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.     

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.     

Cystatin C-cathepsin B axis regulates amyloid beta levels and associated neuronal deficits in an animal model of Alzheimer's disease

Impaired degradation of amyloid beta (Abeta) peptides could lead to Abeta accumulation, an early trigger of Alzheimer's disease (AD). How Abeta-degrading enzymes are regulated remains largely unknown. Cystatin C (CysC, CST3) is an endogenous inhibitor of cysteine proteases, including cathepsin B (CatB), a recently discovered Abeta-degrading enzyme. A CST3 polymorphism is associated with an increased risk of late-onset sporadic AD. Here, we identified CysC as the key inhibitor of CatB-induced Abeta degradation in vivo. Genetic ablation of CST3 in hAPP-J20 mice significantly lowered soluble Abeta levels, the relative abundance of Abeta1-42, and plaque load. CysC removal also attenuated Abeta-associated cognitive deficits and behavioral abnormalities and restored synaptic plasticity in the hippocampus. Importantly, the beneficial effects of CysC reduction were abolished on a CatB null background, providing direct evidence that CysC regulates soluble Abeta and Abeta-associated neuronal deficits through inhibiting CatB-induced Abeta degradation.     

Proteases and lipoprotein receptors in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.     

Internalized antibodies to the Abeta domain of APP reduce neuronal Abeta and protect against synaptic alterations

Immunotherapy against beta-amyloid peptide (Abeta) is a leading therapeutic direction for Alzheimer disease (AD). Experimental studies in transgenic mouse models of AD have demonstrated that Abeta immunization reduces Abeta plaque pathology and improves cognitive function. However, the biological mechanisms by which Abeta antibodies reduce amyloid accumulation in the brain remain unclear. We provide evidence that treatment of AD mutant neuroblastoma cells or primary neurons with Abeta antibodies decreases levels of intracellular Abeta. Antibody-mediated reduction in cellular Abeta appears to require that the antibody binds to the extracellular Abeta domain of the amyloid precursor protein (APP) and be internalized. In addition, treatment with Abeta antibodies protects against synaptic alterations that occur in APP mutant neurons.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.     

Release of acetylcholinesterase (AChE) from beta-amyloid plaques assemblies improves the spatial memory impairments in APP-transgenic mice

The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid-beta-peptide (Abeta). Amyloid deposits contain "chaperone molecules" which play critical roles in amyloid formation and toxicity. In the present work, we test an analog of hyperforin (IDN 5706) which releases the AChE from both the Abeta fibrils and the AChE-Abeta burdens in transgenic mice. Hyperforin is an acylphloroglucinol compound isolated from Hypericum perforatum (St. John's Wort), which is able to prevent the Abeta-induced spatial memory impairments and Abeta neurotoxicity. Altogether this gathered evidence indicates the important role of AChE in the neurotoxicity of Abeta plaques and finding new compounds which decrease the AChE-Abeta interaction may be a putative therapeutic agent to fight the disease.     

Glycosylphosphatidylinositol-anchored proteins play an important role in the biogenesis of the Alzheimer's amyloid beta-protein.

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.     

Clearing the brain's amyloid cobwebs.

Elevated cerebral levels of amyloid beta-protein occur universally in Alzheimer's disease, yet only a few patients show evidence of increased Abeta production. Therefore, defects in proteases that degrade Abeta could underlie some or many cases of familial and sporadic AD. This previously neglected topic has begun receiving serious attention. Understanding how proteolysis regulates Abeta levels in the cerebral cortex has implications for both the pathogenesis and the treatment of this protean disorder.     

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.     

I32E and V36K double mutation in beta2-sheet abrogates amyloid beta peptide toxicity

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, wherein, the accumulation of amyloid beta (Abeta) peptide as cytotoxic oligomers leads to neuropathologic changes. Transgenic mice with brain Abeta plaques immunized with aggregated Abeta have reduced amyloid burden and improved cognitive functions. However, such active immunization in humans led to a small but significant occurrence of meningoencephalitis in 6% AD volunteers due to Abeta induced toxicity. In an attempt to develop safer alternative vaccines, the design of a highly soluble peptide homologous to Abeta (Abeta-EK), that has a reduced amyloidogenic potential while maintaining the major immunogenic epitopes of Abeta is reported. More importantly, this homologue has been shown to be non-toxic, as this peptide failed to exert any observable effect on erythrocytes. The results of the present study suggests that immunization with non-toxic Abeta derivative may offer a safer therapeutic approach to AD, instead of using toxic Abeta fibrils.     

