The consequences of reducing expression of the alpha7 nicotinic receptor by RNA interference and of stimulating its activity with an alpha7 agonist in SH-SY5Y cells indicate that this receptor plays a neuroprotective role in connection with the pathogenesis of Alzheimer's disease

In order to examine the neuroprotective effects of the alpha7 nicotinic receptor (nAChR) in relationship to the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that targets specifically towards alpha7 nAChR or exposed to 20microM 3-[2,4-dimethoxybenzylidene] anabaseine (DMXB), a selective agonist of this same receptor. The levels of alpha7 nAChR mRNA and protein were measured by RT-PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs), total APP and the extracellular signal-regulated kinase 1/2 (ERK1/2) were also determined by Western blotting. SH-SY5Y cells transfected with siRNA or exposed to DMXB were then treated with 1microM Abeta(25-35), following which the levels of lipid peroxidation and rate of reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. Compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha7 nAChR mRNA and protein by 81% and 69% lower levels, respectively; exhibited reduced levels of the alphaAPPs and ERK1/2 proteins; and demonstrated enhanced lipid peroxidation and a decreased rate of MTT reduction. In cells exposed to DMXB, the level of alpha7 nAChR protein was elevated by 23%, with no alteration in the content of the corresponding mRNA; the levels of the alphaAPPs and ERK1/2 proteins were increased. Inhibition of the expression of the alpha7 nAChR gene enhanced the toxicity exerted by Abeta, whereas stimulation of this receptor attenuated this toxicity exerted. These findings indicate that alpha7 nAChR may play a significant neuroprotective role by enhancing cleavage of APP by alpha-secretase, regulating signal transduction, improving antioxidant defenses and inhibiting the toxicity of Abeta, which is connected with the pathogenesis of AD.     

Inhibiting gene expression of alpha3 nicotinic receptor in SH-SY5Y cells with the effects on APP metabolism and antioxidation in Alzheimer's disease

In order to examine the effects of alpha3 nicotinic acetylcholine receptor (nAChR) in connection with the pathogenesis of Alzheimer's disease (AD), neuroblastoma (SH-SY5Y) cells were transfected with small interference RNAs (siRNAs) that target specifically towards alpha3 nAChR. The expressions of alpha3 nAChR mRNA and protein were measured by real-time PCR and Western blotting, respectively. The levels of the alpha-form of secreted amyloid precursor protein (alphaAPPs) and total-APP were determined by Western blotting. SH-SY5Y cells transfected with siRNA were then treated with 1muM beta-amyloid peptide (Abeta)(1-42), following which the levels of lipid peroxidation, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the reduction rate of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were characterized by utilizing spectrophotometric procedures. As compared to controls, SH-SY5Y cells transfected with siRNA expressed the decreases in the levels of alpha3 nAChR mRNA and protein by 98% and 66% lower levels, respectively; exhibited reduced level of the alphaAPPs; and demonstrated enhanced lipid peroxidation, decreased rate of MTT reduction, and declined activities of SOD and GSH-Px. Inhibited gene expression of the alpha3 nAChR enhanced the toxicity exerted by Abeta. These results indicate that alpha3 nAChR may improve cleavage of APP by alpha-secretase, enhance antioxidation and inhibit the toxicity of Abeta, suggesting that the receptor might play an important role in AD.     

Protein kinase C regulation of intracellular and cell surface amyloid precursor protein (APP) cleavage in CHO695 cells

Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE.     

Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts

Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts.     

Cleavage of Alzheimer's amyloid precursor protein by alpha-secretase occurs at the surface of neuronal cells.

The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.     

Atorvastatin-induced activation of Alzheimer's alpha secretase is resistant to standard inhibitors of protein phosphorylation-regulated ectodomain shedding

Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or 'shedding' of the large APP ectodomain can occur via one of two competing pathways, the alpha- and beta-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The alpha-secretase pathway cleaves within the amyloidogenic Abeta domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the alpha- and beta-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of alpha-secretase-released, soluble APP (sAPPalpha). However, statin-induced stimulation of sAPPalpha release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPalpha secretion. These results suggest that statins may regulate alpha-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A 'phospho-proteomic' analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.     

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.     

A novel sorting nexin modulates endocytic trafficking and alpha-secretase cleavage of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage.     

