Robert K. K. Lee

Neuronal Expression of β-Amyloid Precursor Protein Alzheimer Mutations Causes Intracellular Accumulation of a C-terminal Fragment Containing Both the Amyloid β and Cytoplasmic Domains

Five different Alzheimer mutations of the β-amyloid precursor protein (APP) were expressed in neurons via recombinant herpes simplex virus (HSV) vectors, and the levels of APP metabolites were quantified. The predominant intracellular accumulation product was a C-terminal fragment of APP that co-migrated with the protein product of an HSV recombinant expressing the C-terminal 100 amino acids (C100) of APP, which is known to cause neurodegeneration. Fractionation studies revealed that the C-terminal fragment generated by expression of the Alzheimer mutations, like C100, partitioned into membrane fractions and was particularly enriched in synaptosomes. The processing abnormality caused by expression of the Alzheimer mutations occurs predominantly in neurons. Expression of these mutations or of C100 alone in neurons caused increased secretion of Aβ relative to that of neurons infected with wild type APP recombinant vectors. These data show that expression of APP mutations that cause familial Alzheimer’s disease increases the intracellular accumulation of potentially amyloidogenic and neurotoxic C-terminal fragments of APP in neurons.

Effect of a 5-HT2C serotonin agonist, dexnorfenfluramine, on amyloid precursor protein metabolism in guinea pigs

Stimulation of serotonin receptor subtypes 5-HT(2A) or 5-HT(2C) in stably transfected 3T3 cells by dexnorfenfluramine (DEXNOR) or serotonin increases secretion of the APP metabolite APP(s). It is not known whether activation of these receptors can also affect APP metabolism in vivo. We examined the effects of a single intraperitoneal (i.p.) injection of DEXNOR on APP(s) levels in cerebrospinal fluid (CSF) of guinea pigs. These levels were significantly (P<0.05) increased by a single dose of DEXNOR (1-4 mg/kg); those of the APP metabolites Abeta(1-40) and Abeta(1-42) were unaffected. The DEXNOR-induced (1 mg/kg) increases in CSF APP(s) were suppressed by ritanserin (1 mg/kg) but not by ketanserin (2 mg/kg). When given alone, ritanserin did not affect CSF levels of APP(s), Abeta(1-40), or Abeta(1-42). Chronic treatment with DEXNOR for 9 days (1 mg/kg bid, i.p.) increased CSF APP(s) levels, measured 2 h after the last injection (P<0.05), and decreased those of CSF Abeta(1-42) (P<0.05). Neither hippocampal nor cortical levels of the APP holoprotein (APP(h)), nor body weight, were affected by DEXNOR. Chronic administration of mCPP (1-(m-chlorophenyl)piperazine) (2 mg/kg bid, i.p.), a 5-HT(2B/2C) agonist, for 9 days also increased CSF APP(s) levels (P<0.5) when measured 2 h after the drugs last administration; hippocampal and cortical APP(h) levels were unaffected. However, mCPP also caused a significant decrease in body weight gain. These data indicate that the pharmacological activation of 5-HT(2C) receptors can stimulate CSF APP(s) secretion and reduce Abeta production in vivo. Hence 5-HT(2C) receptors, which apparently are localized to the brain, may represent useful targets for the development of treatments for Alzheimers disease.

Regulation of APP Synthesis and Secretion by Neuroimmunophilin Ligands and Cyclooxygenase Inhibitors

Abstract: We and others previously showed that both the synthesis of the amyloid precursor protein (APP) and its processing (i.e., to amyloidogenic Aβ peptides; soluble nonamyloidogenic APPs; and other APP fragments) are regulated by neurotransmitters. Transmitters that elevate cellular cAMP levels (like norepinephrine and prostaglandins, which act on β‐adrenergic receptors and prostaglandin E2 receptors respectively) enhance APP synthesis and the formation of amyloidogenic APP holoprotein. Transmitters that stimulate phosphatidylinositol hydrolysis (by activating muscarinic m1 or m3 receptors, serotoninergic 5HT2a or 5HT2c receptors, or metabotropic glutamate receptors of subtypes 1 or 5) increase the conversion of APP to soluble APPs, and decrease the formation of Aβ. These findings suggest that drugs that regulate the activity of neurotransmitter receptors might be useful in preventing the excessive formation of Aβ or other amyloid precursors in Alzheimers disease.

Prostaglandin E2 regulates amyloid precursor protein expression via the EP2 receptor in cultured rat microglia

We investigated the effects of prostaglandin E2 (PGE2) on amyloid precursor protein (APP) expression in cultured rat microglia. PGE2 treatment significantly increased the expression of APP holoprotein and was associated with an elevation in cyclic AMP (cAMP). Direct activation of adenylate cyclase with forskolin also increased APP expression. Co-treatment of microglia with PGE2 and the PKA inhibitor H-89 suppressed the overexpression of APP caused by PGE2 alone. The prostaglandin EP2 receptor is known to be positively coupled to cAMP production. Stimulation of the EP2 receptor with butaprost increased APP holoprotein, whereas co-incubation of the cells with PGE(2) and the EP2 receptor antagonist AH-6809 blocked the effect of PGE2 on APP expression. These data suggest that PGE2 is able to regulate the expression of APP, and that this effect may be mediated by the EP2 receptor and the cAMP signaling cascade.

