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.

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G-protein alpha-subunits (Galphao, Galphai, Galphas, Galphaq) and adenylyl cyclase (AC) I and II isoforms were quantified in hippocampus and entorhinal cortex from 22 cases staged for Alzheimer's disease (AD) pathologies according to Braak and Braak. Hippocampal Galphai levels declined significantly with neurofibrillary staging, whereas AC I levels in this region increased. Significant amyloid stage-related reductions of Galphai were seen in both the hippocampus and entorhinal cortex. The hippocampus also showed a significant reduction of Galphao with amyloid staging. It is concluded that levels of inhibitory G-protein subunits Galphao, and in particular Galphai, decrease in parallel to the extent of AD pathology.

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The binding of [35S]GTP gamma S was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 microM. The specific binding was 98%. Different guanine nucleotides were all able to compete with [35S]GTP gamma S binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [35S]GTP gamma S does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [35S]GTP gamma S binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTP gamma S, did not displace [35S]GTP gamma S binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [35S]GTP gamma S binding was strongly Mg(2+)-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [35S]GTP gamma S in the presence of GTP may reflect functional G-protein activity.