MITOGEN-ACTIVATED PROTEIN KINASE IS INVOLVED IN N-METHYL-d- ASPARTATE RECEPTOR REGULATION OF AMYLOID PRECURSOR PROTEIN CLEAVAGE J. MILLS and P. B. REINER* Kinsmen Laboratory of Neurological Research, Graduate Program in Neuroscience, University of British Columbia, 2255 Westbrook Mall, Vancouver, British Columbia, Canada, V6T 1Z3 Abstract —Glutamate is the principal excitatory neurotransmitter in the mammalian brain. Several lines of evidence suggest that glutamatergic hypoactivity exists in the Alzheimer’s disease brain, where it may contribute to both brain amyloid burden and cognitive dysfunction. Although metabotropic glutamate receptors have been shown to alter cleavage of the amyloid precursor protein, little attention has been paid to the role of N-methyl-d-aspartate receptors in this process. We now report that activation of N-methyl-d-aspartate receptors in transiently transfected human embryonic kidney 293 cells increases production of the soluble amyloid precursor protein derivative. Moreover, using both pharmacological and gene transfer techniques, we show that this effect is largely due to activation of the mitogen-activated protein kinase cascade, specifically the pathway leading to activation of extracellular signal-regulated protein kinase but not other mitogen-activated protein kinases. These observations further our understanding of the pathways that regulate amyloid precursor protein cleavage, and buttress the notion that regulation of amyloid precursor protein cleavage is critically dependent upon the mitogen-activated protein kinase cascade.  1999 IBRO. Published by Elsevier Science Ltd. Key words: Alzheimer’s disease, APP, sAPP, MAPK, NMDA. Several lines of evidence suggest that glutamate plays a role in the pathophysiology of Alzheimer’s disease. Corticocorti- cal connections and the major projection pathways of the hippocampus utilize glutamate as a neurotransmitter and degenerate early in the disease process, 15 and post mortem analysis reveals that glutamate concentrations are significantly decreased in the brains of Alzheimer’s disease patients. 24 Because these brain regions accumulate amyloid deposits and are intimately involved in learning and memory, 45 it has been suggested that glutamatergic hypoactivity may contribute both to increased brain amyloid burden and memory dysfunc- tion. 33 Finally, glutamate receptors are heavily implicated in the molecular mechanisms underlying cognitive function, in particular long-term potentiation, the premier cellular model of learning. 5,34 For these reasons, investigating the role of glutamate receptors in the regulation of amyloid precursor protein (APP) processing 43 is of significant theoretical and clinical importance. Glutamate is the principal excitatory neurotransmitter in the mammalian brain. Glutamate acts upon both iono- tropic receptors, which mediate transmembrane ion fluxes, and G-protein-coupled receptors, which initiate cascades leading to activation of intracellular effectors. 46,56 Gluta- matergic G-protein-coupled receptors linked to the phos- pholipase C/protein kinase C (PKC) signaling system regulate release of the soluble N-terminal ectodomain of APP (sAPP). 27,32,33,49,61 These findings are in keeping with previous data demonstrating that activation of seroton- ergic or cholinergic G-protein-coupled receptors increases sAPP production and concomitantly decreases b-amyloid production. 4,23,33,48,49,60,66 Less attention has been paid to the role of ionotropic gluta- mate receptors in the regulation of APP cleavage. Among the ionotropic glutamate receptors, the N-methyl-d-aspartate (NMDA) receptor is unique in that it is highly permeable to Ca 2+ . 40 Because Ca 2+ -dependent regulation of APP cleavage has been demonstrated in a variety of cell lines, 4,50–52 we reasoned that activation of NMDA receptors might result in altered cleavage of APP. Indeed, stimulation of Ca 2+ -per- meable nicotinic receptors increases sAPP release from PC12 cells. 26 Ca 2+ regulation of sAPP release has been shown to occur in a PKC-independent manner, 4 possibly via acti- vation of tyrosine kinases, 50 but the signaling pathway mediating this effect has not been well characterized. The mitogen-activated protein kinase (MAPK) pathway, involv- ing sequential activation of p21Ras, Raf, mitogen-activated protein kinase kinase (MEK) and an MAPK family member known as extracellular signal-regulated protein kinase (ERK), is a likely candidate as it is activated by Ca 2+ , 13,54 regulates APP cleavage in both a PKC-dependent and -independent manner, 11,22,42 and requires activation of tyrosine kinases. For these reasons, we hypothesized that (i) stimulation of NMDA receptors regulates APP processing, and (ii) ERK is required for NMDA receptor-mediated regulation of APP cleavage. In order to test these hypotheses, we transiently transfected human embryonic kidney 293 (HEK 293) cells with NMDA receptors and inhibited the MAPK pathway using either PD 98059, a selective inhibitor of MEK1, 1,12,31 or transient expression of a kinase-dead MEK1 mutant. 57 NMDA receptor regulation of APP cleavage 1333 1333 Neuroscience Vol. 94, No. 4, pp. 1333–1338, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00381-4 Pergamon *To whom correspondence should be addressed. Tel.: + 1-604-822-7948; fax: + 1-604-822-7981. E-mail address: pbr@unixg.ubc.ca (P. B. Reiner) Abbreviations: APP, amyloid precursor protein; APV, d,l-2-amino-5-phos- phonovalerate; ERK, extracellular signal-regulated protein kinase; HEK, human embryonic kidney; JNK/SAPK, c-Jun N-terminal kinase stress- activated protein kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; NMDA, N-methyl-d- aspartate; PKC, protein kinase C; sAPP, soluble ectodomain of APP.