effective only in cultures at 7 days of age and older. These findings are discussed with reference to the development of various cell types in these cultures.

Tetanus toxin inhibition of K+-stimulated [3H]GABA release from developing cell cultures of the rat cerebellum.

The effect of tetanus toxin pretreatment on K+-stimulated [3H]gamma-aminobutyric acid release from neuron-enriched cerebellar cell cultures at various stages during their development in vitro was assessed. Tetanus toxin had little inhibitory effect on immature (1-3-day-old) cultures, but markedly reduced K+-evoked [3H]gamma-aminobutyric acid release from 7- and 14-day-old cultures (approximately 80% inhibition). It is suggested that cerebellar neurons in culture develop tetanus toxin-sensitive transmitter release mechanisms similar to their in vivo counterparts.

Autoreceptors modify the evoked release of [3H]GABA from cerebellar neurons in dissociated cell culture.

We investigated the effect of GABA, muscimol and THIP on the K+ -stimulated and spontaneous release of [3H]GABA from neuron-enriched cell cultures of the rat cerebellum. Each agonist produced significant reductions in evoked [3H]GABA without causing marked changes in spontaneous release. The agonist-induced inhibition of K+ -stimulated [3H]GABA release was reversed by the GABA antagonists bicuculline and picrotoxin. It is suggested that GABAergic neurons in cerebellar cell cultures possess GABA receptors which are involved in the regulation of evoked transmitter release.

Potassium-stimulated, calcium-dependent release of [3H]GABA from neuron- and glia-enriched cultures of cells dissociated from rat cerebellum.

The release of [3H]GABA from cultures of cells dissociated from the rat cerebellum was investigated. The culture system contained a population of neurons (stellate and basket cells) which were capable of accumulating [3H]GABA and releasing it in a calcium-dependent manner in response to 50 mM potassium. In addition, the release of [3H]GABA for cultured astrocytes was found to be insensitive to potassium depolarization.

A simple perfusion chamber for studying neurotransmitter release from cells maintained in monolayer culture.

A perfusion chamber is described for studying the efflux of putative neurotransmitters from CNS cells maintained in monolayer culture. We have used this apparatus to investigate the efflux of newly accumulated [3H]GABA from cell cultures of the early postnatal rat cerebellum.