Metabotropic Glutamate Receptors Regulate APP Processing in Hippocampal Neurons and Cortical Astrocytes Derived from Fetal Ratsa

It has previously been shown that stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol (DAG) and activating protein kinase C (PKC), accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, non‐amyloidogenic peptides (APPs). This relationship has been demonstrated in human glioma and neuroblastoma cells as well as in transfected human embryonic kidney (HEK) cells and PC12 cells.1,2

Amyloid precursor protein and membrane phospholipids in primary cortical neurons increase with development, or after exposure to nerve growth factor or Aβ1–40

Abstract We examined the relationships between membrane phospholipid levels, the secretion and expression of the amyloid precursor protein (APP), and the responses of both to nerve growth factor (NGF), Aβ 1–40 or Aβ 40–1 in developing cortical neurons cultured from rat embryos. Neuronal membrane phospholipid levels per cell, and phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine increased individually between the first and seventh days of culturing. The amounts of APP holoprotein and APP mRNAs in the cells, as well as the amounts of soluble APP (APPs) secreted by them, also increased during neuronal development in vitro. The increases in APPs exceeded the increases in APP which, in turn, exceed those in phospholipid levels. The levels of APP holoprotein, but not of phospholipids, increased when neurons were grown in a choline-free medium, suggesting that increases in APP are not sufficient to stimulate changes in membrane phospholipids. Treatment of neuron cultures for four days with NGF or Aβ 1–40 , but not with Aβ 40–1 , dose-dependently increased membrane phospholipids, tau and GAP-43, as well as APP holoprotein and secreted APPs. These results indicate that agents, like NGF or Aβ 1–40 , which enhance membrane phospholipid levels may promote neurite formation, APP expression and APPs secretion in primary neuronal cultures.

Platelet-activating factor increases prostaglandin E2 release from astrocyte-enriched cortical cell cultures

The phospholipid mediator platelet-activating factor (PAF) increased the release of prostaglandin E(2) (PGE(2)) from astrocyte-enriched cortical cell cultures in a concentration- and time-dependent manner. The nonhydrolyzable PAF analog methylcarbamyl-PAF (mc-PAF), the PAF intermediate lyso-PAF, and arachidonic acid (AA) also produced this effect. In contrast, phosphatidlycholine (PC) and lyso-PC, lipids that are structurally similar to PAF and lyso-PAF, had no effect on PGE(2) production, suggesting that PAF-induced PGE(2) release is not the consequence of nonspecific phospholipid-induced membrane perturbation. Antagonism of intracellular PAF binding sites completely abolished the ability of mc-PAF and lyso-PAF to mobilize PGE(2,) and attenuated the AA effect. Antagonism of the G-protein-coupled PAF receptor in plasma membranes had no significant effect on mc-PAF, lyso-PAF or AA-induced PGE(2) release. Based on the present findings, we hypothesize that intracellular PAF is a physiologic stimulus of PGE(2) production in astrocytes.

Intracellular and cell-surface distribution of amyloid precursor protein in cortical astrocytes.

Amyloid peptides that aggregate to form plaques in Alzheimers disease are derived from secretory processing of the amyloid precursor protein (APP). Transport of APP to the cell surface may be prerequisite for non-amyloidogenic APP processing and the secretion of soluble APP (APPs), while missorting or reinternalization of APP to intracellular compartments can promote amyloid formation. In cultured astrocytes, APP mRNA and holoprotein are increased by elevations in cAMP levels, and 8-Bromo-cAMP promotes process formation on these cells. We now report that treatment of cultured astrocytes with 8-Bromo-cAMP increased intracellular and cell surface APP in the soma and perinuclear region as detected by immunolabeling with monoclonal antibody 22C11 and polyclonal antibody Kunitz-type protease inhibitor (KPI) (against the N-terminus and KPI domain of APP, respectively) and led to intense but discontinuous labelling of APP on the surface of astrocytic processes. Northern and Western blot analyses confirmed that 8-Bromo-cAMP treatment of cultured astrocytes also increased APP mRNA and KPI-containing APP holoprotein, implying that the intense APP immunolabeling observed in 8-Bromo-cAMP treated astrocytes was not derived from truncated forms of APP (e.g., APPs), but reflected high levels of APP holoprotein containing intact amyloid peptides. Discontinuous cell surface staining in process-bearing astrocytes may be caused by miscompartmentalization of APP related to rearrangement of the cytoskeleton. Inasmuch as intracellular APP is not accessible for non-amyloidogenic processing, we suggest that the increased immunoreactivity of intracellular APP in process-bearing astrocytes may predispose the cells to increased amyloid production.

Choline plus cytidine stimulate phospholipid production, and the expression and secretion of amyloid precursor protein in rat PC12 cells

The amyloid precursor protein (APP) is a transmembrane protein anchored in the membrane lipid bilayer. Choline and cytidine are major precursors of cell membranes, and are regulatory elements in membrane biosynthesis. We examined the levels of cellular APP holoprotein and secreted APPs when rat PC12 cells are stimulated to undergo increase in membrane phospholipids by choline+cytidine (2+2, 5+5, 10+10 or 50+50 microM) treatment. We now show that as phospholipids levels are increased by supplemental choline and cytidine treatment, the levels of cell-associated APP also rise stoichiometrically; these treatments also caused major (up to 6. 8-fold) increases in the amounts of secreted APP released into the cell medium, and also stimulated increased process formation. These results show that choline plus cytidine increase both phospholipid levels, and the expression and secretion in PC12 cells. It appears that agents that stimulate cellular membrane biosynthesis may be used to stimulate the secretion of neurotrophic APPs and neurite formation in neurodegenerative disorders such as Alzheimers disease.