guanosine-5--o-(3-thiotriphosphate) and Nerve-Degeneration

We report the effects of amyloid precursor protein (APP) fragment 714-723 (APP(714-723); peptide P1) and its V717F and V717G mutants (peptides P2 and P3, respectively) on G-protein activity ([35S]GTPgammaS binding) in membranes from postmortem human control and Alzheimer's disease (AD) brains. The peptides P1, P2, and P3 revealed a significant stimulatory effect on [35S]GTPgammaS binding in control temporal cortex. The most potent stimulator, P3, at 10 microM concentration enhanced [35S]GTPgammaS binding by 500%. The effect was threefold stronger than that for wild-type P1 and twofold stronger than that for P2. In sporadic AD, the stimulatory effect of P1, P2, and P3 on G-proteins was reduced significantly whereas in Swedish familial AD (SFAD), only P1 elicited marked stimulation (at 10 microM by 50%). In control sensory postcentral cortex, the stimulation of G-proteins by P3 was 1.5-fold lower than that in control temporal cortex, whereas in AD and SFAD the effect showed no remarkable regional difference. Treatment of membranes with H2O2 produced 1.5-fold higher stimulation in [35S]GTPgammaS binding to temporal cortex than that in binding to sensory postcentral cortex. In AD and SFAD, the stimulation by H2O2 revealed no significant regional difference. Glutathione, desferrioxamine (DFO), and 17beta-estradiol markedly decreased the strong stimulatory effect by P3 on [35S]GTPgammaS binding to control temporal cortex, with the protective effect by DFO being most potent. The G(alphaO)-protein levels were not changed in AD or SFAD brain membranes as compared to levels in control membranes. We suggest that strong G-protein stimulation by P3 in the human brain implies the specific (per)oxidation mechanism that might be affected by regional content of peroxidizing substrates and antioxidants.

Topics: Aged; Alzheimer Disease; Amyloid beta-Protein Precursor; Antioxidants; Cell Membrane; Cerebral Cortex; Deferoxamine; Estradiol; Female; Glutathione; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Hydrogen Peroxide; Lipid Peroxidation; Male; Mutation; Nerve Degeneration; Oxidative Stress; Peptide Fragments; Protein Binding

The neuropeptide galanin is expressed in the mammalian central nervous system and has been implicated in neurotrophic actions. Central galanin administration induces cognitive deficits in rodents and inhibits the release of acetylcholine in the hippocampus. In addition, a galanin hyperinnervation of the basal forebrain cholinergic cells in Alzheimer's disease patients has been reported. To evaluate the effect of galanin treatment on galanin and muscarinic cholinergic receptor G protein coupling, galanin was administered into the lateral ventricle of rats via an implanted cannula. Galanin or muscarinic receptor functional coupling to G proteins was quantified by galanin or carbachol stimulation of guanosine 5'-(gamma-[35S]thio)triphosphate binding in rat brain slices. Guanosine 5'-(gamma-[35S]thio)triphosphate basal binding in nucleus basalis of Meynert and thalamic nuclei was increased in the vehicle treated group. This effect was reverted by galanin treatment and indicates that the surgery increased receptor functional coupling to G proteins, which is restored by a possible neurotrophic action mediated by galanin. In addition, in galanin administered animals, galanin-stimulated binding was increased in the amygdala but decreased in the diagonal band, whilst binding stimulation mediated by carbachol was found to be increased in the amygdala, thalamic nuclei and diagonal band. These findings indicate that galanin treatment modulates the coupling of galanin and muscarinic cholinergic receptors to G proteins in specific regions of the rat central nervous system.

