C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

Gamma-secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.     

The presenilin 1 deltaE9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.     

Abeta46 is processed to Abeta40 and Abeta43, but not to Abeta42, in the low density membrane domains

Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites referred to as gamma-, epsilon-, and zeta-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide gamma-secretase inhibitor, causes differential accumulation of longer amyloid beta-proteins (Abetas) within Chinese hamster ovary cells co-expressing beta C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Abeta46 localized exclusively to low density membrane (LDM) domains. Incubating the Abeta46-accumulating LDM domains at 37 degrees C produced Abeta40, Abeta42, Abeta43, and beta-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented beta-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Abeta46 and the concomitant appearance of Abeta40 and Abeta43 but not Abeta42. Further addition of DAPT suppressed the production of Abeta40/43 and abolished the decrease in the amount of Abeta46. These data indicate that preaccumulated Abeta46 is processed by gamma-secretase to Abeta40/43 but not to Abeta42 in the LDM domains. The amount of newly produced Abeta40 and Abeta43 was roughly equivalent to the decrease in the amount of Abeta46. Temporal profiles did not show a maximal concentration for Abeta43, suggesting that Abeta46 is processed to Abeta40 and Abeta43 through a nonsuccessive process.     

Co-expressed presenilin 1 NTF and CTF form functional gamma-secretase complexes in cells devoid of full-length protein

The enzyme gamma-secretase catalyzes the intramembrane proteolytic cleavage that generates the amyloid beta-peptide from the beta-amyloid precursor protein. The presenilin (PS) protein is one of the four integral membrane protein components of the mature gamma-secretase complex. The PS protein is itself subjected to endoproteolytic processing, generating stable N- and C-terminal fragment (NTF and CTF, respectively) heterodimers. Here we demonstrate that coexpression of PS1 NTF and CTF functionally mimics expression of the full-length PS1 protein and restores gamma-secretase activity in PS-deficient mammalian cells. The coexpressed fragments re-associate with each other inside the cell, where they also interact with nicastrin, another gamma-secretase complex component. Analysis of gamma-secretase activity following the expression of mutant forms of NTF and CTF, under conditions bypassing endoproteolysis, indicated that the putatively catalytic Asp257 and Asp385 residues have a direct effect on gamma-secretase activity. Moreover, we demonstrate that expression of the wild-type CTF rescues endoproteolytic cleavage of C-terminally truncated PS1 molecules that are otherwise uncleaved and inactive. Recovery of cleavage is critically dependent on the integrity of Asp385. Taken together, our findings indicate that ectopically expressed NTF and CTF restore functional gamma-secretase complexes and that the presence of full-length PS1 is not a requirement for proper complex assembly.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Presenilin-1 D257A and D385A mutants fail to cleave Notch in their endoproteolyzed forms, but only presenilin-1 D385A mutant can restore its gamma-secretase activity with the compensatory overexpression of normal C-terminal fragment

The enzyme gamma-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the gamma-secretase complex and is processed endoproteolytically, generating N- and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other gamma-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of gamma-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their gamma-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349-467). However, the defective PS-1 D257A mutant could not restore their gamma-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the gamma-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.     

Wild-type presenilin 1 protects against Alzheimer disease mutation-induced amyloid pathology

Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism.     

Conserved residues within the putative active site of gamma-secretase differentially influence enzyme activity and inhibitor binding

Gamma-secretase performs the final processing step in the generation of amyloid-beta (Abeta) peptides, which are believed to be causative for Alzheimer's disease. Presenilins (PS) are required for gamma-secretase activity and the presence of two essential intramembranous aspartates (D257 and D385) has implicated this region as the putative catalytic centre of an aspartyl protease. The presence of several key hydrogen-bonding residues around the active site of classical aspartyl proteases led us to investigate the role of both the critical aspartates and two nearby conserved hydrogen bond donors in PS1. Generation of cell lines stably overexpressing the D257E, D385E, Y256F and Y389F engineered mutations has enabled us to determine their role in enzyme catalysis and binding of a transition state analogue gamma-secretase inhibitor. Here we report that replacement of either tyrosine residue alters gamma-secretase cleavage specificity, resulting in an increase in the production of the more pathogenic Abeta42 peptide in both cells and membranous enzyme preparations, without affecting inhibitor binding. In contrast, replacement of either of the aspartate residues precludes inhibitor binding in addition to inactivation of the enzyme. Together, these data further incriminate the region around the intramembranous aspartates as the active site of the enzyme, targeted by transition state analogue inhibitors, and highlight the roles of individual residues.     

Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.     

Presenilin/gamma-secretase-mediated cleavage of the voltage-gated sodium channel beta2-subunit regulates cell adhesion and migration

The voltage-gated sodium channel beta2-subunit (beta2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that beta2 undergoes ectodomain shedding followed by presenilin (PS)-dependent gamma-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an alpha-secretase enzyme, ADAM10, resulted in ectodomain cleavage of beta2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (beta2-CTF) was independently inhibited by three specific gamma-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. gamma-Secretase inhibitor treatment also increased endogenous beta2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free gamma-secretase assay, we detected gamma-secretase activity-dependent generation of a 12 kDa beta2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of beta2 processing by gamma-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific gamma-secretase inhibitor, would alter beta2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing beta2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased beta2-CTF levels, we also tested whether beta2-CTFs or beta2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of beta2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that beta2 is a PS/gamma-secretase substrate and gamma-secretase mediated cleavage of beta2-CTF is required for cell-cell adhesion and migration of beta2-expressing cells.     

