Selective effects of the APOE ε4 allele on presynaptic cholinergic markers in the neocortex of Alzheimer's disease

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Abstract

The effects of the APOE ε4 allele on a range of pre- and postsynaptic cholinergic markers were studied in a cohort of community-based Alzheimer's disease (AD) patients. Compared with age-matched controls, the postmortem AD neocortex showed decreased choline acetyltransferase (ChAT) and acetyl cholinesterase activities, lower muscarinic M2, and nicotinic α4β2 receptor densities, as well as reduced M1 receptor coupling to G-proteins. However, the ε4 allele was dose-dependently correlated only with higher losses of ChAT activities. AD patients with two ε4 alleles also had more β-amyloid containing senile plaques in the temporal cortex compared to patients with 0/1 ε4. This study suggests that APOE ε4 selectively affects presynaptic cholinergic function which may contribute to the clinical and neuropathological features of AD.

Introduction

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized neuropathologically by extracellular β-amyloid (Aβ)-containing senile plaques (SP), intracellular neurofibrillary tangles (NFT) consisting of hyperphosphorylated forms of microtubule associated protein τ, and neuronal degeneration. Although degeneration in the source nuclei of several neurotransmitter systems has been found in AD, one of the earliest and most consistently affected is the basal forebrain cholinergic neuron (Davies and Maloney, 1976, Whitehouse et al., 1982). Accompanying the loss of cholinergic neurons are neurochemical alterations including reduced choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities, reduced acetylcholine release, as well as losses of muscarinic M2 and nicotinic receptors in the neocortex and hippocampus (Francis et al., 1985, Mash et al., 1985, Perry et al., 1977, Perry et al., 1987, Sims et al., 1983, Wilcock et al., 1982). Postsynaptic M1 receptors appeared to be preserved in AD (Mash et al., 1985) but was subsequently shown to be defective in coupling to G-proteins (Flynn et al., 1991, Ladner et al., 1995). Other studies also reported deficits in postsynaptic signaling such as decreases in phosphoinositide hydrolysis and protein kinase C (PKC) activities (Cole et al., 1988, Ferrari-DiLeo and Flynn, 1993, Jope et al., 1997) which are activated by M1 and M3 receptors (Caulfield, 1993). Taken together, these studies suggest a state of profound presynaptic as well as postsynaptic cholinergic dysfunction in AD that may underlie both the cognitive and behavioral symptoms of AD (Cummings and Kaufer, 1996, Lai et al., 2001, Minger et al., 2000, Perry et al., 1978, Tsang et al., in press, Wilcock et al., 1982).

Various gene mutations have been linked to familial forms of AD, such as those encoding amyloid precursor protein (APP), presenilin 1 and presenilin 2, which result in altered APP processing and increased production of plaque-forming Aβ species (Selkoe, 2001). For sporadic AD which constitutes the majority of cases, the major genetic risk factor is APOE which encodes apolipoprotein E (apoE), a 34 kDa lipid carrier protein. Of the three APOE alleles (ε2, ε3, and ε4 encoding apoE2, apoE3, and apoE4 isoforms, respectively), the ε4 is dose-dependently associated with increased risk of developing AD compared with ε3 and ε2 (Corder et al., 1993, Poirier et al., 1993, Saunders et al., 1993). Much research effort has been expanded into elucidating the pathogenic mechanisms of E4 in AD. In addition to its role as a key regulator of plasma lipid levels, apoE may be involved in neuronal repair and plasticity in the CNS, with the E4 being less effective than the E3 isoform (Mahley and Huang, 1999, White et al., 2001). E4 has also been shown to facilitate Aβ deposition and τ hyperphosphorylation more potently than E2 and E3 in vitro (Holtzman et al., 2000, Tesseur et al., 2000). Additionally, AD patients with one or two ε4 showed higher plaque and, to a lesser extent, higher tangle burden than non-ε4 carriers (Nagy et al., 1995, Olichney et al., 1996, Schmechel et al., 1993). Finally, apoE may affect cholinergic neurons and signaling in an isoform-specific manner. For example, some studies demonstrated that ε4 positive AD patients had more extensive deficits in ChAT activities compared with non-ε4 carriers (Poirier et al., 1995, Soininen et al., 1995, Beffert and Poirier, 1996) while others did not (Corey-Bloom et al., 2000, Svensson et al., 1997, Tiraboschi et al., 2004). The effect of APOE genotype on other cholinergic markers is less clear. M1, M2, and nicotinic receptors do not seem to differ between patients with or without ε4 (Reid et al., 2001, Poirier et al., 1995, Svensson et al., 1997). However, the radioligands used to label nicotinic sites in these studies are not subunit-specific, and it is not known whether APOE genotype is correlated with specific losses of nicotinic receptor subpopulations, such as those containing α4β2 subunits (Warpman and Nordberg, 1995). Furthermore, although studies have shown selective impairment of muscarinic receptor-mediated phosphoinositide hydrolysis by the apoE4 isoform (Cedazo-Minguez and Cowburn, 2001), it is not known whether apoE4 affected the coupling of G-proteins to M1 receptors in AD. In this study, we correlated APOE genotype in a cohort of AD patients with a range of pre- and postsynaptic cholinergic neurochemical markers in the postmortem frontal and temporal cortex in order to test the hypothesis that deficits or alterations in cholinergic neurotransmission in the AD neocortex are influenced by APOE ε4 genotype.

Section snippets

Patients and neuropathological assessments

A maximum of 40 AD subjects and 20 elderly controls were included in this study. The AD subjects were derived from an autopsied subset of a cohort of community-based dementia patients from Oxfordshire, UK enrolled in a longitudinal study of behavior in dementia (Hope et al., 1999). The inclusion/exclusion criteria as well as point-of-entry characteristics have been previously described in detail (Hope et al., 1997, Hope et al., 1999). Complete drug histories were recorded, and none of the

Demographic, disease, and neurochemical variables in control vs. AD

Table 1 shows that demographic variables were matched between the AD subjects and controls (Student's P > 0.05) except for lower pH in AD, possibly an indication of more severe acidosis due to prolonged agonal state (Hardy et al., 1985). However, pH, as well as other demographic variables listed in Table 1, did not correlate with neurochemical variables (Pearson P > 0.05). As shown in Table 1, ChAT and AChE activities as well as M2 and α4β2 nicotinic receptor densities are reduced in one or

Discussion

The ε4 allele of APOE is at present the only well-established susceptibility gene for sporadic AD, with risk of disease development at a particular age increasing with ε4 allele dose. The widely replicable nature of these findings has led to intense research efforts toward uncovering the mechanisms by which ApoE4 affect the neurodegenerative process of AD. Here, using a well-characterized cohort of AD patients, we investigated whether inheritance of the ε4 allele was associated with cholinergic

Acknowledgments

This study is supported by the Wellcome Trust, UK. In Singapore, the work was supported by the Department of Clinical Research, Singapore General Hospital, as well as the Biomedical Research Council (BMRC/03/1/21/17/259). In Spain, this work was funded through the “UTE project CIMA”, the Secretaria de Estado de Educacion y Universidades and the Fondo Social Europeo (EX2004-0250). APOE genotyping was performed by J. Stewart, Department of Neuropathology, University of Glasgow, UK. M. K. P. Lai

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