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.     

Transglutaminase induces protofibril-like amyloid beta-protein assemblies that are protease-resistant and inhibit long-term potentiation

An increasing body of evidence suggests that soluble assemblies of amyloid beta-protein (Abeta) play an important role in the initiation of Alzheimer disease (AD). In vitro studies have found that synthetic Abeta can form soluble aggregates through self-assembly, but this process requires Abeta concentrations 100- to 1000-fold greater than physiological levels. Tissue transglutaminase (TGase) has been implicated in neurodegeneration and can cross-link Abeta. Here we show that TGase induces rapid aggregation of Abeta within 0.5-30 min, which was not observed with chemical cross-linkers. Both Abeta40 and Abeta42 are good substrates for TGase but show different aggregation patterns. Guinea pig and human TGase induced similar Abeta aggregation patterns, and oligomerization was observed with Abeta40 concentrations as low as 50 nm. The formed Abeta40 species range from 5 to 6 nm spheres to curvilinear structures of the same width, but up to 100 nm in length, that resemble the previously described self-assembled Abeta protofibrils. TGase-induced Abeta40 assemblies are resistant to a 1-h incubation with either neprilysin or insulin degrading enzyme, whereas the monomer is rapidly degraded by both proteases. In support of these species being pathological, TGase-induced Abeta40 assemblies (100 nm) inhibited long term potentiation recorded in the CA1 region of mouse hippocampus slices. Our data suggest that TGase can contribute to AD by initiating Abeta oligomerization and aggregation at physiological levels, by reducing the clearance of Abeta due to the generation of protease-resistant Abeta species, and by forming Abeta assemblies that inhibit processes involved in memory and learning. Our data suggest that TGase might constitute a specific therapeutic target for slowing or blocking the progression of AD.     

Cellular cofactors potentiating induction of stress and cytotoxicity by amyloid beta-peptide

Insights into factors underlying causes of familial Alzheimer's disease (AD), such as mutant forms of beta-amyloid precursor protein and presenilins, and those conferring increased risk of sporadic AD, such as isoforms of apolipoprotein E and polymorphisms of alpha2-macroglobulin, have been rapidly emerging. However, mechanisms through which amyloid beta-peptide (Abeta), the fibrillogenic peptide most closely associated with neurotoxicity in AD, exerts its effects on cellular targets have only been more generally outlined. Late in the course of AD, when Abeta fibrils are abundant, non-specific interactions of amyloid with cellular elements are likely to induce broad cytotoxicity. However, early in AD, when concentrations of Abeta are much lower and extracellular deposits are infrequent, mechanisms underlying cellular dysfunction have not been clearly defined. The key issue in elucidating the means through which Abeta perturbs cellular properties early in AD is the possibility that protective therapy at such times may prevent cytotoxicity at a point when damage is still reversible. This brief review focusses on two cellular cofactors for Abeta-induced cellular perturbation: the cell surface immunoglobulin superfamily molecule RAGE (receptor for advanced glycation endproducts) and ABAD (Abeta binding alcohol dehydrogenase). Although final proof for the involvement of these cofactors in cellular dysfunction in AD must await the results of further in vivo experiments, their increased expression in AD brain, as well as other evidence described below, suggests the possibility of specific pathways for Abeta-induced cellular perturbation which could provide future therapeutic targets.     

