Molecular subtypes of muscarinic receptors (m1–m5) are novel targets for cholinergic replacement therapies in Alzheimer's disease. However, the status of these receptors in human brain and Alzheimer's disease is incompletely understood. The m1–m5 receptors in brains from control subjects and Alzheimer's disease patients were examined using a panel of specific antisera and radioligand binding. Quantitative immunoprecipitation demonstrated a predominance of the m1, m2, and m4 receptor subtypes in cortical and subcortical regions in control subjects. In Alzheimer's disease, normal levels of m1 receptors measured by radioligand binding contrasted with decreased m1 receptor immunoreactivity, suggesting that the m1 receptor is altered in Alzheimer's disease. The m2 immunoreactivity was decreased, consistent with the loss of m2 binding sites and the location of this receptor subtype on presynaptic cholinergic terminals. The m4 receptor was up‐regulated significantly and may offer a target for new memory‐enhancing drugs. Differential alterations of molecular subtypes of muscarinic receptors may contribute to the cholinergic component of Alzheimer's disease dementia.

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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.

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Equilibrium binding assays support four (M1–M4) and molecular cloning studies have identified five (m1–m5) muscarinic receptor subtypes in the brain and peripheral tissues. However, the overlapping affinities of virtually all of the available muscarinic antagonists have permitted the unambiguous direct labeling of only two of the subclasses of muscarinic receptors, the M1 and M2 subtypes. Thus, the major obstacle to providing a detailed map of the distribution of muscarinic receptor subtypes in the brain, a tissue that expresses all five receptor proteins, has been the lack of muscarinic receptor subtype‐specific ligands. Recent studies have exploited the distinct kinetic binding properties of muscarinic receptor subtypes and have demonstrated that a combination of both kinetic and equilibrium labeling approaches affords selective labeling of the five muscarinic receptors. Application of these novel labeling strategies has permitted for the first time a comparison of the distinct autoradiographic localization patterns of the M1–M5 receptor subtypes in the brain. These new labeling techniques compare well with previous pharmacological, immunological, and molecular methods for localizing and quantifying muscarinic receptor subtypes, and provide further evidence that the pharmacologically defined M1–M5 receptors correspond to the molecularly defined m1–m5 proteins. While unequivocal distribution profiles of each of the five muscarinic receptors awaits the development of more subtype‐selective ligands, the labeling strategies described here provide an alternative, versatile approach for studying muscarinic receptor subtype distribution in the brain. Drug Dev. Res. 40:104–116, 1997. © 1997 Wiley‐Liss, Inc.