BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice

Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE1), the enzyme that initiates Abeta production, and other Abeta-lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta-lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non-transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full-length Abeta. A newly developed ELISA detected a significant reduction of full-length soluble Abeta 1-40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x-40 Abeta was moderately reduced due to detection of non-full-length Abeta and compensatory activation of alpha-secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non-transgenic mice provide more accurate evaluation of Abeta-reducing strategies than was previously feasible.     

Abeta42 is essential for parenchymal and vascular amyloid deposition in mice

Considerable circumstantial evidence suggests that Abeta42 is the initiating molecule in Alzheimer's disease (AD) pathogenesis. However, the absolute requirement for Abeta42 for amyloid deposition has never been demonstrated in vivo. We have addressed this by developing transgenic models that express Abeta1-40 or Abeta1-42 in the absence of human amyloid beta protein precursor (APP) overexpression. Mice expressing high levels of Abeta1-40 do not develop overt amyloid pathology. In contrast, mice expressing lower levels of Abeta1-42 accumulate insoluble Abeta1-42 and develop compact amyloid plaques, congophilic amyloid angiopathy (CAA), and diffuse Abeta deposits. When mice expressing Abeta1-42 are crossed with mutant APP (Tg2576) mice, there is also a massive increase in amyloid deposition. These data establish that Abeta1-42 is essential for amyloid deposition in the parenchyma and also in vessels.     

Hydrogen peroxide promotes Abeta production through JNK-dependent activation of gamma-secretase

Accumulation of senile plaques composed of amyloid beta-peptide (Abeta) is a pathological hallmark of Alzheimer disease (AD), and Abeta is generated through the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretase. Although oxidative stress has been implicated in the AD pathogenesis by inducing Abeta production, the underlying mechanism remains elusive. Here we show that the pro-oxidant H(2)O(2) promotes Abeta production through c-Jun N-terminal kinase (JNK)-dependent activation of gamma-secretase. Treatment with H(2)O(2) induced significant increase in the levels of intracellular and secreted Abeta in human neuroblastoma SH-SY5Y cells. Although gamma-secretase-mediated cleavage of APP or C99 was enhanced upon H(2)O(2) treatment, expression of APP or its alpha/beta-secretase-mediated cleavage was not affected. Silencing of the stress-activated JNK by small interfering RNA or the specific JNK inhibitor SP600125 reduced H(2)O(2)-induced gamma-secretase-mediated cleavage of APP. JNK activity was augmented in human brain tissues from AD patients and active JNK located surrounding the senile plaques in the brain of AD model mouse. Our data suggest that oxidative stress-activated JNK may contribute to senile plaque expansion through the promotion of gamma-secretase-mediated APP cleavage and Abeta production.     

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.     

Interaction of human and mouse Abeta peptides

Transgenic mice over-expressing mutant human amyloid precursor protein have become an important tool for research on Alzheimer's disease (AD) and, in particular, for therapeutic screening. Many models have reported formation of amyloid plaques with age as is detected in AD. However, the plaques generated in transgenic mice are more soluble than human plaques. Differences in solubility may occur for a number of reasons; one proposal is the presence of murine Abeta peptides within the CNS milieu. Here, we report the interaction of human and murine Abeta peptides, Abeta40 and Abeta42, utilizing a fluorescence assay to monitor formation of mixed pre-fibrillar aggregates, electron microscopy to examine morphological characteristics and detergent solubility to monitor stability. Our results demonstrate that interspecies Abeta aggregates and fibres are readily formed and are more stable than homogenous human fibres. Furthermore, these results suggest that the presence of endogenous murine Abeta in human APP transgenic mice does not account for the increased solubility of plaques.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Mitochondrial Abeta: a potential cause of metabolic dysfunction in Alzheimer's disease

Deficits in mitochondrial function are a characteristic finding in Alzheimer's disease (AD), though the mechanism remains to be clarified. Recent studies revealed that amyloid beta peptide (Abeta) gains access into mitochondrial matrix, which was much more pronounced in both AD brain and transgenic mutant APP mice than in normal controls. Abeta progressively accumulates in mitochondria and mediates mitochondrial toxicity. Interaction of mitochondrial Abeta with mitochondrial enzymes such as amyloid beta binding alcohol dehydrogenase (ABAD) exaggerates mitochondrial stress by inhibiting the enzyme activity, releasing reactive oxygen species (ROS), and affecting glycolytic, Krebs cycle and/or the respiratory chain pathways through the accumulation of deleterious intermediate metabolites. The pathways proposed may play a key role in the pathogenesis of this devastating neurodegenerative disorder, Alzheimer's disease.     

