guanosine-triphosphate and Alzheimer-Disease

Increased interest in the aging and Alzheimer's disease (AD)-related impairments in autophagy in the brain raise important questions about regulation and treatment. Since many steps in endocytosis and autophagy depend on GTPases, new measures of cellular GTP levels are needed to evaluate energy regulation in aging and AD. The recent development of ratiometric GTP sensors (GEVALS) and findings that GTP levels are not homogenous inside cells raise new issues of regulation of GTPases by the local availability of GTP. In this review, we highlight the metabolism of GTP in relation to the Rab GTPases involved in formation of early endosomes, late endosomes, and lysosomal transport to execute the autophagic degradation of damaged cargo. Specific GTPases control macroautophagy (mitophagy), microautophagy, and chaperone-mediated autophagy (CMA). By inference, local GTP levels would control autophagy, if not in excess. Additional levels of control are imposed by the redox state of the cell, including thioredoxin involvement. Throughout this review, we emphasize the age-related changes that could contribute to deficits in GTP and AD. We conclude with prospects for boosting GTP levels and reversing age-related oxidative redox shift to restore autophagy. Therefore, GTP levels could regulate the numerous GTPases involved in endocytosis, autophagy, and vesicular trafficking. In aging, metabolic adaptation to a sedentary lifestyle could impair mitochondrial function generating less GTP and redox energy for healthy management of amyloid and tau proteostasis, synaptic function, and inflammation.

Topics: Alzheimer Disease; Autophagy; Brain; Endocytosis; Guanosine Triphosphate; Humans; rab GTP-Binding Proteins

Transglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. During the last two decades its additional enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, protein kinase) and non-enzymatic (multiple interactions in protein scaffolds) activities, which do not require Ca(2+) , have been recognized. It became a prevailing view that TG2 is silent as a transamidase, except in extreme stress conditions, in the intracellular environment characterized by low Ca(2+) and high GTP concentrations. To counter this presumption a critical review of the experimental evidence supporting the role of this enzymatic activity in cellular processes is provided. It includes the structural basis of TG2 regulation through non-canonical Ca(2+) binding sites, mechanisms making it sensitive to low Ca(2+) concentrations, techniques developed for the detection of protein transamidation in cells and examples of basic cellular phenomena as well as pathological conditions influenced by this irreversible post-translational protein modification.

Topics: Aging; Alternative Splicing; Alzheimer Disease; Amino Acid Sequence; Animals; Binding Sites; Calcium; Cross-Linking Reagents; Enzyme Activation; Extracellular Space; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Huntington Disease; Molecular Sequence Data; Oxidation-Reduction; Protein Glutamine gamma Glutamyltransferase 2; Protein Processing, Post-Translational; Sequence Alignment; Transglutaminases

Topics: Administration, Inhalation; Alzheimer Disease; Animals; Binding Sites; Brain; Chelating Agents; Edetic Acid; Egtazic Acid; Glutamic Acid; Guanosine Triphosphate; Humans; Isotope Labeling; Mercury; Microtubules; Nucleotides; Rats; Tubulin

A major cytoskeletal and molecular change of yet unknown mechanism is the accumulation of argentophilic neurofibrillary tangles in certain selected neurons of the aged human brain, especially the Alzheimer disease/senile dementia of the Alzheimer type (AD/SDAT). Increasing evidence suggests an association between neurofibrillary tangles and a defective microtubule (MT) assembly in AD/SDAT brain. Tubulin, the basic unit of MT is an alpha-beta heterodimer protein of about 55,000 molecular weight each. The beta subunit of tubulin has an exchangeable (E) binding site for guanosine triphosphate (GTP) which in the presence of microtubule-associated proteins (MAPS) promotes the MT assembly. The 8-azido GTP, a photoaffinity analog of GTP, which has been proven to mimic the natural ligand in promoting MT assembly, was used to examine the GTP binding of beta- tubulin in AD brain. Preliminary studies revealed a minimal photoinsertion into the beta-subunit of tubulin in AD/SDAT brain homogenate. Furthermore, the exchangeable GTP binding site was also unavailable in brain tubulin isolated from most of the AD/SDAT cases.

