The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

The bad seed in Alzheimer's disease

In this issue of Neuron, McGowan et al. report on a new mouse model of amyloid deposition as occurs in Alzheimer's disease. Unlike previous models in which overexpression of the amyloid precursor protein results in amyloid plaque formation, McGowan et al. have produced mice that overexpress only Abeta40 or Abeta42 and prove that Abeta42 is critical for the formation of amyloid deposits in vivo.     

Cholesterol-dependent generation of a seeding amyloid beta-protein in cell culture.

Deposition of aggregated amyloid beta-protein (Abeta), a proteolytic cleavage product of the amyloid precursor protein (Abeta ), is a critical step in the development of Alzheimer's disease(Abeta++). However, we are far from understanding the molecular mechanisms underlying the initiation of Abeta polymerization in vivo. Here, we report that a seeding Abeta, which catalyzes the fibrillogenesis of soluble Abeta, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this Abeta depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel Abeta and our recent study (3), it is suggested that a conformationally altered form of Abeta, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.     

Regulation of beta-amyloid secretion by FE65, an amyloid protein precursor-binding protein

The principal component of Alzheimer's amyloid plaques, Abeta, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear. We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both alphaAPPs and Abeta secretion. The dramatic (4-fold) FE65-dependent increase in Abeta secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Abeta secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.     

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.     

Ceramide stabilizes beta-site amyloid precursor protein-cleaving enzyme 1 and promotes amyloid beta-peptide biogenesis

The lipid second messenger ceramide regulates several biochemical events that occur during aging. In addition, its level is highly elevated in the amyloid-burdened brains of Alzheimer's disease patients. Here, we analyzed the impact of aberrant ceramide levels on amyloid beta-peptide (Abeta) generation by using a cell-permeable analog of ceramide, C6-ceramide, and several biochemical inhibitors of the sphingomyelin/glycosphingolipid biosynthetic pathway. We found that C6-ceramide increased the biogenesis of Abeta by affecting beta-but not gamma-cleavage of the amyloid precursor protein. Similarly to C6-ceramide, increased levels of endogenous ceramide induced by neutral sphingomyelinase treatment also promoted the biogenesis of Abeta. Conversely, fumonisin B1, which inhibits the biosynthesis of endogenous ceramide, reduced Abeta production. Exogenous C6-ceramide restored both intracellular ceramide levels and Abeta generation in fumonisin B1-treated cells. These events were specific for amyloid precursor protein and were not associated with apoptotic cell death. Pulse-chase and time-course degradation experiments showed that ceramide post-translationally stabilizes the beta-secretase BACE1. Taken together, these data indicate that the lipid second messenger ceramide, which is elevated in the brains of Alzheimer's disease patients, increases the half-life of BACE1 and thereby promotes Abeta biogenesis.     

The use of seldi proteinchip arrays to monitor production of Alzheimer's betaamyloid in transfected cells.

beta-Amyloid (Abeta), a 39-43 residue peptide, is the principal component of senile plaques found in the brains of patients with Alzheimer's disease (AD). There are two main lines of evidence that its deposition is the cause of neurodegeneration. First, mutations found in three genes in familial Alzheimer's cases give rise to increased production of the longest, most toxic, form, Abeta 1-42. Second. aggregated Abeta is toxic to neuronal cells in culture. Inhibitors of the proteases involved in its release from the amyloid precursor protein are, therefore, of major therapeutic interest. The best candidates for the releasing proteases are both aspartyl proteases, which are integrated into the membranes of the endoplasmic reticulum and Golgi network. A sensitive assay using Ciphergen's Seldi system has been developed to measure all the variants of Abeta in culture supernatants, which will be of great value in screening inhibitors of these proteases. With this assay, it has been shown that increasing intracellular cholesterol increases the activities of both beta-secretase, and gamma-secretase 42. Moreover, changing the intracellular targeting of amyloid precursor glycoprotein (APP) yields increased alpha-secretase cleavage, and increases in the amounts of oxidized/nitrated forms of Abeta.     

