G-proteins and adenylyl cyclase in Alzheimer's disease postmortem brain

Alzheimer's disease (AD) is the most common type of dementia in the elderly. The major pathological changes seen in the disease are the formation of neurofibrillary tangles, of which the main component is the hyperphosphorylated form of tau, and the over accumulation of plaques formed by beta-amyloid. However, it is still unknown how these changes relate to neurodegeneration. The degenerative process in AD is accompanied by deficits in several neurotransmitters.

Although receptors are fairly well preserved, there is evidence that receptor function may be compromised at the level of signal transduction. The adenylyl cyclase (AC) complex has been shown to be disrupted at different levels, from receptor-G-protein coupling to the enzyme. Heterotrimeric GTP-binding proteins (G-proteins), playing an essential role in linking many cell-surface receptors to effector proteins at the plasma membrane, have been shown to be altered, both m terms of levels and coupling. However most studies have examined cases with clinical AD, showing end-stage pathology. Thus, very little is known about how these changes evolve during the course of the disease, and whether they may occur prior to the appearance of clinical symptoms.

The aim of this project was to study changes in G-proteins and AC at different stages of AD pathology from early to severe. This was done by determining the correlation between alterations in G-proteins and AC and the localisation and degree of neurofibrillary changes and amyloid deposition in human postmortem brain. We focused on the hippocampal formation, which is affected early in the disease and where the pathology has been shown to occur in a predictive and region-specific manner. The main technique used m this project was autoradiography, by which it is possible, not only to localise and quantify signal transduction components, but also to study the function and coupling of them.

The objective of the first part of this thesis was to characterise [35S]GTPyS and [3H]forskolin binding for the subsequent studies in human brain. In study I we found that GIP was able to stimulate [35S]GTP-gamma-S binding in grey matter areas. This effect was dependent on Mg2+ and correlated with the known distribution of low Kin GTPase, suggesting that it reflects the rate of GIP hydrolysis. Study H showed that the inhibition, but not the stimulation, of [3H]forskolin binding, brought about by low and high concentrations of the GTP-analogue Gpp[NH]p respectively, was abolished by NEM. This indicates that the inhibition is mediated by Gi, whereas the stimulation is mediated through G.

The results obtained from the [3H]forskolin autoradiography in postmortem human brain showed that AC levels may be largely preserved in AD. However, the loss of the inhibitory effect of Gpp[NH]p in the entorhinal cortex indicates that there is an impaired Gi-AC coupling or loss/impairment of Ca2+/CaM dependent isoforms with increasing AD pathology. Autoradiography showed no evidence of any major change in G-protein levels, but the loss of the stimulatory effect of GIP on [35S]GTP-gamma-S binding indicates a decrease in G-protein GIP-hydrolysis. This was observed only in the regions most affected by the neurofibrillary changes, but was detected already at stages prior to those related to clinical symptoms. Immunoblotting of homogenates from the same tissues revealed a significant decrease in G, in hippocampus, and an increase in the Ca2+/CaM dependent AC isoform 1.

In conclusion, the results presented in this thesis indicate that the neuropathological changes seen in AD are not accompanied by extensive changes in the levels of G-proteins and AC. However, there appear to be more subtle changes restricted to the areas most affected by the pathology. These alterations late with the staging for neurofibrillary changes and may occur already at stages prior those related to clinical symptoms of the disease. Our results indicate that these changes include the inhibitory Gi-proteins and involve both decreased levels and impaired function/coupling. These alterations could lead to dysfunction of several components downstream m the cell and possibly play a role in the pathogenesis of the disease.

List of scientific papers

I. Garcia-Jimenez A, Cowburn RF, Winblad B, Fastbom J (1997). Autoradiographic characterisation of [35S]GTP gamma S binding sites in rat brain. Neurochem Res. 22(8): 1055-63.
https://pubmed.ncbi.nlm.nih.gov/9239762

II. Garcia-Jimenez A, Cowburn RF, Einblad B, Fastbom J (1998). Inhibition of [3H]forskolin binding by Gpp[NH]p may reflect adenylyl cyclase coupling to Gi. Neuroreport. 9(14): 3329-33.
https://pubmed.ncbi.nlm.nih.gov/9831472

III. Garcia-Jimenez A, Cowburn RF, Ohm TG, Bogdanovic N, Winblad B, Fastbom J (1999). Quantitative autoradiography of [3H]forskolin binding sites in post-mortem brain staged for Alzheimers disease neurofibrillary changes and amyloid deposits. Brain Res. 850(1-2): 104-17.
https://pubmed.ncbi.nlm.nih.gov/10629754

IV. Garcia?Jimenez A, Cowburn RF, Ohm TG, Lasn H, Winblad B, Bogdanovic N, Fastbom J (2002). Loss of stimulatory effect of guanosine triphosphate on [(35)S]GTP?S binding correlates with Alzheimers disease neurofibrillary pathology in entorhinal cortex and CA1 hippocampal subfield. J Neurosci Res. 67(3): 388-398.
https://pubmed.ncbi.nlm.nih.gov/11813244

V. Garcia-Jimenez A, Fastbom J, Ohm TG, Cowburn RF (2002). Decreased G-alpha-i-subunit levels in hippocampus of brains staged for Alzheimers disease neurofibrillary pathology. [Manuscript]