Morphological and functional abnormalities in neuromuscular junctions of Drosophila melanogaster induced by the expression of human APP gene

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in the increased production of amyloid-beta-protein (Abeta). To gain insights into synaptic effects of APP, we expressed APP, mutant form APP-Swedish and BACE in the motor neurons of fly larvae. We have shown that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons causes significant morphological and functional changes in neuromuscular junctions (NMJs): a dramatic increase in the number of synaptic buttons and changes in exocytosis as revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed for APP (APP-Swedish) and human beta-secretase (BACE) resulting in secretion of amyloid beta protein (Abeta). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from neurotoxic effects of Abeta.     

{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE

Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD.     

Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.     

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.     

Correlation between beta-amyloid peptide production and human APP-induced neuronal death

The production of amyloid peptide (Abeta) from its precursor (APP) plays a key role in Alzheimer's disease (AD). However, the link between Abeta production and neuronal death remains elusive. We studied the biological effects associated with human APP expression and metabolism in rat cortical neurons. Human APP expressed in neurons is processed to produce Abeta and soluble APP. Moreover, human APP expression triggers neuronal death. Pepstatin A, an inhibitor of aspartyl proteases that reduces Abeta production, protects neurons from APP-induced neurotoxicity. This suggests that Abeta production is likely to be the critical event in the neurodegenerative process of AD.     

Rab5-stimulated up-regulation of the endocytic pathway increases intracellular beta-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Abeta production

We previously identified abnormalities of the endocytic pathway in neurons as the earliest known pathology in sporadic Alzheimer's disease (AD) and Down's syndrome brain. In this study, we modeled aspects of these AD-related endocytic changes in murine L cells by overexpressing Rab5, a positive regulator of endocytosis. Rab5-transfected cells exhibited abnormally large endosomes immunoreactive for Rab5 and early endosomal antigen 1, resembling the endosome morphology seen in affected neurons from AD brain. The levels of both Abeta40 and Abeta42 in conditioned medium were increased more than 2.5-fold following Rab5 overexpression. In Rab5 overexpressing cells, the levels of beta-cleaved amyloid precursor protein (APP) carboxyl-terminal fragments (betaCTF), the rate-limiting proteolytic intermediate in Abeta generation, were increased up to 2-fold relative to APP holoprotein levels. An increase in beta-cleaved soluble APP relative to alpha-cleaved soluble APP was also observed following Rab5 overexpression. BetaCTFs were co-localized by immunolabeling to vesicular compartments, including the early endosome and the trans-Golgi network. These results demonstrate a relationship between endosomal pathway activity, betaCTF generation, and Abeta production. Our findings in this model system suggest that the endosomal pathology seen at the earliest stage of sporadic AD may contribute to APP proteolysis along a beta-amyloidogenic pathway.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

beta-Amyloid peptide vaccination results in marked changes in serum and brain Abeta levels in APPswe/PS1DeltaE9 mice, as detected by SELDI-TOF-based ProteinChip technology.

Although the pathogenesis of Alzheimer's disease (AD) is not fully understood, growing evidence indicates that the deposition of beta-amyloid (Abeta) and the local reactions of various cell types to this protein play major roles in the development of the disease. Immunization with the Abeta 1-42 peptide has been reported to decrease Abeta deposits in the brains of mutant amyloid precursor protein (APP/V717F) transgenic (tg) mice (Schenk et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173-177). We have replicated this finding in APPswe/PS1DeltaE9 tg mice, which also develop Abeta deposits in the brain. The immunized animals developed high titers of antibodies against Abeta 1-42 in serum, and Abeta deposits in the brains were significantly reduced. Using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry and ProteinChip((R)) technology, we detected trends toward increased soluble Abeta peptide in the brain and a decrease in assayable Abeta peptide in the serum of immunized compared with control animals. This last finding raises the possibility that anti-Abeta antibodies in the periphery sequester Abeta peptides or target them for degradation and in this way contribute to the enhanced Abeta clearance from the brain in immunized animals.     

Coumarin derivatives protection against ROS production in cellular models of Abeta toxicities

The role of oxidative stress and free radicals in the development of Alzheimer's disease (AD) has been the focus of many recent studies. The role of hydrogen peroxide (H(2)O(2)) in AD is thought to be associated with Abeta (amyloid - beta) damage in cells. A number of coumarin derivatives were previously found to be potent anti-inflammatory and antioxidant agents. Herein, these coumarin derivatives were tested as H(2)O(2) scavengers with the DCF assay using two types of neuronal cells: (a) wild type (N2a) neuroblastoma cells and (b) APP/PS1 transgenic cell line expressing Abeta. Their scavenging activity was varied between the types of cell cultures and it was found to be concentration and time dependent in the mutant cells. Their protective role against cell death further supports this notion. These results suggest that these compounds could be used as a template in the design of new molecules with a possible role in AD.     