Biophysical characterization of longer forms of amyloid beta peptides: possible contribution to flocculent plaque formation

Alzheimer's disease is characterized by amyloid deposits in the parenchyma and vasculature of the brain. The plaques are mainly composed of amyloid beta (Abeta) peptides ending in residues 40 and 42. Novel longer Abeta peptides were found in brain homogenates of mouse models of Alzheimer's disease and human brain tissue of patients carrying the familial amyloid precursor protein V717F mutation. The biophysical characteristics of these longer Abeta peptides and their role in plaque formation are not understood. We chose to focus our studies on Abeta peptides ending in residues Ile45, Val46 and Ile47 as these peptides were identified in human brain tissue. A combination of circular dichroism and electron microscopy was used to characterize the secondary and tertiary structures of these peptides. All three longer Abeta peptides consisted mainly of a beta-sheet secondary structure. Electron microscopy demonstrated that these beta-structured peptides formed predominantly amorphous aggregates, which convert to amyloid fibres over extended time periods. As these longer peptides may act as seeds for the nucleation of fibrils composed predominantly of shorter amyloid peptides, these interactions were studied. All peptides accelerated the random to beta-structural transitions and fibril formation of Abeta40 and 42.     

Alzheimer beta-amyloid peptides: structures of amyloid fibrils and alternate aggregation products

The amyloidoses are a heterogeneous group of diseases, which are characterized by the local or systemic deposition of amyloid. At the root of these diseases are changes in protein conformation where normal innocuous proteins transform into insoluble amyloid fibrils and deposit in tissues. The amyloid fibrils of Alzheimer's disease are composed of the Abeta peptide and deposit in the form of senile plaques. Neurodegeneration surrounds the amyloid deposits, indicating that neurotoxic substances are produced during the deposition process. Whether the neurotoxic species is the amyloid fibril or a fibril precursor is a current area of active research. This review focuses on advancements made in elucidating the molecular structures of the Abeta amyloid fibril and alternate aggregation products of the Abeta peptide formed during fibrillogenesis.     

Presenilin-1 280Glu-->Ala mutation alters C-terminal APP processing yielding longer abeta peptides: implications for Alzheimer's disease

Presenilin (PS) mutations enhance the production of the Abeta42 peptide that is derived from the amyloid precursor protein (APP). The pathway(s) by which the Abeta42 species is preferentially produced has not been elucidated, nor is the mechanism by which PS mutations produce early-onset dementia established. Using a combination of histological, immunohistochemical, biochemical, and mass spectrometric methods, we examined the structural and morphological nature of the amyloid species produced in a patient expressing the PS1 280Glu-->Ala familial Alzheimer's disease mutation. Abundant diffuse plaques were observed that exhibited a staining pattern and morphology distinct from previously described PS cases, as well as discreet amyloid plaques within the white matter. In addition to finding increased amounts of CT99 and Abeta42 peptides, our investigation revealed the presence of a complex array of Abeta peptides substantially longer than 42/43 amino acid residue species. The increased hydrophobic nature of longer Abeta species retained within the membrane walls could impact the structure and function of plasma membrane and organelles. These C-terminally longer peptides may, through steric effects, dampen the rate of turnover by critical amyloid degrading enzymes such as neprilysin and insulin degrading enzyme. A complete understanding of the deleterious side effects of membrane bound Abeta as a consequence of gamma-secretase alterations is needed to understand Alzheimer's disease pathophysiology and will aid in the design of therapeutic interventions.     

Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.     

