The neuronal adaptor protein X11alpha reduces Abeta levels in the brains of Alzheimer's APPswe Tg2576 transgenic mice

Increased production and deposition of the 40-42-amino acid beta-amyloid peptide (Abeta) is believed to be central to the pathogenesis of Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP), but the mechanisms that regulate APP processing to produce Abeta are not fully understood. X11alpha (also known as munc-18-interacting protein-1 (Mint1)) is a neuronal adaptor protein that binds APP and modulates APP processing in transfected non-neuronal cells. To investigate the in vivo effect of X11alpha on Abeta production in the brain, we created transgenic mice that overexpress X11alpha and crossed these with transgenics harboring a familial Alzheimer's disease mutant APP that produces increased levels of Abeta (APPswe Tg2576 mice). Analyses of Abeta levels in the offspring generated from two separate X11alpha founder mice revealed a significant, approximate 20% decrease in Abeta(1-40) in double transgenic mice expressing APPswe/X11alpha compared with APPswe mice. At a key time point in Abeta plaque deposition (8 months old), the number of Abeta plaques was also deceased in APPswe/X11alpha mice. Thus, we report here the first demonstration that X11alpha inhibits Abeta production and deposition in vivo in the brain.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo

Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease

beta-site APP cleaving enzyme 1 (BACE1) is the beta-secretase enzyme required for generating pathogenic beta-amyloid (Abeta) peptides in Alzheimer's disease (AD). BACE1 knockout mice lack Abeta and are phenotypically normal, suggesting that therapeutic inhibition of BACE1 may be free of mechanism-based side effects. However, direct evidence that BACE1 inhibition would improve cognition is lacking. Here we show that BACE1 null mice engineered to overexpress human APP (BACE1(-/-).Tg2576(+)) are rescued from Abeta-dependent hippocampal memory deficits. Moreover, impaired hippocampal cholinergic regulation of neuronal excitability found in the Tg2576 AD model is ameliorated in BACE1(-/-).Tg2576(+) bigenic mice. The behavioral and electrophysiological rescue of deficits in BACE1(-/-).Tg2576(+) mice is correlated with a dramatic reduction of cerebral Abeta40 and Abeta42 levels and occurs before amyloid deposition in Tg2576 mice. Our gene-based approach demonstrates that lower Abeta levels are beneficial for AD-associated memory impairments, validating BACE1 as a therapeutic target for AD.     

Duration and specificity of humoral immune responses in mice vaccinated with the Alzheimer's disease-associated beta-amyloid 1-42 peptide.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by overproduction of beta-amyloid (Abeta), which is formed from amyloid precursor protein (APP), with the subsequent pathologic deposition of Abeta in regions of the brain important for memory and cognition. Recently, vaccination of murine models of AD that exhibit Abeta deposition has halted or delayed the usual progression of the pathology of AD. Our group has demonstrated that vaccination of a doubly transgenic mouse model (expressing mutant APP and presenilin-1) with the Abeta 1-42 peptide protects these mice from the memory deficits they would ordinarily develop. This report further characterizes the Abeta 1-42 peptide vaccine in mice. Anti-Abeta response time course analysis indicated that at least three vaccinations (each 100 microg) were necessary to elicit a significant anti-Abeta titer. Subsequent vaccinations resulted in half-maximal antibody titers of at least 10,000, and these titers were maintained for at least 5 months after the final boost. Peptide binding competition studies indicated that the highest humoral responses are generated against the N terminus of the Abeta peptide. Also, measurement of specific murine Ig isotypes in Abeta-vaccinated mice demonstrated a predominant IgG(1) and IgG(2b) response, suggesting a type 2 (Th2) T-helper cell immune response, which drives humoral immunity. Finally, lymphocyte proliferation assay experiments using Abeta peptides and splenocytes from vaccinated mice demonstrated that the vaccine specifically stimulates T-cell epitopes present within the Abeta peptide.     