Enhancement of alpha-secretase cleavage of amyloid precursor protein by a metalloendopeptidase nardilysin

Amyloid-beta (Abeta) peptide, the principal component of senile plaques in the brains of patients with Alzheimer's disease, is derived from proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Alternative cleavage of APP by alpha-secretase occurs within the Abeta domain and precludes generation of Abeta peptide. Three members of the ADAM (a disintegrin and metalloprotease) family of proteases, ADAM9, 10 and 17, are the main candidates for alpha-secretases. However, the mechanism that regulates alpha-secretase activity remains unclear. We have recently demonstrated that nardilysin (EC 3.4.24.61, N-arginine dibasic convertase; NRDc) enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of ADAM17. In this study, we show that NRDc enhances the alpha-secretase activity of ADAMs, which results in a decrease in the amount of Abeta generated. When expressed with ADAMs in cells, NRDc dramatically increased the secretion of alpha-secretase-cleaved soluble APP and reduced the amount of Abeta peptide generated. A peptide cleavage assay in vitro also showed that recombinant NRDc enhances ADAM17-induced cleavage of the peptide substrate corresponding to the alpha-secretase cleavage site of APP. A reduction of endogenous NRDc by RNA interference was accompanied by a decrease in the cleavage by alpha-secretase of APP and increase in the amount of Abeta generated. Notably, NRDc is clearly expressed in cortical neurons in human brain. Our results indicate that NRDc is involved in the metabolism of APP through regulation of the alpha-secretase activity of ADAMs, which may be a novel target for the treatment of Alzheimer's disease.     

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42)

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.     

ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein

Recently, we have shown that green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Abeta levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the alpha-COOH-terminal fragment (alpha-CTF) of APP and corresponding elevation of the NH(2)-terminal APP product, soluble APP-alpha (sAPP-alpha). These beneficial effects were associated with increased alpha-secretase cleavage activity, but no significant alteration in beta-or gamma-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidate alpha-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with "Swedish" mutant human APP (SweAPP N2a cells) leads to markedly elevated active ( approximately 60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased alpha-CTF cleavage, and elevated sAPP-alpha. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated alpha-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (alpha-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease.     

Intracellular copper deficiency increases amyloid-beta secretion by diverse mechanisms

In Alzheimer's disease there is abnormal brain copper distribution, with accumulation of copper in amyloid plaques and a deficiency of copper in neighbouring cells. Excess copper inhibits Abeta (amyloid beta-peptide) production, but the effects of deficiency have not yet been determined. We therefore studied the effects of modulating intracellular copper levels on the processing of APP (amyloid precursor protein) and the production of Abeta. Human fibroblasts genetically disposed to copper accumulation secreted higher levels of sAPP (soluble APP ectodomain)alpha into their medium, whereas fibroblasts genetically manipulated to be profoundly copper deficient secreted predominantly sAPPbeta and produced more amyloidogenic beta-cleaved APP C-termini (C99). The level of Abeta secreted from copper-deficient fibroblasts was however regulated and limited by alpha-secretase cleavage. APP can be processed by both alpha- and beta-secretase, as copper-deficient fibroblasts secreted sAPPbeta exclusively, but produced primarily alpha-cleaved APP C-terminal fragments (C83). Copper deficiency also markedly reduced the steady-state level of APP mRNA whereas the APP protein level remained constant, indicating that copper deficiency may accelerate APP translation. Copper deficiency in human neuroblastoma cells significantly increased the level of Abeta secretion, but did not affect the cleavage of APP. Therefore copper deficiency markedly alters APP metabolism and can elevate Abeta secretion by either influencing APP cleavage or by inhibiting its degradation, with the mechanism dependent on cell type. Overall our results suggest that correcting brain copper imbalance represents a relevant therapeutic target for Alzheimer's disease.     

Differential expression of cholesterol hydroxylases in Alzheimer's disease

Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (CYP46A1) and cholesterol 27-hydroxylase (CYP27A1). Immunocytochemical studies show that CYP46A1 and CYP27A1 are expressed in neurons and some astrocytes in the normal brain, and CYP27A1 is present in oligodendrocytes. In Alzheimer's disease (AD), CYP46A1 shows prominent expression in astrocytes and around amyloid plaques, whereas CYP27A1 expression decreases in neurons and is not apparent around amyloid plaques but increases in oligodendrocytes. Although previous studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols have yet to be examined. To understand the role of cholesterol oxidation in AD, we compared the effects of 24(S)- and 27-hydroxycholesterol on the processing of APP and analyzed the cell-specific expression patterns of the two cholesterol hydroxylases in the human brain. Both oxysterols inhibited production of Abeta in neurons, but 24(S)-hydroxycholesterol was approximately 1000-fold more potent than 27-hydroxycholesterol. The IC(50) of 24(S)-hydroxycholesterol for inhibiting Abeta secretion was approximately 1 nm. Both oxysterols induced ABCA1 expression with IC(50) values similar to that for inhibition of A beta secretion, suggesting the involvement of liver X receptor. Oxysterols also inhibited protein kinase C activity and APP secretion following stimulation of protein kinase C. The selective expression of CYP46A1 around neuritic plaques and the potent inhibition of APP processing in neurons by 24(S)-hydroxycholesterol suggests that CYP46A1 affects the pathophysiology of AD and provides insight into how polymorphisms in the CYP46A1 gene might influence the pathophysiology of this prevalent disease.     