Topics: Alzheimer Disease; Binding Sites; Brain; Guanosine Triphosphate; Humans; Microtubules; Neurofibrils; Tubulin

The endocytic pathway is a system of dynamically communicating vesicles, known as early endosomes, that internalize, sort, and traffic nutrients, trophic factors, and signaling molecules to sites throughout the cell. In all eukaryotic cells, early endosome functions are regulated by Rab5 activity, dependent upon its binding to GTP, whereas Rab5 bound to GDP represents the biologically inactive form. An increasing number of neurodegenerative diseases are associated with endocytic dysfunction and, in the case of Alzheimer's disease (AD) and Down syndrome (DS), an early appearing highly characteristic reflection of endocytic pathway dysfunction is an abnormal enlargement of Rab5 positive endosomes. In AD and DS, endosome enlargement accompanying accelerated endocytosis and fusion, upregulated transcription of endocytosis-related genes, and aberrant signaling by endosomes are caused by pathological Rab5 overactivation. In this chapter, we describe a battery of methods that have been used to assess Rab5 activation in models of AD/DS and are applicable to other cell and animal disease models. These methods include (1) fluorescence recovery after photobleaching (FRAP) assay; (2) quantitative measurement of endosome size by light, fluorescence and electron microscopy; (3) detection of GTP-Rab5 by in situ immunocytochemistry in vitro and ex vivo; (4) immunoprecipitation and GTP-agarose pull-down assay; (5) biochemical detection of Rab5 in endosome-enriched subcellular fractions obtained by OptiPrep™ density gradient centrifugation of mouse brain.

Topics: Alzheimer Disease; Animals; Down Syndrome; Endocytosis; Endosomes; Guanosine Triphosphate; Mice; rab5 GTP-Binding Proteins

β-Amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer's disease (AD). Endosomes are highly active APP processing sites, and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates rab5 overactivation in Down syndrome (DS) and AD, which is caused by elevated levels of the β-cleaved carboxy-terminal fragment of APP (βCTF). βCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. βCTF levels are also elevated in AD brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 overactivation through βCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.

Topics: Adaptor Proteins, Signal Transducing; Aged; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Brain; Cell Line, Tumor; Cells, Cultured; Down Syndrome; Endosomes; Female; Fibroblasts; Gene Knockdown Techniques; Guanosine Triphosphate; HEK293 Cells; Humans; Male; Mice, Inbred C57BL; Middle Aged; Neurons; rab5 GTP-Binding Proteins

The aim of the present work was to analyze the status of metabotropic glutamate receptors (mGluRs) in the frontal cortex (area 8) from ten cases with common form DLB (cDLB) and eleven cases with pure AD in comparison with five age-matched controls. mGluRs, determined by radioligand binding assays, were significantly decreased in cerebral cortex in cDLB. This decrease was already present in cases with early AD changes not involving the frontal cortex, but dramatically correlated with AD neuropathological changes, at its greatest in isocortical stages, which was associated with a decrease in the expression levels of mGluR1 detected by Western blotting. Moreover, mGluRs analyzed in pure AD were lower than those obtained in cDLB and also correlated with progression of illness. On the other hand, the expression levels of phospholipase Cbeta1 (PLCbeta1) isoform, which is the effector of group I mGluRs, was decreased in parallel in cDLB cases. Finally, the PLCbeta1 decrease was associated with reduced GTP- and l-glutamate-stimulated PLC activity in both cDLB and AD cases. These results show that group I mGluRs/PLC signaling are down-regulated and desensitized in the frontal cortex in cDLB and AD cases and that these modifications worsen with progression of AD changes in the cerebral neocortex. Therefore, group I mGluR dysfunction may be implicated in the pathogenesis of cognitive impairment and dementia in common form of DLB and pure AD.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Binding, Competitive; Disease Progression; Down-Regulation; Enzyme Activation; Female; Frontal Lobe; Glutamic Acid; Guanosine Triphosphate; Humans; Isoenzymes; Lewy Body Disease; Male; Middle Aged; Neurons; Phospholipase C beta; Radioligand Assay; Receptors, Metabotropic Glutamate; Signal Transduction; Type C Phospholipases

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.