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.     

BACE1 suppression by RNA interference in primary cortical neurons

Extracellular deposition of amyloid-beta (Abeta) aggregates in the brain represents one of the histopathological hallmarks of Alzheimer's disease (AD). Abeta peptides are generated from proteolysis of the amyloid precursor proteins (APPs) by beta- and gamma-secretases. Beta-secretase (BACE1) is a type I integral membrane glycoprotein that can cleave APP first to generate C-terminal 99- or 89-amino acid membrane-bound fragments containing the N terminus of Abeta peptides (betaCTF). As BACE1 cleavage is an essential step for Abeta generation, it is proposed as a key therapeutic target for treating AD. In this study, we show that small interfering RNA (siRNA) specifically targeted to BACE1 can suppress BACE1 (but not BACE2) protein expression in different cell systems. Furthermore, BACE1 siRNA reduced APP betaCTF and Abeta production in primary cortical neurons derived from both wild-type and transgenic mice harboring the Swedish APP mutant. The subcellular distribution of APP and presenilin-1 did not appear to differ in BACE1 suppressed cells. Importantly, pretreating neurons with BACE1 siRNA reduced the neurotoxicity induced by H2O2 oxidative stress. Our results indicate that BACE1 siRNA specifically impacts on beta-cleavage of APP and may be a potential therapeutic approach for treating AD.     

RNA interference silencing of the adaptor molecules ShcC and Fe65 differentially affect amyloid precursor protein processing and Abeta generation

The amyloid precursor protein (APP) and its pathogenic by-product amyloid-beta protein (Abeta) play central roles in Alzheimer disease (AD) neuropathogenesis. APP can be cleaved by beta-secretase (BACE) and alpha-secretase to produce APP-C99 and APP-C83. These C-terminal fragments can then be cleaved by gamma-secretase to produce Abeta and p3, respectively. p3 has been reported to promote apoptosis, and Abeta is the key component of senile plaques in AD brain. APP adaptor proteins with phosphotyrosine-binding domains, including ShcA (SHC1), ShcC (SHC3), and Fe65 (APBB1), can bind to and interact with the conserved YENPTY motif in the APP-C terminus. Here we have described for the first time the effects of RNA interference (RNAi) silencing of ShcA, ShcC, and Fe65 expression on APP processing and Abeta production. RNAi silencing of ShcC led to reductions in the levels of APP-C-terminal fragments (APP-CTFs) and Abeta in H4 human neuroglioma cells stably overexpressing full-length APP (H4-FL-APP cells) but not in those expressing APP-C99 (H4-APP-C99 cells). RNAi silencing of ShcC also led to reductions in BACE levels in H4-FL-APP cells. In contrast, RNAi silencing of the homologue ShcA had no effect on APP processing or Abeta levels. RNAi silencing of Fe65 increased APP-CTF levels, although also decreasing Abeta levels in H4-FL-APP cells. These findings suggest that pharmacologically blocking interaction of APP with ShcC and Fe65 may provide novel therapeutic strategies against AD.     

Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer's disease

Alzheimer's disease (AD) is a late-onset dementia that is characterized by the loss of memory and an impairment of multiple cognitive functions. Advancements in molecular, cellular, and animal model studies have revealed that the formation of amyloid beta (Abeta) and other derivatives of the amyloid precursor protein (APP) are key factors in cellular changes in the AD brain, including the generation of free radicals, oxidative damage, and inflammation. Recent molecular, cellular, and gene expression studies have revealed that Abeta enters mitochondria, induces the generation of free radicals, and leads to oxidative damage in post-mortem brain neurons from AD patients and in brain neurons from cell models and transgenic mouse models of AD. In the last three decades, tremendous progress has been made in mitochondrial research and has provided significant findings to link mitochondrial oxidative damage and neurodegenerative diseases such as AD. Researchers in the AD field are beginning to recognize the possible involvement of a mutant APP and its derivatives in causing mitochondrial oxidative damage in AD. This article summarizes the latest research findings on the generation of free radicals in mitochondria and provides a possible model that links Abeta proteins, the generation of free radicals, and oxidative damage in AD development and progression.