Effect of tumor necrosis factor-alpha converting enzyme (TACE) and metalloprotease inhibitor on amyloid precursor protein metabolism in human neurons

Tumor necrosis factor-alpha (TNF-alpha) is implicated in inflammatory processes and much effort is being directed at inhibiting the release of TNF-alpha for treatment of inflammatory conditions. In this context, the drug CP-661,631 has been developed to inhibit the TNF-alpha converting enzyme (TACE). However, TACE is also implicated in amyloid precursor protein secretion. Amyloid precursor protein (APP) undergoes constitutive and regulated secretion by alpha-secretase endoproteolytic cleavage within the amyloid beta peptide (Abeta) domain. Alternative cleavage at the N- and C-terminus of the Abeta domain by beta- and gamma-secretases results in the production of Abeta. In many cellular and in vivo animal models, increased secretion of APP results in a concomitant decrease in the production of Abeta suggesting that the two pathways are intricately linked. However, in human primary neuron cultures, increased APP secretion is not associated with a decrease in total Abeta production. To determine if the use of CP-661,631 may enhance amyloidogenic processing in human brain, we have assessed the effect of CP-661,631 on APP metabolism in primary cultures of human neurons. Our results show that CP-661,631 effectively prevents regulated APP secretion but does not increase total Abeta levels in human primary neuron cultures.     

Oxidation of cholesterol by amyloid precursor protein and beta-amyloid peptide

Alzheimer's disease (AD) is characterized by accumulation of the neurotoxic peptide beta-amyloid, which is produced by proteolysis of amyloid precursor protein (APP). APP is a large membrane-bound copper-binding protein that is essential in maintaining synaptic function and may play a role in synaptogenesis. beta-Amyloid has been shown to contribute to the oxidative stress that accompanies AD. Later stages of AD are characterized by neuronal apoptosis. However, the biochemical function of APP and the mechanism of the toxicity of beta-amyloid are still unclear. In this study, we show that both beta-amyloid and APP can oxidize cholesterol to form 7beta-hydroxycholesterol, a proapoptotic oxysterol that was neurotoxic at nanomolar concentrations. 7beta-Hydroxycholesterol inhibited secretion of soluble APP from cultured rat hippocampal H19-7/IGF-IR neuronal cells and inhibited tumor necrosis factor-alpha-converting enzyme alpha-secretase activity but had no effect on beta-site APP-cleaving enzyme 1 activity. 7beta-Hydroxycholesterol was also a potent inhibitor of alpha-protein kinase C, with a K(i) of approximately 0.2 nm. The rate of reaction between cholesterol and beta-amyloid was comparable to the rates of cholesterol-metabolizing enzymes (k(cat) = 0.211 min(-)1). The rate of production of 7beta-hydroxycholesterol by APP was approximately 200 times lower than by beta-amyloid. Oxidation of cholesterol was accompanied by stoichiometric production of hydrogen peroxide and required divalent copper. The results suggest that a function of APP may be to produce low levels of 7-hydroxycholesterol. Higher levels produced by beta-amyloid could contribute to the oxidative stress and cell loss observed in Alzheimer's disease.     

Protein kinase C epsilon suppresses Abeta production and promotes activation of alpha-secretase

Deposition of plaques containing Abeta is considered important in the pathogenesis of Alzheimer's disease. Phorbol esters that activate protein kinase C (PKC) promote alpha-secretase-mediated processing of the beta amyloid precursor protein (APP), which generally reduces formation of Abeta. To determine which PKC isozymes mediate this process, we studied CHO cells that express human APP751. Phorbol 12-myristate, 13-acetate (PMA)-stimulated APP secretion, which was reduced by a general PKC inhibitor bisindoylmaleimide I, but not by G? 6976, which inhibits PKCalpha, beta, gamma, and mu. Since PKCdelta and epsilon were the only other PMA-sensitive isozymes present, we studied cells that express selective peptide inhibitors of these isozymes. Expression of the PKCepsilon inhibitor inhibited PMA-induced APPs secretion and suppression of Abeta production. In contrast, the PKCdelta inhibitor had no effect. These results provide evidence that PKCepsilon decreases Abeta production by promoting alpha-secretase mediated cleavage of APP.     

