Characterization and expression of subtypes of nicotinic receptors in brain and adrenal medulla : with focus on development, Alzheimer's disease and transgenic animal models

Abstract

The cholinergic nicotinic receptors (nAChRs) seem to play an important role in numerous important physiological processes in brain as well as peripheral tissues of man. In first trimester human brain mRNA levels for the alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 nAChR subunits and binding sites were detected at 5 weeks of gestation. A significant decrease in both number of 3 Hcytisine binding sites (alpha4 ligand) and mRNA level for the alpha4 nAChR while no change in 3 Hepibatidine binding sites (alpha3 and alpha4 ligand) or level of alpha3 mRNA was seen in the aged brain compared to fetal brain. In contrast, a significantly decreased number of the 3 H-epibatidine binding sites was observed in the aged adrenal medulla compared to fetal adrenal medulla.

The relative mRNA level of alpha3 nAChR subunit was found to be 1000% higher in adult mice adrenal medulla compared to brain while a corresponding -40% and -29% lower mRNA expression of alpha4 and alpha7 nAChR subunits was observed in mice adrenal medulla compared to brain. A significantly higher protein level for the alpha3 nAChR subunit was observed of the rat adrenal medulla while the alpha4 subunit protein was much lower. Saturation binding studies With 3 H-epibatidine in homogenates of rat adrenal medulla revealed the presence of solely one high affinity binding site suggesting a high abundance of the alpha3 nAChR subtype in comparison to the alpha4 and alpha7 nAChR subunits in rodent adrenal medulla. An age-related reduction in receptor protein levels were observed for the alpha3, alpha4, alpha7 and beta2 nAChR subunits in both brain and adrenal medulla of rat.

Changes in nAChR subtypes were compared in the adrenal medulla and brain of two different transgenic mice models i.e. the APPSWETg, which overexpress human beta-amyloid and hAChE-Tg which over- express human acetylcholine- esterase. Both transgenic mice models show impairment in cognitive function although the underlying causative mechanisms are probably different. An increased relative mRNA level for the alpha7 subunit was observed in adrenal medulla of both APPSWETg (+53%) and hAChE-Tg (+56%) while no significant change in alpha7 mRNA was observed in the brain in the transgenic mice compared to non-transgenic mice. On the other hand, opposite changes were observed for the alpha3 mRNA in adrenal medulla where an increase (+18%) was observed in hAChE-Tg mice while a decrease (-28%) was observed in APPSWETg mice. Thus, different underlying pathological mechanisms can cause different changes in the expression levels of various niches in brain and adrenal medulla.

Changes in nAChR subtypes were investigated in the autopsy brain tissues from Patients with Alzheimer's disease (AD). A significant decrease in protein levels of the nAChR subtypes and number of nAChR binding sites were observed while no significant change was observed in the corresponding subunit mRNA levels except for the alpha7 subunit where an increase in relative mRNA level was observed in the hippocampus. The findings suggest that the changes in nAChRs in AD seem mainly to occur at posttranscriptional level.

Nicotine is well known to induce an upregulation of the nAChRs in brain. In rat sub-chronical treatement with nicotine caused no upregulation in the either 3 H-nicotine or 3 H-epibatidine binding sites in the adrenal medulla while a significant increase in number of binding sites of both ligands was measured in the brain. The findings suggest both receptor subtype and organ-specific effects of nicotine exposure. Investigation of post-mortem brain tissue from smoking AD patients and controls showed an upregulation of the alpha4 nAChR protein and binding sites compared to non-smoking subjects. No significant change was detected in the mRNA levels for the various nAChRs subunits either in smoking controls or in smoking AD patients. In addition significantly lower levels of both soluble and insoluble Abeta 1-40 and AP 1-42 were observed in frontal and temporal cortex of smoking AD subjects compared to non-smoking AD subjects.

From the findings of this thesis it is evident that different nAChRs with different subtype specificity and density express in brain and adrenal medulla. Internal and external factors including development, ageing, pathological mechanism and drug exposure differently regulate the expression levels of the various nAChR subtypes in adrenal medulla compared to brain.