Topics: Alzheimer Disease; Animals; Disease Models, Animal; Galanin; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Nerve Degeneration; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Galanin; Receptors, Muscarinic; Sulfur Radioisotopes

A1 adenosine receptors (A1ARs) are widely expressed in the brain during development. To examine whether A1AR activation can alter postnatal brain formation, neonatal rats from postnatal days 3 to 14 were treated with the A1AR agonist N6-cyclopentyladenosine (CPA) in the presence or absence of the peripheral A1AR antagonist 8-(p-sulfophenyl)-theophylline (8SPT). CPA or CPA + 8SPT treatment resulted in reductions in white matter volume, ventriculomegaly, and neuronal loss. Quantitative electron microscopy revealed reductions in total axon volume following A1AR agonist treatment. We also observed reduced expression of myelin basic protein in treated animals. Showing that functional A1ARs were present over the ranges of ages studies, high levels of specific [3H]CCPA binding were observed at PD 4, 7 and 14, and receptor-G protein coupling was present at each age. These observations show that activation of A1ARs with doses of CPA that mimic the effects of high adenosine levels results in damage to the developing brain.

Topics: Adenosine; Animals; Animals, Newborn; Body Weight; Cell Count; Cerebral Cortex; Cerebral Ventricles; Drug Combinations; Drug Interactions; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Hippocampus; Microscopy, Electron; Myelin Basic Protein; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroglia; Neurons; Presynaptic Terminals; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P1; Telencephalon; Theophylline

Prosaposin-derived peptides have been proposed as potential therapeutics for neurodegenerative diseases. Previously, we reported that the minimal length for bioactivity was 12 amino acids, and key amino acids were described based on interspecies conservation. In this article, we have further investigated the sequence requirements for bioactive Prosaptide (Myelos Corporation) peptides in terms of length and amino acid identity. The use of Prosaptide peptides for treatment of central nervous system (CNS) disorders requires that they are stable in vivo. Although robust effects of our prototypical peptide Prosaptide TX14(A) have been shown in the peripheral nervous system, minimal success has been achieved when treating the CNS in rats and this may be due to instability of Prosaptide TX14(A) in brain. Herein, we demonstrate that, indeed, Prosaptide TX14(A) is rapidly degraded in the brain and we have attempted to design prosaptides with increased CNS stability. One peptide, Prosaptide TX15-2, shows increased stability in brain and may be of use in the treatment of CNS disorders. With the aim of designing Prosaptide peptides that may be systemically administered for CNS treatment, we have investigated the blood-brain barrier permeability of Prosaptide TX14(A) and TX15-2. Both of these peptides cross the blood-brain barrier via a nonspecific mechanism.

Topics: Animals; Biological Transport; Blood-Brain Barrier; Brain; Cells, Cultured; Drug Design; Glycoproteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Nerve Degeneration; Neurites; Neurons; Neuroprotective Agents; Protein Precursors; Rats; Rats, Sprague-Dawley; Saposins; Structure-Activity Relationship

Weaver mutant mice have a selective degeneration of the nigrostriatal dopamine pathway arising between 7-21 days after birth. The goal of this study was to investigate the effects of this mutation on different parameters of the nigrostriatal and mesolimbic dopamine system: apparent D1 and D2 receptor binding sites as well as their signal transduction pathway. Using quantitative autoradiography of ligands for dopamine D1, D2 receptors and the dopamine uptake site, we found a significant loss in apparent D1 receptor binding sites throughout the neostriatum, significant increase of apparent D2 receptor binding in the dorsal aspect of the neostriatum, and almost complete loss of DA uptake sites in these regions of the weaver mouse. In contrast to the neostriatum, the density of dopamine receptors and uptake sites in the nucleus accumbens of the weaver mouse did not differ from controls. Despite alterations in the binding of apparent D1 and D2 receptors, there was no significant difference in either basal, DA stimulated or GTPgammaS stimulated cAMP production. These findings suggest the down-regulation of apparent D1 receptor binding sites reported in this model, probably does not reflect an important physiological mechanism through which these animals compensate for loss of dopamine innervation.