Effects of statins on alpha7 nicotinic receptor, cholinesterase and alpha-form of secreted amyloid precursor peptide in SH-SY5Y cells

In order to reveal the neuroprotective effects of statins that could be of interest for the prevention and treatment of Alzheimer's disease (AD), we investigated the expression of nicotinic acetylcholine receptors (nAChRs) detected by RT-PCR, the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by colorimetric determination, and the levels of the alpha-form of secreted beta-amyloid precursor protein (alphaAPPs) by Western blotting in neuroblastoma (SH-SY5Y) cells exposed to lovastatin, atorvastatin, rosuvastatin and simvastatin, respectively. The results indicated that all statins studied, both lipophilic and hydrophilic, induced high expression of alpha7 nAChR, decreased cholinesterase activities, and increased alphaAPPs, which suggests that statins might play important neuroprotective roles in AD treatment.     

Estrogen-induced cell signalling in a cellular model of Alzheimer's disease

Alzheimer's disease (AD) is characterised by deposition of a 4 kDa amyloid-beta peptide (Abeta) into senile plaques of the affected brain. Abeta is a proteolytic product of the membrane protein, amyloid precursor protein (APP). An alternative cleavage pathway involves alpha-secretase activity and results in secretion of a 100 kDa non-amyloidogenic APP (sAPPalpha) and therefore a potential reduction in Abeta secretion. We have shown that estrogen induces alpha-cleavage and therefore results in the secretion of sAPPalpha. This secretion is signalled via MAP-kinase and PI-3 kinase signal-transduction pathways. These pathways also have the potential to inhibit the activation of glycogen synthase kinase 3beta (GSK), a protein involved in cell death. Therefore, the aim of this work was to further elucidate the estrogen-mediated signaling pathways involved in APP processing, with particular emphasis on GSK activity. By stimulating rat hypothalamic neuronal GT1-7 cells with estradiol, we found that estrogen decreases the activation state of GSK via the MAP kinase pathway. Moreover, the inhibition of GSK activity by LiCl causes enhanced sAPPalpha secretion in a pattern similar to that seen in response to estrogen, suggesting a pivotal role for this deactivation in APP processing. Further, inactivation of GSK by estrogen can be confirmed in an in vivo model. Elucidation of the signaling pathways involved in APP processing may help to understand the pathology of AD and may also prove beneficial in developing therapeutic strategies to combat AD.     

Protein kinase C-dependent alpha-secretase competes with beta-secretase for cleavage of amyloid-beta precursor protein in the trans-golgi network

The release of amyloidogenic amyloid-beta peptide (Abeta) from amyloid-beta precursor protein (APP) requires cleavage by beta- and gamma-secretases. In contrast, alpha-secretase cleaves APP within the Abeta sequence and precludes amyloidogenesis. Regulated and unregulated alpha-secretase activities have been reported, and the fraction of cellular alpha-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated alpha-secretase cleavage of APP has been shown to occur at the cell surface, we sought to identify the intracellular site of PKC-regulated alpha-secretase APP cleavage. To accomplish this, we measured levels of secreted ectodomains and C-terminal fragments of APP generated by alpha-secretase (sAPPalpha) (C83) versus beta-secretase (sAPPbeta) (C99) and secreted Abeta in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. We found that PKC stimulation increased sAPPalpha but decreased sAPPbeta levels by altering the competition between alpha- versus beta-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for beta-secretase activity prompted us to hypothesize that PKC-regulated alpha-secretase(s) also reside in this organelle. To test this hypothesis, we performed studies demonstrating proteolytically mature TACE intracellularly, and we also showed that regulated alpha-secretase APP cleavage occurs in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated alpha-secretase APP cleavage in the TGN by TACE/ADAM-10, we reveal ADAM activity in a novel location. Finally, the competition between TACE/ADAM-10 and beta-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.     

Reversible and irreversible acetylcholinesterase inhibitors cause changes in neuronal amyloid precursor protein processing and protein kinase C level in vitro

The alternative routes of cleavage of the amyloid precursor protein (APP) result in the generation and secretion of both soluble APP and beta-amyloid, the latter being the main component of the amyloid deposits in the brains of individuals with Alzheimer's disease (AD). This study examined the question of whether acetylcholinesterase (AChE) inhibitors can alter the processing of APP and the level of protein kinase C (PKC) in primary rat basal forebrain cultures. Western blotting was used to test two AChE inhibitors (reversible and irreversible) for their ability to enhance the release of APP and PKC content. These inhibitors were ambenonium (AMB) and metrifonate (MTF), at different concentrations. A significant increase was found in the cell-associated APP level in a basal forebrain neuronal culture, and there was an elevation of the APP release into the medium. Increases were similarly observed in the PKC levels after AMB or MTF treatment. The results suggest that these AChE inhibitors promote the non-amyloidogenic route of APP processing, which may be due to their stimulatory effects on PKC. The PKC activation may enhance the alpha-secretase activity and consequently the production of the N-terminal APP. Since both a decreased level of APP secretion and a low activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the administration of AChE inhibitors to AD patients may facilitate the memory processes and exert a neuroprotective effect.