Topics: Alzheimer Disease; Animals; Cell Line; Cytoskeleton; Guanosine Triphosphate; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Mice; Microglia; Monocytes; Phagocytosis; Protein Prenylation; rho GTP-Binding Proteins

We examined the quantity and quality of G proteins in membrane preparations of post-mortem human brain, i.e. in parietal, temporal and occipital cortical regions, from normal subjects over age (17-89 years old) and with Alzheimer's disease (AD) in comparison with aged-matched controls. In normal aging, the immunoreactivities determined of G(ialpha), G(qalpha) and G(beta) were inversely correlated with age. The function of G proteins was examined by photoaffinity GTP analogue [azidoanilido GTP (AAGTP)] labelling. AAGTP labelling to G(salpha) and G(i/oalpha), and the ratio of G(salpha) to G(i/oalpha) AAGTP labelling showed no age-dependent changes. In AD compared to age-matched controls, there were no significant differences in the levels of G(sHalpha), G(sLalpha), G(ialpha), G(oalpha), G(qalpha) and G(beta) subunits. Functional effects of G proteins, however, as measured by AAGTP labelling to G(salpha), but not to G(i/oalpha), was significantly decreased in AD compared to controls in the parietal and temporal cortex, but not in the occipital cortex. These results suggest that the disturbances of post-receptor trans-membrane signalling in AD can be attributed to functional changes of G(salpha), and these are independent of alterations in the level for those proteins in normal aging.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Alzheimer Disease; Azides; Case-Control Studies; Cerebral Cortex; GTP-Binding Protein alpha Subunits, Gs; Guanosine Triphosphate; Humans; Immunoblotting; Middle Aged; Photoaffinity Labels; Postmortem Changes; Synaptic Membranes; Time Factors

To determine changes in signal transduction from the muscarinic acetylcholine receptor (mAChR) to G protein in brain tissue of dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), we investigated GTP-sensitive agonist high affinity binding, which is considered an index of the formation of the mAChR-G protein complex. Brain tissue was obtained at necropsy from eight patients with DLB, nine patients with Alzheimer's disease and seven patients as controls. Membrane fractions were prepared from frontal and temporal cerebral tissues. Displacement curves of [(3)H]l-quinuclidinyl benzilate (QNB) binding by carbamylcholine were analyzed by the nonlinear least-squares methods. The proportion of and affinity for the agonist in GTP-sensitive agonist high affinity binding were estimated. The percentages GTP-sensitive agonist high affinity bindings were significantly decreased in DLB (P<0.01) and Alzheimer's disease (P<0.05) only in the frontal lobe. There were no significant differences in the temporal lobe. The ratio of agonist affinity (Kd value of low affinity component/Kd value of high affinity component) did not significantly differ among groups in either the frontal lobe or temporal lobe. The concentration of mAChR-G protein complex is considered reduced in the frontal lobe of brains with DLB and Alzheimer's disease. Therefore, signal transduction from mAChR to G protein was disturbed in the frontal lobe in these diseases.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Analysis of Variance; Autopsy; Binding, Competitive; Brain; Carbachol; Cholinergic Agonists; Female; Guanosine Triphosphate; Humans; Lewy Body Disease; Male; Middle Aged; Muscarinic Antagonists; Postmortem Changes; Quinuclidinyl Benzilate; Receptors, Muscarinic

Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G-proteins) couple many different cell surface receptor types to intracellular effector mechanisms. Uncoupling between receptors and G-proteins and between G-proteins and adenylyl cyclase (AC) and phospholipase C (PLC) has been described for Alzheimer's disease (AD) brain. However, there is little information on whether altered G-protein signaling in AD is just an end-stage phenomenon or is important for the progression of disease pathology. Here we used [(35)S]GTPgammaS autoradiography to study G-protein distribution in sections of entorhinal cortex and hippocampus from 23 cases staged for neurofibrillary changes and amyloid deposits according to Braak and Braak (Acta Neuropathol. [1991] 82:239-259). We also studied the effects of GTP, which has been found to increase [(35)S]GTPgammaS binding in an Mg(2+)-dependent manner. Results show that the ability of GTP (3 microM) to stimulate [(35)S]GTPgammaS binding declined significantly with staging for neurofibrillary changes in the entorhinal cortex (P < 0.05, ANOVA) and CA1 subfield of the hippocampus (P < 0.05, ANOVA). No significant changes were seen for [(35)S]GTPgammaS binding in the absence of GTP. Our results suggest a decrease in G-protein GTP hydrolysis, which correlates with the progression of AD neurofibrillary changes, in the regions most affected by this pathology. These alterations appear to occur prior to stages corresponding to clinical disease and could lead to an impaired regulation of several signaling systems in AD brain.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid; Autoradiography; Binding, Competitive; Disease Progression; Entorhinal Cortex; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hippocampus; Humans; Magnesium; Male; Middle Aged; Neurofibrillary Tangles; Severity of Illness Index; Sulfur Radioisotopes; Tissue Distribution