Topics: Adenylyl Cyclases; Animals; Autoradiography; Benzamides; Benzazepines; Cocaine; Corpus Striatum; Cyclic AMP; Dopamine; Dopamine Antagonists; Down-Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Limbic System; Mice; Mice, Neurologic Mutants; Nerve Degeneration; Nucleus Accumbens; Receptors, Dopamine D1; Receptors, Dopamine D2; Reference Values; Signal Transduction; Substantia Nigra; Tritium

Considerable interest has recently focused on the weaver mutation, which causes inward rectifier channel alterations leading to profound impairment of neuronal differentiation and to severe motor dysfunction in mice (Hess, 1996). The principal targets of mutation are cerebellar granule cells, most of which fail to differentiate and degenerate in a premigratory position (Rakic and Sidman, 1973a,b). Two hypotheses have been put forward to explain the pathogenetic role of mutant inward rectifier channels: namely that inward rectifier channel activity is either lacking (Surmeier et al., 1996) or altered (Kofuji et al., 1996; Silverman et al., 1996; Slesinger et al., 1996). We have examined this question by recording inward rectifier currents from cerebellar granule cells in situ at different developmental stages in wild-type and weaver mutant mice. In wild-type mice, the inward rectifier current changed from a G-protein-dependent activation to a constitutive activation as granule cells developed from premigratory to postmigratory stages. In weaver mutant mice, G-protein-dependent inward rectifier currents were absent in premigratory granule cells. A population of putative granule cells in the postmigratory position expressed a constitutive inward rectifier current with properties compatible with mutated GIRK2 channels expressed in heterologous systems. Because granule cells degenerate at the premigratory stage (Smeyne and Goldowitz, 1989), the loss of inward rectifier current and its regulation of membrane potential are likely to play a key role in the pathogenesis of weaver neuronal degeneration.

Topics: Anesthetics, Local; Animals; Cell Movement; Cerebellum; Female; G Protein-Coupled Inwardly-Rectifying Potassium Channels; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Lidocaine; Male; Membrane Potentials; Mice; Mice, Neurologic Mutants; Nerve Degeneration; Patch-Clamp Techniques; Potassium Channels; Potassium Channels, Inwardly Rectifying; Receptors, Muscarinic

Chemicals which affect different steps of the phototransduction cascade were used to identify the site of action of the rdgB gene product of Drosophila. In the rdgB mutant, the photoreceptor cells degenerate after several days of exposure to light, whereas raising the flies in the dark largely prevents the degeneration. In the rdgBKS222 mutant, which was used in the present studies, the light-induced degeneration is characterized by (1) selective degeneration of the peripheral but not the central photoreceptor cells; (2) random distribution of degenerated cells among ommatidia; and (3) the degeneration is specific to the rdgB but not the wild-type photoreceptor cells. In the present study, we show that application of specific chemical agents to the eyes of rdgBKS222 flies in the dark mimics the effects of light and causes retinal degeneration indistinguishable from light. The agents used in these studies are the metabolically stable GTP analogs GTP gamma S and Gpp(NH)p as well as fluoride ions, which are known to activate the transducing guanine nucleotide binding protein (G-protein of fly photoreceptors). It is unlikely that the chemically induced retinal degeneration is mediated by effects on energy metabolism, since application of the metabolic inhibitors CN- and 2-deoxy-D-glucose did not increase the extent of retinal degeneration over that observed in control flies treated with Ringer solution. The GDP analog GDP beta S, which inhibits G-protein activity, greatly reduced the extent of retinal degeneration in the dark, over that observed in control flies treated with Ringer solution. These results suggest that activation of the G-protein precedes the step in the transduction cascade that leads to retinal degeneration and provides a powerful tool to investigate the molecular mechanism of light-induced degeneration in the rdgB mutant.

Topics: Animals; Darkness; Drosophila melanogaster; Electrophysiology; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanylyl Imidodiphosphate; Mutation; Nerve Degeneration; Retina