Lawrence M. Refolo

Nerve and epidermal growth factors induce the release of the Alzheimer amyloid precursor from PC 12 cell cultures.

Antisera against specific sites of the Alzheimer beta Amyloid protein precursor (beta APP) were used to study the effects of nerve and epidermal growth factors on the expression and processing of this protein in PC12 cell cultures. Two major beta APP proteins (140 and 105 kDa) both containing the Kunitz-protease inhibitor insert (KPI), were detected in cell extracts of naive PC12 cells. Treatment of these cultures with nerve growth factor (NGF) induced the release of two beta APP species 125 and 120 kDa, both of which contained the KPI domain and lacked the carboxy-terminal portion of the precursor. The released beta APP contained O-linked sugars. Only one of the released beta APP proteins bound to the lectin Concanavalin A indicating that they differ in their glycosylation. Epidermal growth factor (EGF) also induced the release of beta APP proteins into the culture medium with similar electrophoretic mobilities as those released by NGF.

Differential brain expression of the Alzheimer's amyloid precursor protein.

The expression of the Alzheimer amyloid protein precursor (AAPP) was examined in human, monkey, dog and rat brains. Two proteins, one identified as AAPP695 and the other as AAPP751, were immunoprecipitated from the in vitro translation of human, dog and rat brain polysomes. The AAPP751 to AAPP695 ratio was highest in human, intermediate in dog and lowest in rat brain polysomes. Human cerebral cortex contained higher levels of the AAPP751 mRNA than either dog or rat cortex. AAPP695 was detected in both cerebral cortex and cerebellum of all species examined. In contrast, AAPP751 was detected predominantly in the cortex of human, monkey and to a lesser extent dog brains while it was not detected in rat brain. These findings indicate that the amyloid precursors are differentially expressed in different mammalian brains and suggest that AAPP751 is mainly expressed in the brain regions involved in plaque formation.

Intracellular Cyclic AMP Inhibits Constitutive and Phorbol Ester‐Stimulated Secretory Cleavage of Amyloid Precursor Protein

Abstract: α‐Secretase cleaves the full‐length Alzheimers amyloid precursor protein (APP) within the amyloid β peptide sequence, thus precluding amyloid formation. The resultant soluble truncated APP is constitutively secreted. This nonamyloidogenic processing of APP is increased on stimulation of the phospholipase C/protein kinase C pathway by phorbol esters. Here we used C6 cells transfected with APP751 to examine whether the α‐secretase cleavage is regulated by the adenylate cyclase signal transduction pathway. Forskolin, an activator of adenylate cyclase, inhibited both the constitutive and phorbol ester‐stimulated secretion of nexin II (NXII), the secreted product of the α‐secretase cleavage of APP751. At 1 µM, forskolin inhibited secretion of NXII by ∼50% without affecting either the intracellular levels of total APP or the secretion of secretory alkaline phosphatase. In contrast, 1,9‐dideoxyforskolin, an inactive analogue of forskolin, did not affect secretion of NXII. These results indicated that forskolin specifically inhibited the α‐secretase cleavage of APP751. Forskolin treatment increased the intracellular concentration of cyclic AMP (cAMP), suggesting that the forskolin effects on APP cleavage may be mediated by cAMP. In support of this suggestion, both dibutyryl cAMP, a cAMP analogue, and isoproterenol, an activator of adenylate cyclase, also inhibited secretion of NXII. These data indicate that forskolin inhibition of the nonamyloidogenic cleavage of APP is mediated by the second messenger cAMP, which together with the protein kinase C signal transduction pathway modulates the secretory cleavage of APP.

The Alzheimer's amyloid precursor is cleaved intracellularly in the trans-golgi network or in a post-golgi compartment