Kinetics of inhibition of beta-amyloid aggregation by transthyretin

Deposition of beta-amyloid (Abeta) fibrils is an early event in the neurodegenerative processes associated with Alzheimer's disease. According to the "amyloid cascade" hypothesis, Abeta aggregation, and its subsequent deposition as fibrils, is the underlying cause of disease. Abeta is a proteolytic product of amyloid precursor protein (APP); several mutations in APP have been identified that are associated with early onset of disease. Transgenic mice overexpressing APP with the Swedish mutation develop numerous plaques but, surprisingly, lack the neurofibrillary tangles and neuronal loss characteristic of Alzheimer's disease, in apparent contradiction of the amyloid cascade hypothesis. However, recent studies suggest that coproduction of sAPPalpha, an alternative proteolytic product of APP, increases synthesis of transthyretin that, in turn, interacts directly with Abeta to inhibit its toxicity. Here we report results from biophysical analysis of Abeta aggregation kinetics in the presence of transthryetin. At substoichiometric ratios, transthyretin drastically decreased the rate of aggregation without affecting the fraction of Abeta in the aggregate pool. Detailed analysis of the data using a mathematical model demonstrated that the decrease in aggregation rate was due to both a decrease in the rate of elongation relative to the rate of initiation of filaments and a decrease in lateral association of filaments to fibrils. Tryptophan quenching data indicated that transthyretin binds weakly to Abeta, with an estimated apparent KS of 2300 M-1. Taken together, the data support a hypothesis wherein transthyretin preferentially binds to aggregated rather than monomeric Abeta and arrests further growth of the aggregates.     

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.     

Molecular biology and genetics of Alzheimer's disease

Like several other adult onset neurodegenerative diseases, Alzheimer's disease is a multifactorial illness with both genetic and non-genetic causes. Recent genetic studies have identified four genes associated with inherited risk for AD (presenilin 1, presenilin 2, amyloid precursor protein, and apolipoprotein E). These genes account for about half of the total genetic risk for Alzheimer's disease. It is suspected that several other Alzheimer's disease-susceptibility genes exist, and their identification is the subject of ongoing research. Nevertheless, biological studies on the effects of mutations in the four known genes has led to the conclusion that all of these genes cause dysregulation of amyloid precursor protein processing and in particular dysregulation of the handling of a proteolytic derivative termed Abeta. The accumulation of Abeta appears to be an early and initiating event that triggers a series of downstream processes including misprocessing of the tau protein. This cascade ultimately causes neuronal dysfunction and death, and leads to the clinical and pathological features of Alzheimer's disease. Knowledge of this biochemical cascade now provides several potential targets for the development of diagnostics and therapeutics.     

Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death

Converging evidence suggests that the accumulation of cerebral amyloid beta-protein (Abeta) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Abeta thus represents a novel therapeutic approach to lowering steady-state Abeta levels, but the consequences of sustained upregulation in vivo have not been studied. Here we show that transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons significantly reduces brain Abeta levels, retards or completely prevents amyloid plaque formation and its associated cytopathology, and rescues the premature lethality present in amyloid precursor protein (APP) transgenic mice. Our findings demonstrate that chronic upregulation of Abeta-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo.     

Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP).

The familial Alzheimer's disease gene product amyloid beta precursor protein (APP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical pathway leading to Abeta formation has been extensively studied since extracellular aggregates of Abeta peptides are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of APP processing is unknown. Cleavage of APP by gamma-secretase releases, together with Abeta, a COOH-terminal APP intracellular domain, termed AID. This peptide has recently been identified in brain tissue of normal control and patients with sporadic Alzheimer's disease. We have previously shown that AID acts as a positive regulator of apoptosis. Nevertheless, the molecular mechanism by which AID regulates this process remains unknown. Hoping to gain clues about the function of APP, we used the yeast two-hybrid system to identify interaction between the AID region of APP and JNK-interacting protein-1 (JIP1). This molecular interaction is confirmed in vitro, in vivo by fluorescence resonance energy transfer (FRET), and in mouse brain lysates. These data provide a link between APP and its processing by gamma-secretase, and stress kinase signaling pathways. These pathways are known regulators of apoptosis and may be involved in the pathogenesis of Alzheimer's disease.     