Selecting cells with different Alzheimer's disease gamma-secretase activity using FACS. Differential effect on presenilin exon 9 gamma- and epsilon-cleavage.

The ultimate step in Alzheimer's disease Abeta generation involves gamma-secretase, which releases Abeta from its membrane-bound precursor. A similar presenilin-dependent proteolytic activity is implicated in the release of the Notch intracellular domain. We have developed a novel assay for gamma-secretase activity based on green fluorescent protein detection. This involves cotransfection of a substrate-activator based on the amyloid precursor protein or the Notch sequence and a fluorescent reporter gene. Stable fluorescent cell populations were selected by fluorescent activated cell sorting and characterized. This assay enabled the identification and sorting of populations, which differ in their levels of gamma-secretase activity, with high fluorescent cells producing more Abeta than low fluorescent cells. Specific gamma-secretase inhibitors, L-685,458 and MW167, reduced cell fluorescence in a dose-dependent manner that paralleled inhibition of Abeta secretion. Overexpression of presenilin 1 increased the cell fluorescence. Cells expressing presenilin with different aspartate mutations (D257A, D385A and D257A/D385A) or exon 9 deletion mutation showed reduced fluorescence. The single aspartate mutations showed a concomitant reduction in Abeta secretion, whereas the D257A/D385A and DeltaE9 mutations had no effect on Abeta secretion.     

PS1 activates PI3K thus inhibiting GSK-3 activity and tau overphosphorylation: effects of FAD mutations

Phosphatidylinositol 3-kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimer's disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by gamma-secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1-induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase-3 (GSK-3), suppresses GSK-3-dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1-dependent PI3K/Akt activation, thus promoting GSK-3 activity and tau overphosphorylation at AD-related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.     

Characterization of the reconstituted gamma-secretase complex from Sf9 cells co-expressing presenilin 1, nicastrin [correction of nacastrin], aph-1a, and pen-2

Gamma-secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in Abeta42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in Abeta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile.     

Carboxyl-terminal fragments of Alzheimer beta-amyloid precursor protein accumulate in restricted and unpredicted intracellular compartments in presenilin 1-deficient cells

Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved COOH-terminal fragments of betaAPP (alpha- or beta-CTFs). The major COOH-terminal fragment (CTF) in brain was identified as betaAPP-CTF-(11-98), which is consistent with the observation that cultured neurons generate primarily Abeta-(11-40). In PS1(-/-) murine neurons and fibroblasts expressing the loss-of-function PS1(D385A) mutant, CTFs accumulated in the endoplasmic reticulum, Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1(-/-) neurons as compared with PS1(D385A) mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of endoplasmic reticulum-to-Golgi trafficking indicated that in PS1(-/-) neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, although we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g. an activator of gamma-secretase, a substrate adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments).     

Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.     

Dimerization of presenilin-1 in vivo: suggestion of novel regulatory mechanisms leading to higher order complexes

A growing body of evidence indicates that presenilins could exist and be active as oligomeric complexes. Using yeast two-hybrid and cell culture analysis, we provide evidence that presenilin-1 (PS1) may self-oligomerize giving rise to specific full-length/full-length homodimers. When expressed in N2A and HEK239T cultured cells, full-length PS1-wt and 5(')myc-PS1-wt form specific homodimers corresponding to twice their molecular weight. The Alzheimer's disease-associated PS1 mutations Y115H, M146L, L392V, deltaE10(PS1(1-289/320-467)), the gamma-secretase dominant negative mutant D257A, and the PS1 polymorphism mutant E318G do not affect their ability to self-oligomerize. Under non-denaturing conditions, endogenous PS1 forms specific homo-oligomers in human cultured cells. The results obtained herein suggest that PS1 associates intramolecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or gamma-secretase-associated activity.     

Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. Here, we demonstrate that FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing,an important feature of AD pathology. These observations provide molecular insights into the "Ca2+ dysregulation" hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.     

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling

Here, we examined whether amyloid-beta (Abeta) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin-1 (PS-1) knock-in mice, and APP/PS-1 double Tg mice]. ELISA revealed that the insoluble form of Abeta(1-40) was markedly accumulated in the retinas of APP and APP/PS-1, but not PS-1 Tg, mice (vs. wild-type mice). In APP Tg and APP/PS-1 Tg mice, immunostaining revealed accumulations of intracellular Abeta(1-42) in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS-1 Tg, but not PS-1 Tg, mice had less NMDA-induced retinal damage than wild-type mice, and the reduced damage in APP/PS-1 Tg mice was diminished by the pre-treatment of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, a gamma-secretase inhibitor. Furthermore, the number of TUNEL-positive cells was significantly less in ganglion cell layer of APP/PS-1 Tg mice than PS-1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin-dependent protein kinase IIalpha (CaMKIIalpha), but not total CaMKIIalpha or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non-treated retinas of APP/PS-1 Tg mice (vs. wild-type mice). CaMKIIalpha and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS-1 Tg mice as compared with wild-type mice. The NMDA-induced increase in p-CaMKIIalpha in the retina was also lower in APP/PS-1 Tg mice than in wild-type mice. In electroretinogram and visual-evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS-1 mice versus wild-type mice. Hence, Abeta may impair retinal function by reducing activation of NMDA-receptor signaling pathways.