Altered processing of the amyloid precursor protein (APP) is a central event in the formation of amyloid deposits in the brains of individuals with Alzheimer's disease. To investigate whether cellular APP processing is controlled by cell-surface neurotransmitter receptors, human embryonic kidney (293) cell lines were transfected with the genes for human brain muscarinic acetylcholine receptors. Stimulation of m1 and m3 receptor subtypes with carbachol increased the basal release of APP derivatives within minutes of treatment, indicating that preexisting APP is released in response to receptor activation. Receptor-activated APP release was blocked by staurosporine, suggesting that protein kinases mediate neurotransmitter receptor-controlled APP processing.

To determine whether neurodegeneration in Alzheimer disease brain is associated with degradation of structural cell membrane molecules, we measured tissue levels of the major membrane phospholipids and their metabolites in three cortical areas from postmortem brains of Alzheimer disease patients and matched controls. Among phospholipids, there was a significant (P < 0.05) decrease in phosphatidylcholine and phosphatidylethanolamine. There were significant (P < 0.05) decreases in the initial phospholipid precursors choline and ethanolamine and increases in the phospholipid deacylation product glycerophosphocholine. The ratios of glycerophosphocholine to choline and glycerophosphoethanolamine to ethanolamine were significantly increased in all examined Alzheimer disease brain regions. The activity of the glycerophosphocholine-degrading enzyme glycerophosphocholine cholinephosphodiesterase was normal in Alzheimer disease brain. There was a near stoichiometric relationship between the decrease in phospholipids and the increase of phospholipid catabolites. These data are consistent with increased membrane phospholipid degradation in Alzheimer disease brain. Similar phospholipid abnormalities were not detected in brains of patients with Huntington disease, Parkinson disease, or Down syndrome. We conclude that the phospholipid abnormalities described here are not an epiphenomenon of neurodegeneration and that they may be specific for the pathomechanism of Alzheimer disease.Brain lesions characteristic of Alzheimer disease (AD) include amyloid deposition, the formation of neurofibrillary tangles, and neuronal degeneration. The etiology and pathophysiology of neuronal death in AD are unknown. Cell membrane lipid abnormalities have been described in AD brain, and it has been hypothesized that these contribute to amyloid deposition (1, 2) and neuronal dysfunction (3). Initial evidence for a biochemical abnormality in the metabolism of phospholipids came from in vitro 31P NMR spectroscopic studies showing that the ratio of glycerophosphocholine (GPC) to glycerophosphoethanolamine (GPE) as well as levels of glycerophosphodiesters and phosphomonoesters were increased in AD brain (4-6). Quantitative HPLC analysis has demonstrated that the increase in glycerophosphodiesters was due to accumulation of the phospholipid catabolites GPC and GPE (7, 8). The biochemical mechanisms accounting for the increase of phospholipid catabolites are unknown. To investigate the abnormalities in the phospholipid metabolic pathways, we examined levels of the parent phospholipids, their precursors, and their catabolites (see Fig. 1) in three cortical brain areas obtained at autopsy from AD patients and matched controls. To rule out the possibility that the elevation in GPC reflects a slowing in its degradation,

3. Antagonist pA2 values (the negative logarithm of the dissociation equilibrium constant) were determined while recording from individual neurones. pA2 values were: pirenzepine 7 0, hyoscine 8-9, and 4-DAMP 8&7.4. I.p.s.p.s in the submucous plexus were also depressed by muscarinic agonists, and this was competitively reversed by pirenzepine and 4-DAMP, with apparent pA2 values of 6-9 and 8-7 respectively. Muscarinic antagonists alone increased the amplitude ofthe i.p.s.p. evoked either by single or repeated stimuli. This enhancement was observed with low concentrations of antagonists and did not become greater when the concentrations were increased.5. Muscarinic agonists depolarized about one-quarter of myenteric and submucous plexus neurones. Low concentrations of pirenzepine antagonized these depolarizations; the pA2 value was 8-4. Cholinergic slow e.p.s.p.s recorded in some myenteric neurones were depressed or abolished by pirenzepine; concentrations that caused 50 % inhibition (IC.50) for this action ranged from 10 to 60 nM.6. It is concluded that presynaptic muscarinic receptors, activation of which inhibits the release of acetylcholine and noradrenaline, are the m2 type. Post-synaptic muscarinic receptors, activation of which depolarizes the membrane, are of the ml R. A. NORTH, B. E. SLACK AND A. SURPRENANT type. The results also suggest that acetylcholine may exert a tonic inhibition of noradrenaline release in the submucous plexus through m2 receptors, and mediates the slow e.p.s.p. in the myenteric plexus through ml receptors.

