Evidence that glypican is a receptor mediating beta-amyloid neurotoxicity in PC12 cells.

Docking of beta-amyloid fibrils to neuronal or glial cell membranes may be an early, necessary and intervenable step during the progression of Alzheimer's disease. Formation of neurofibrillary tangles and amyloid plaques as well as neurotoxicity and inflammation may be direct or indirect consequences. In an attempt to find a receptor that mediates those effects, we assessed rat pheochromocytoma PC12 cell 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction after addition of beta-amyloid to the culture medium. Presence of competitive substances in the medium, cell-surface treatment and specific block of cellular synthesis pathways helped to identify the heparan sulphate moiety of a glycosylphosphatidylinositol-anchored protein likely to represent glypican as a possible receptor mediating beta-amyloid neurotoxicity.

Regulated secretion of beta-amyloid precursor protein in rat brain.

The beta-amyloid precursor protein (APP) is a ubiquitous, highly conserved secretory glycoprotein that is expressed at high levels in mammalian brain by neurons, astrocytes, and activated microglia. Secreted APP (APPs) is generated by the cleavage of APP within the beta-amyloid (A beta) portion of its ectodomain. The formation and secretion of APPs can be increased by activation of particular neurotransmitter receptors and subsequent protein phosphorylation. We found that tissue slices from rat cortex, hippocampus, striatum, and cerebellum secrete APPs in vitro. APPs secretion was enhanced by electrical stimulation, but was not associated with a general increase in the release of total protein, lactate dehydrogenase (LDH) activity, or neuronal cell adhesion molecules. The pharmacological profile of stimulation-induced APPs secretion suggests complex interactions between muscarinic receptor subtypes in the tissue slices: in the unstimulated state, activation of Muscarinic M1 receptors increased APPs release while nonspecific activation of multiple muscarinic receptors had little effect on APPs release; in electrically stimulated slices, nonspecific inhibition of muscarinic receptors blunted the increase in APPs secretion. The nonspecific muscarinic agonist carbachol increased APPs secretion only in the presence of an M2 receptor antagonist, suggesting that activation of M2 receptors suppresses APPs formation. These data indicate that secretory APP processing in brain includes depolarization-enhanced cleavage of the cell-associated holoprotein within its ectodomain, and that the net effect of depolorization involves several subtypes of acetylcholine receptors.

Regulation of proteolytic processing of the amyloid beta-protein precursor of Alzheimer's disease in transfected cell lines and in brain slices.

beta A4 is the principal component of Alzheimer's disease brain amyloid. It is derived from proteolytic processing of amyloid beta-protein precursors (APP), a family of transmembrane glycoproteins. Secretion of APPs, a secreted proteolytic derivative that is cleaved within the beta A4 domain of APP, is increased many-fold by the activation of cell-surface receptors, like the muscarinic m1 and m3 receptor subtypes, which are coupled to protein kinase C. Concomitantly, their activation decreases the formation of both secreted soluble beta A4 and of endosomal-lysosomal C-terminal APP derivatives. These data suggest that muscarinic m1 and m3 receptors accelerate non-amyloidogenic APP processing and depress the formation of potentially amyloidogenic derivatives. Other receptors that stimulate APPs secretion include those for bradykinin, vasopressin, and interleukin-1 receptors. A similar control mechanism is present in rat brain tissue slices, in which the release of both APPs and endogenous neurotransmitters is increased by electrical depolarization. This increase is tetrodotoxin-sensitive and frequency-dependent, suggesting that APPs release may normally depend on neuronal activity. Taken together, our findings suggest that specific receptor agonists might be effective in reducing the formation of potentially amyloidogenic APP derivatives in vivo.

Receptor-coupled amyloid precursor protein processing.

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.