The amyloid fl-protein (@)l is the major component of the neuritic plaque cores and cerebrovascular amyloid observed in the brains of Alzheimer disease (AD) patients.2J Its sequence is part of at least three distinct precursor proteins termed APPH5, APP75,, and APP?70.’-9 Both APP,,, and APP770 contain a 56-amino acid insert with high homology to the Kunitz-type serine protease inhibitors (KPI).7-9 APP has been shown to undergo extensive posttranslational modifications including glycosylation, sulfation, and phosphorylation.10-12 In addition, APP was shown to be the core protein of a chondroitin sulfate proteoglycan.’j APP proteins are integral membrane proteins containing a large extracellular domain, a transmembrane region, and a small cytoplasmic p o r t i ~ n . ~ . ~ The @ corresponds to APP751 sequence 653 to about 695 and comprises portions of extracellular and membrane-spanning domains of APP. Secreted APP forms are produced after full-length APP is cleaved by the putative enzyme APP secretase at the C-terminal site of Lysof APP751, which corresponds to AJ3 residue 16.” These secreted forms have been detected in cell cultures, in human cerebrospinal fluid, in serum, and in platelet granules, but since the secretion mechanism involves cleavage within the AJ3 sequence, these APP forms are not amyloidogenic. @ must arise from alternative proteolytic pathways in which its sequence remains intact.15J6 In this paper we present evidence that KPIcontaining APP is cleaved by APP secretase intracellularly either in the TGN or in a post-Golgi compartment, and the resultant soluble APP (nexin 11) is transported in vesicles and exocytosed. In addition, nexin I1 production is not affected by inhibitors of lysosomal proteolysis such a leupeptin and chloroquine, indicating that APP secretase is not a lysosomal enzyme.

Chondroitin sulfate proteoglycan form of cellular and cell-surface Alzheimer amyloid precursor

The biological function of the amyloid precursor protein (APP) is still not fully understood. Recently, we reported that secreted truncated APP occurs in a chondroitin sulfate proteoglycan form. Here we present evidence that full length APP-chondroitin sulfate proteoglycan is present on the cell surface of C6 glioma cells. In addition, densitometric quantitation of Western blots showed that approximately 50% of the mature cell-associated full length APP is in the proteoglycan form. These findings suggest that the proteoglycan nature of APP may be important for the implementation of its biological function.

Heat-shock induces abnormalities in the cellular distribution of amyloid precursor protein (APP) and APP fusion proteins

The heat shock or stress response may play a role in the pathogenesis of Alzheimers disease. We conducted experiments to visualize microscopically the distribution of wild type amyloid precursor protein (APP) and the behavior of an APP deletion mutant under stress. This was achieved by heat-shock treatment of cells expressing fusion recombinant APP proteins tagged with secreted placental alkaline phosphatase (SEAP). The fusion proteins were cleaved and secreted in a manner similar to wild type APP in unstressed control cells. SEAP activity was detected by cytochemical methods within the cytoplasm in less than 10% of transfected unstressed cells. Heat shocked cells showed a striking difference from the control cells in that over 90% of the stressed cells displayed strong intracytoplasmic SEAP activity occurring with Golgi-like pattern and/or membranous distribution. The effects of heat shock were not due to a peculiar behavior of the clones and depended on the APP portion of the constructs. This study shows miscompartmentalization of APP under stress. Such cellular changes may bear important implications in the processing of APP.

Cellular Processing and Proteoglycan Nature of Amyloid Precursor Proteinsa

Amyloid β protein (β/A4 or Aβ), the main protcinaceous component of the amyloid depositions of the Alzheimers brain, derives from the proteolytic processing of the amyloid precursor protein (APP). Cleavage of the amyloid precursor by at least two distinct secretase activities produces soluble secreted APP. The major secretase cleavage (site I) takes place between Aβ 16 and 17, while the minor cleavage (site II) takes place after Aβ Lys 28 and may produce potentially amyloidogenic secreted APP. Full‐length cellular APP is cleaved by secretase intracellularly in the Trans‐Golgi Network (TGN) or in post‐Golgi vesicles. The resultant soluble APP is transported to the plasma membrane and exocytosed.

Effects of nerve growth factor on the expression and post-translational processing of the beta-amyloid precursor proteins in PC12 cells

236 Alzheimer’s disease (AD) is characterized by extensive cholinergic denervation in both cortex and hippocampus. However, cholinergic therapies are of limited value. We investigated choline@ mechanisms in hippocampus which was denervated by a fimbriafomix lesion. Denervated pyramidal cells were found to be hypersensitive to carbachol, responding to a given dose with larger depolarizing currems and with a more pronounced blockade of the after-hyperpolarizing (AHP) current. Also, in denervated hippocampus, depolarization blockade was induced by lower concentrations of carbachol. In some cells this blockade could be reversed by addition of clonidine. It is suggested that treatment failure in AD is due to the induction of depolarization blockade and that combined treatment with a hyperpolarizing agent may enhance the therapeutic effects of cholinergic agents in this disease.