Stimulation of m1 and m3 muscarinic acetylcholine receptors, which are coupled to phosphoinositide hydrolysis and protein kinase C activation, has been shown to increase the release of soluble amyloid precursor protein derivatives (APPs). The effect is mimicked by phorbol esters, which directly activate protein kinase C. Using human embryonic kidney cells expressing individual muscarinic receptor subtypes, we found that stimulation of APPs release by the muscarinic agonist carbachol was only partially reduced by a specific inhibitor of protein kinase C (the bisindolylmaleimide GF 109203X), while the response to phorbol 12-myristate 13-acetate (PMA) was abolished. The increase in APPs release elicited by carbachol and PMA was accompanied by elevated tyrosine phosphorylation of several proteins and reduced by tyrosine kinase inhibitors; GF 109203X significantly reduced the stimulation of tyrosine phosphorylation by carbachol and PMA. Inhibition of protein tyrosine phosphatases by vanadyl hydroperoxide markedly increased cellular tyrosine phosphorylation and enhanced APPs release as effectively as PMA and carbachol. Direct phosphorylation of amyloid precursor protein on tyrosine residues following treatment with carbachol, PMA, or vanadyl hydroperoxide was not observed. The results implicate both tyrosine phosphorylation and protein kinase C-dependent mechanisms in the regulation of APPs release by G protein-coupled receptors, and suggest that carbachol and PMA increase APPs release from human embryonic kidney cells expressing m3 muscarinic receptors via partially divergent pathways that converge at a tyrosine phosphorylation-dependent step.

Choline is an essential nutrient for humans. It is a precursor of membrane phospholipids (e.g., phosphatidylcholine (PC)), the neurotransmitter acetylcholine, and via betaine, the methyl group donor S-adenosylmethionine. High choline intake during gestation and early postnatal development in rat and mouse models improves cognitive function in adulthood, prevents age-related memory decline, and protects the brain from the neuropathological changes associated with Alzheimer’s disease (AD), and neurological damage associated with epilepsy, fetal alcohol syndrome, and inherited conditions such as Down and Rett syndromes. These effects of choline are correlated with modifications in histone and DNA methylation in brain, and with alterations in the expression of genes that encode proteins important for learning and memory processing, suggesting a possible epigenomic mechanism of action. Dietary choline intake in the adult may also influence cognitive function via an effect on PC containing eicosapentaenoic and docosahexaenoic acids; polyunsaturated species of PC whose levels are reduced in brains from AD patients, and is associated with higher memory performance, and resistance to cognitive decline.

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells.

Background: The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β-and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxylterminus by Ϸ8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated ( These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.Substance P (SP), a member of the tachykinin family of peptides, is involved in many physiological processes, including exocrine gland secretion, vasodilation, pain transmission, and neurogenic inflammation (1). Molecular cloning established that the receptor for SP belongs to the G protein-coupled receptor family that possesses seven putative transmembrane domains (2, 3). Results from crosslinking and reconstitution studies have provided evidence that the SP receptor, also known as the neurokinin-1 receptor, couples to a subgroup of G proteins, Gq͞ 11 (4, 5). It has been shown in many tissues that SP activates phospholipase C ␤ (PLC ␤ ) which results in a transient increase in intracellular inositol 1,4,5-trisphosphate (IP 3 ) and cytosolic calcium concentration (6-9). The SP receptor, when stably expressed in Chinese hamster ovary (CHO) cells, also couples to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis (10, 11).Biochemical studies in which the SP receptors in a membrane preparation of rat submaxillary glands were covalently labeled with a photoreactive SP analog (12, 13) have demonstrated the presence of two SP receptor isoforms that differ in the length of the cytoplasmic carboxyl-terminus by approximately 8 kDa. The photolabeling of the full-length SP receptor isoform (molecular mass, 46 kDa following deglycosylation) and the short isoform (molecular mass, 37 kDa following deglycosylation) are inhibited by SP with the same IC 50 (1 nM), indicating that both receptor isoforms bind SP with a similar affinity. A cDNA encoding a carboxyl-terminally truncated SP receptor with a calculated molecular weight of 35,797 has been cloned (14). When expressed in COS cells, this truncated receptor, in contrast to the receptor detected by photoaffinity labeling, has a binding affinity at least 10-fold less than that of the full-length receptor. Therefore, it is unlikely that the short receptor isoform identified in the rat salivary gland is derived from this spliced variant.To study the functional properties of the short SP receptor isoform identified in the rat salivary gland by photoaffinity labeling, a rat SP receptor mutant (324 amino acids; molecular weight, 37,360) carboxyl-terminally truncated to approximate the size of this short receptor isoform (13) was stably expressed in CHO cells (15). In this report, we have co...