Rabbits fed cholesterol-enriched diets exhibit pathological features of inclusion body myositis

Sporadic inclusion body myositis (IBM) is the most common age-related muscle disease in humans; however, its etiology is unknown, there are few animal models for this disease, and effective treatments have not been identified. Similarities between pathological findings in Alzheimer's disease brain and IBM skeletal muscle include increased levels of amyloid precursor protein (APP) and amyloid beta-protein (Abeta). Moreover, there have been suggestions that elevated levels of free cholesterol might participate in the pathogenesis of Alzheimer's disease and IBM due, in part, to its role in Abeta generation. Here, we tested the hypothesis that rabbits fed cholesterol-enriched diets might faithfully exhibit human-like IBM pathological features. In skeletal muscle of one-third of the female rabbits fed cholesterol-enriched diet but not control diet, we found features of IBM, including vacuolated muscle fibers, increased numbers of mononuclear inflammatory cells, increased intramuscular deposition of Abeta, hyperphosphorylated tau, and increased numbers of muscle fibers immunopositive for ubiquitin. The cholesterol-enriched diet increased mRNA and protein levels of APP, increased the protein levels of betaAPP cleaving enzyme, and shifted APP processing in favor of Abeta production. Our study has demonstrated that increased ingestion of high levels of dietary cholesterol can result in pathological features that resemble IBM closely and thus may serve as an important new model with which to study this debilitating disorder.     

Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease

The processing of amyloid precursor protein (APP) generates amyloid-beta (Abeta) peptides 1-40 and 1-42. The latter is neurotoxic and its accumulation results in amyloid fibril formation and the generation of senile plaques, the hallmark of Alzheimer's disease (AD). Whilst there has been considerable progress made in understanding the generation of Abeta by alpha-, beta- and gamma-secretase activity on APP, recently enzymes involved in the degradation of Abeta have been identified including neprilysin and insulin-degrading enzyme (IDE). We review the pathways involved in proteolytic processing of APP and discuss the potential implications of aberrant proteolysis on neurodegeneration. It is conceivable that single nucleotide polymorphisms (SNPs) in the regulatory regions of genes in these proteolytic cascades, which alter their expression, could contribute to some of the age-related changes seen in AD.     

Secretases as targets for the treatment of Alzheimer's disease

Alzheimer's disease (AD) is the major cause of dementia in most developed countries. Treatment to modify this disease is currently unavailable, but needed urgently. The amyloid-cascade hypothesis proposes that amyloid beta-peptide (Abeta), found in the plaques characteristic of AD, plays an early, critical role in the disease process. It follows that preventing the generation of Abeta could be therapeutically useful in all cases of AD. Inhibition of the secretases that produce Abeta from a large precursor protein is the main approach to achieve this goal.     

Selectively 2H-labeled Glu/Asp: application to pKa measurements in Abeta amyloid peptides.

Human Abeta peptides have been linked to Alzheimer's disease, and it is hypothesized that formation of amyloid as well as neurotoxicity are important events in the etiology of the disease. Previous studies have shown that the soluble precursor to Alzheimer's amyloid undergoes a pH-dependent folding transition as the self-assembly activity appears, and based upon inter-residue proximities, it was suspected that stabilization of the soluble form might rely upon formation of an intramolecular salt-bridge. However, pKa studies on a model 17-residue Abeta fragment supported an electrostatic model where a solvation imperative for charged side-chain atoms drives the folding process. To explore this model in an active 26-residue fragment as well as the full-length 40-residue Abeta peptide, pKa measurements were performed via 1H and 2H NMR. To overcome issues related to sensitivity and spin system degeneracy, specifically deuterated allyl protected-Fmoc amino acids were synthesized for incorporation into a series of peptides, and a high sensitivity 2H observe NMR probe was constructed.