Expression of tau reduces secretion of Abeta without altering the amyloid precursor protein content in CHOsw cells

Insoluble deposits of tau and amyloid precursor protein (APP) peptides Abeta characterize Alzheimer's disease. We studied the role of tau in the metabolism of APP in cells stably expressing APP Swedish mutation (CHOsw). Transient expression of tau in CHOsw cells caused morphological changes, bundling of microtubules and perinuclear aggregation of Golgi-derived vesicles. It also reduced the secretion of Abeta(1-40) and Abeta(1-42) without altering the APP steady state levels. This was accompanied by a reduction in the gamma-secretase and an increase in the insulin degrading enzyme activities. Our results suggest that tau may play an inhibitory role in the amyloidogenic activity of APP.     

Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease

Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid beta-peptide (Abeta). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Abeta, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Abeta production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Abeta. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo.     

Calpain inhibitor I increases beta-amyloid peptide production by inhibiting the degradation of the substrate of gamma-secretase. Evidence that substrate availability limits beta-amyloid peptide production

The calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) has been reported to have complex effects on the production of the beta-amyloid peptide (Abeta). In this study, the effects of ALLN on the processing of the amyloid precursor protein (APP) to Abeta were examined in 293 cells expressing APP or the C-terminal 100 amino acids of APP (C100). In cells expressing APP or low levels of C100, ALLN increased Abeta40 and Abeta42 secretion at low concentrations, decreased Abeta40 and Abeta42 secretion at high concentrations, and increased cellular levels of C100 in a concentration-dependent manner by inhibiting C100 degradation. Low concentrations of ALLN increased Abeta42 secretion more dramatically than Abeta40 secretion. ALLN treatment of cells expressing high levels of C100 did not alter cellular C100 levels and inhibited Abeta40 and Abeta42 secretion with similar IC50 values. These results suggest that C100 can be processed both by gamma-secretase and by a degradation pathway that is inhibited by low concentrations of ALLN. The data are consistent with inhibition of gamma-secretase by high concentrations of ALLN but do not support previous assertions that ALLN is a selective inhibitor of the gamma-secretase producing Abeta40. Rather, Abeta42 secretion may be more dependent on C100 substrate concentration than Abeta40 secretion.     

Altered amyloid-beta metabolism and deposition in genomic-based beta-secretase transgenic mice

Amyloid-beta (Abeta) the primary component of the senile plaques found in Alzheimer's disease (AD) is generated by the rate-limiting cleavage of amyloid precursor protein (APP) by beta-secretase followed by gamma-secretase cleavage. Identification of the primary beta-secretase gene, BACE1, provides a unique opportunity to examine the role this unique aspartyl protease plays in altering Abeta metabolism and deposition that occurs in AD. The current experiments seek to examine how modulating beta-secretase expression and activity alters APP processing and Abeta metabolism in vivo. Genomic-based BACE1 transgenic mice were generated that overexpress human BACE1 mRNA and protein. The highest expressing BACE1 transgenic line was mated to transgenic mice containing human APP transgenes. Our biochemical and histochemical studies demonstrate that mice overexpressing both BACE1 and APP show specific alterations in APP processing and age-dependent Abeta deposition. We observed elevated levels of Abeta isoforms as well as significant increases of Abeta deposits in these double transgenic animals. In particular, the double transgenics exhibited a unique cortical deposition profile, which is consistent with a significant increase of BACE1 expression in the cortex relative to other brain regions. Elevated BACE1 expression coupled with increased deposition provides functional evidence for beta-secretase as a primary effector in regional amyloid deposition in the AD brain. Our studies demonstrate, for the first time, that modulation of BACE1 activity may play a significant role in AD pathogenesis in vivo.     

Truncated carboxyl-terminal fragments of beta-amyloid precursor protein are processed to amyloid beta-proteins 40 and 42

We previously showed that beta-amyloid precursor protein (APP) is cleaved not only in the middle of the membrane (gamma-cleavage) but also at novel cleavage sites close to the membrane/cytoplasmic boundary (epsilon-cleavage), releasing APP intracellular domains (AICDs) 49-99 and 50-99. To learn more about the relationship between gamma- and epsilon-cleavage, C-terminally truncated carboxyl-terminal fragments (CTFs) of APP, especially CTFs1-48 and 1-49 (the postulated products that are generated by epsilon-cleavage), were transiently expressed in CHO cells. Most importantly, the cells expressing CTF1-49 secreted predominantly amyloid beta-protein (Abeta) 40, while those expressing CTF1-48 secreted preferentially Abeta42. This supports our assumption that epsilon-cleavage precedes Alphabeta production and that preceding epsilon-cleavage determines the preference for the final Abeta species. The gamma-secretase inhibitors, L-685,458 and DAPT, suppressed Abeta production from CTF1-49. Regarding Abeta production from CTF1-48, L-685,458 suppressed it, but DAPT failed to do so. A dominant negative mutant of presenilin 1 suppressed the production of Abeta40 and 42 from both CTFs1-48 and 1-49. These data should shed significant light into the mechanism of Abeta production.     