The YWK-II cDNA, RSD-2, encoding a sperm membrane protein was isolated from a rat testis cDNA expression library. Using the RSD-2 insert in combination with rapid amplification of cDNA ends (RACE), the corresponding human gene was isolated from a human testis cDNA expression library. The human testis cDNA, HSD-2, is 3654 bp in length and contains an open reading frame of 763 codons. Hydropathicity analysis showed that the deduced polypeptide is a single strand transmembrane protein. The deduced polypeptide has partial homology with the amyloid precursor protein (APP) and high homology with the amyloid precursor homologue, APLP2/APPH. The YWK-II gene was mapped and assigned to human chromosome locus: 11q24-25. Northern blotting of various human tissue RNAs using the HSD-2 cDNA as a probe showed that the gene is transcribed ubiquitously. The cytoplasmic domain of HSD-2 was expressed in Escherichia coli. In-vitro studies showed that the recombinant polypeptide bound to a GTP-binding protein (G(o)) and was phosphorylated by protein kinase C and cdc2 kinase. In mammalian F11 cells, the recombinant polypeptide was found to be coupled to G(o). Thus, the YWK-II component has the characteristics of a G(o)-coupled receptor and may be involved in G(o)-mediated signal transduction pathway. Protein kinase C and cdc2 kinase may regulate this pathway in spermatozoa by phosphorylating the cytoplasmic domain of the YWK-II component.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Base Sequence; Binding Sites; Chromosome Mapping; Chromosomes, Human, Pair 11; Cytoplasm; DNA, Complementary; Gene Expression; Glutathione Transferase; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Humans; Male; Membrane Proteins; Molecular Sequence Data; Nerve Tissue Proteins; Phosphorylation; Recombinant Fusion Proteins; Spermatozoa; Sulfur Radioisotopes; Tissue Distribution

Hg2+ interacts with brain tubulin and disassembles microtubules that maintain neurite structure. Since it is well known that Hg vapor (Hg0) is continuously released from "silver" amalgam tooth fillings and is absorbed into brain, rats were exposed to Hg0 4h/day for 0, 2, 7, 14 and 28 d at 250 or 300 micrograms Hg/m3 air, concentrations present in mouth air of some humans with many amalgam fillings. Average rat brain Hg concentrations increased significantly (11-47 fold) with duration of Hg0 exposure. By 14 d Hg0 exposure, photoaffinity labelling on the beta-subunit of the tubulin dimer with [alpha 32P] 8N3 GTP in brain homogenates was decreased 41-74%, upon analysis of SDS-PAGE autoradiograms. The identical neurochemical lesion of similar or greater magnitude is evident in Alzheimer brain homogenates from approximately 80% of patients, when compared to human age-matched neurological controls. Total tubulin protein levels remained relatively unchanged between Hg0 exposed rat brains and controls, and between Alzheimer brains and controls. Since the rate of tubulin polymerization is dependent upon binding of GTP to tubulin dimers, we conclude that chronic inhalation of low-level Hg0 can inhibit polymerization of brain tubulin essential for formation of microtubules.

Topics: Administration, Inhalation; Alzheimer Disease; Animals; Autoradiography; Blotting, Western; Brain Chemistry; Dental Amalgam; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Humans; Male; Mercury; Protein Binding; Rats; Rats, Sprague-Dawley; Tubulin

Cortical M1 muscarinic receptor-G-protein coupling, high-affinity, guanine nucleotide-sensitive agonist binding (Flynn et al., 1991; Warpman et al., 1993) and muscarinic receptor-stimulated [3H]PIP2 hydrolysis (Ferrari-DiLeo and Flynn, 1993) are known to be defective in Alzheimer disease. Whether this defect reflects an alteration in the M1 muscarinic receptor, its respective guanine nucleotide binding (G) protein or both is not known. This study compares the number and both basal and muscarinic receptor-mediated function of G-proteins in synaptosomal membranes from cerebral cortical samples of age-matched control subjects and Alzheimer disease patients. Immunoblotting with anti-G alpha q/11 and anti-G beta antibodies demonstrated no alteration in the number of these G-protein subunits in Alzheimer disease. Basal [35S]GTP gamma S binding and hydrolysis of [gamma-32P]GTP by high-affinity GTPase also were not significantly altered in Alzheimer disease compared to control membrane samples. However, muscarinic agonist-stimulated GTP gamma S binding and GTP hydrolysis were significantly reduced (80-100%) in Alzheimer disease cortical samples. Diminished agonist-stimulated GTP gamma S binding and GTP hydrolysis correlated with the loss of guanine nucleotide-sensitive, high-affinity agonist binding (KL/KH) ratio) to the M1 receptor subtype. These data provide further evidence for the loss of muscarinic receptor-G protein coupling in Alzheimer disease and support the hypothesis that muscarinic receptor-mediated cortical activation may be compromised in Alzheimer disease.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Carbachol; Cerebral Cortex; Choline O-Acetyltransferase; Female; Frontal Lobe; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Male; Middle Aged; Oxotremorine; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Pirenzepine; Receptors, Muscarinic; Reference Values; Regression Analysis; Synaptic Membranes; Synaptosomes