Secretion and intracellular generation of truncated Abeta in beta-site amyloid-beta precursor protein-cleaving enzyme expressing human neurons

Insoluble pools of the amyloid-beta peptide (Abeta) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta. Thus, we used human NT2N neurons to investigate the production of Abeta11-40/42 from amyloid-beta precursor protein (APP) by beta-site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta1-40/42 and Abeta11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta1-40/42 and Abeta11-40/42. Therefore, we conclude that Abeta11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.     

Amyloid suppresses induction of genes critical for memory consolidation in APP + PS1 transgenic mice

Mice transgenic for mutated forms of the amyloid precursor protein (APP) plus presenilin-1 (PS1) genes (APP + PS1 mice) gradually develop memory deficits which correlate with the extent of amyloid deposition. The expression of several immediate-early genes (IEGs: Arc, Nur77 and Zif268) and several other plasticity-related genes (GluR1, CaMKIIalpha and Na-K- ATPase alphaIII) critical for learning and memory was normal in young APP + PS1 mice preceding amyloid deposition, but declined as mice grew older and amyloid deposits accumulated. Gene repression was less in APP + PS1 mouse brain regions that contain less Abeta and in APP mice compared with APP + PS1 mice, further linking the extent of amyloid deposition and the extent of gene repression. Critically, we demonstrated that amyloid deposition led specifically to impaired induction of the IEGs with no effects on basal expression using exposure to a novel environment 30 min prior to being killed to induce IEGs. These data imply that Abeta deposition can selectively reduce expression of multiple genes linked to synaptic plasticity, and provide a molecular basis for memory deficiencies found in transgenic APP mice and, most likely, in early stage Alzheimer's disease (AD). Presumably, pharmacological agents blocking the Abeta-related inhibition of gene expression will have benefit in AD.     

Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.     

Peptidomimetic probes and molecular modeling suggest that Alzheimer's gamma-secretase is an intramembrane-cleaving aspartyl protease

The amyloid beta-protein (Abeta), implicated in the pathogenesis of Alzheimer's disease (AD), is a proteolytic metabolite generated by the sequential action of beta- and gamma-secretases on the amyloid precursor protein (APP). The two main forms of Abeta are 40- and 42-amino acid C-terminal variants, Abeta40 and Abeta42. We recently described a difluoro ketone peptidomimetic (1) that blocks Abeta production at the gamma-secretase level [Wolfe, M. S., et al. (1998) J. Med. Chem. 41, 6-9]. Although designed to inhibit Abeta42 production, 1 also effectively blocked Abeta40 formation. Various amino acid changes in 1 still resulted in inhibition of Abeta40 and Abeta42 production, suggesting relatively loose sequence specificity by gamma-secretase. The alcohol counterparts of selected difluoro ketones also lowered Abeta levels, indicating that the ketone carbonyl is not essential for activity and suggesting that these compounds inhibit an aspartyl protease. Selected compounds inhibited the aspartyl protease cathepsin D but not the cysteine protease calpain, corroborating previous suggestions that gamma-secretase is an aspartyl protease with some properties similar to those of cathepsin D. Also, since the gamma-secretase cleavage sites on APP are within the transmembrane region, we consider the hypothesis that this region binds to gamma-secretase as an alpha-helix and discuss the implications of this model for the mechanism of certain forms of hereditary AD.     

Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.     

Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.     

Lack of ABCA1 considerably decreases brain ApoE level and increases amyloid deposition in APP23 mice

ABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Abeta immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Abeta fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble beta-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD.     

RAC1 inhibition targets amyloid precursor protein processing by gamma-secretase and decreases Abeta production in vitro and in vivo

beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.     

Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42.

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.