In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated tau accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of 32P-labeled 8-azidoguanosine triphosphate ([gamma-32P]8N3GTP), the photoaffinity analogue of GTP to the beta-subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the alpha- and beta-subunits of tubulin between Alzheimer's disease and control brains obtained 2-7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2-7 h postmortem and stored at -75 degrees C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE-dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated tau, whereas no abnormal tau was detected in the control brain preparations. Addition of bovine tau to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the beta-subunit. An alkaline phosphatase-treated paired helical filament-enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) tau protein promotes the GTP binding to the beta-subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of tau which depresses the GTP binding.

Topics: Affinity Labels; Aged; Aged, 80 and over; Alzheimer Disease; Azides; Brain; Enzyme-Linked Immunosorbent Assay; Guanosine Triphosphate; Humans; Microtubules; Middle Aged; Phosphorylation; Reference Values; tau Proteins; Tubulin

Adenylyl cyclase (AC) activity was studied in post mortem hippocampus and cerebellum from eight patients with Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and seven non-demented control patients. AC was stimulated via stimulatory guanine nucleotide binding proteins (Gs) using guanosine triphosphate (GTP) and GppNHp (both 10(-4) M) or directly with either forskolin (10(-4) M) or Mn2+ (10(-2) M). Inhibition of AC via A1-receptors was performed with N6-cyclohexyladenosine (CHA) under basal conditions and in the presence of forskolin (10(-5) M). In both brain regions AC activity was significantly reduced in AD/SDAT when compared to controls. Under basal conditions and after stimulation via Gs mean reduction in hippocampus and cerebellum was 47.7% and 58.2%, respectively. The reduction was less pronounced after direct activation of the AC, amounting to 21.8% in hippocampus and 28.1% in cerebellum. CHA inhibited basal and forskolin-stimulated AC concentration-dependently by about 20% (basal) and 30% (forskolin). Inhibition by CHA was similar in hippocampus and cerebellum and tended to be more pronounced in AD/SDAT than in controls. Since the reduction of AC activity in AD/SDAT is greater after stimulation via Gs than after direct activation of the catalytic subunit, we suggest that both Gs and the catalytic subunit seem to be impaired. The fact that CHA-mediated inhibition of AC is not significantly different in AD/SDAT and controls, indicates that in contrast to Gs-, inhibitory G-proteins (Gi) coupling to AC remains intact in Alzheimer's disease.

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Aged; Aged, 80 and over; Alzheimer Disease; Brain; Cerebellum; Colforsin; Enzyme Activation; Female; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hippocampus; Humans; Male; Manganese; Middle Aged; Neurotransmitter Agents; Purinergic P1 Receptor Antagonists; Signal Transduction

Guanosine triphosphate (GTP) is an absolute requirement for tubulin polymerization in situ. The nucleotide photoaffinity probe 8-azidoguanosine 5'-triphosphate (8N3GTP) has been shown to be a biological mimic of GTP in this system and, also, an effective active site probe of the exchangeable GTP binding site. Using [32P]8N3GTP we demonstrate that the exchangeable GTP site of the beta subunit of tubulin is available to added guanine nucleotide in normal aged brain homogenates, whereas it is variably unavailable in Alzheimer's diseased brain. Inability of 8N3GTP to photolabel beta tubulin appears to be associated with neurofibrillary tangle density. These results support the hypothesis that microtubule formation is abnormal in brains affected by Alzheimer's disease.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Azides; Female; Guanosine Triphosphate; Humans; Male; Molecular Weight; Neurofibrils; Tubulin