Age-dependent high-density clustering of GM1 ganglioside at presynaptic neuritic terminals promotes amyloid beta-protein fibrillogenesis

The deposition of amyloid beta-protein (Abeta) is an invariable feature of Alzheimer's disease (AD); however, the biological mechanism underlying Abeta assembly into fibrils in the brain remains unclear. Here, we show that a high-density cluster of GM1 ganglioside (GM1), which was detected by the specific binding of a novel peptide (p3), appeared selectively on synaptosomes prepared from aged mouse brains. Notably, the synaptosomes bearing the high-density GM1 cluster showed extraordinary potency to induce Abeta assembly, which was suppressed by an antibody specific to GM1-bound Abeta, an endogenous seed for AD amyloid. Together with evidence that Abeta deposition starts at presynaptic terminals in the AD brain and that GM1 levels significantly increase in amyloid-positive synaptosomes prepared from the AD brain, our results suggest that the age-dependent high-density GM1 clustering at presynaptic neuritic terminals is a critical step for Abeta deposition in AD.     

Heterologous amyloid seeding: revisiting the role of acetylcholinesterase in Alzheimer's disease

Neurodegenerative diseases associated with abnormal protein folding and ordered aggregation require an initial trigger which may be infectious, inherited, post-inflammatory or idiopathic. Proteolytic cleavage to generate vulnerable precursors, such as amyloid-beta peptide (Abeta) production via beta and gamma secretases in Alzheimer's Disease (AD), is one such trigger, but the proteolytic removal of these fragments is also aetiologically important. The levels of Abeta in the central nervous system are regulated by several catabolic proteases, including insulysin (IDE) and neprilysin (NEP). The known association of human acetylcholinesterase (hAChE) with pathological aggregates in AD together with its ability to increase Abeta fibrilization prompted us to search for proteolytic triggers that could enhance this process. The hAChE C-terminal domain (T40, AChE(575-614)) is an exposed amphiphilic alpha-helix involved in enzyme oligomerisation, but it also contains a conformational switch region (CSR) with high propensity for conversion to non-native (hidden) beta-strand, a property associated with amyloidogenicity. A synthetic peptide (AChE(586-599)) encompassing the CSR region shares homology with Abeta and forms beta-sheet amyloid fibrils. We investigated the influence of IDE and NEP proteolysis on the formation and degradation of relevant hAChE beta-sheet species. By combining reverse-phase HPLC and mass spectrometry, we established that the enzyme digestion profiles on T40 versus AChE(586-599), or versus Abeta, differed. Moreover, IDE digestion of T40 triggered the conformational switch from alpha- to beta-structures, resulting in surfactant CSR species that self-assembled into amyloid fibril precursors (oligomers). Crucially, these CSR species significantly increased Abeta fibril formation both by seeding the energetically unfavorable formation of amyloid nuclei and by enhancing the rate of amyloid elongation. Hence, these results may offer an explanation for observations that implicate hAChE in the extent of Abeta deposition in the brain. Furthermore, this process of heterologous amyloid seeding by a proteolytic fragment from another protein may represent a previously underestimated pathological trigger, implying that the abundance of the major amyloidogenic species (Abeta in AD, for example) may not be the only important factor in neurodegeneration.     

Determination of beta-amyloid peptide signatures in cerebrospinal fluid using immunoprecipitation-mass spectrometry

Early pathogenic events in Alzheimer's disease (AD) involve increased production and/or reduced clearance of beta-amyloid (Abeta), especially the 42 amino acid fragment Abeta1-42. The Abeta1-42 peptide is generated through cleavage of the amyloid precursor protein by beta- and gamma-secretase and is catabolised by a variety of proteolytic enzymes such as insulin-degrading enzyme and neprilysin. Here, we describe a method that employs immunoprecipitation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to determine the pattern of C-terminally truncated Abeta peptides in cerebrospinal fluid (CSF). Using antibodies coupled to magnetic beads, we have detected 18 C-terminally and 2 N-terminally truncated Abeta peptides in CSF. By determining the identity and profile of the truncated Abeta peptides, more insight may be gained about differences in the metabolism and structural properties of Abeta in AD. Finally, the Abeta fragment signatures may prove useful as a diagnostic test for AD.     

Protein aging hypothesis of Alzheimer disease.

Alzheimer disease (AD), the most common form of aging-related neurodegenerative disorders, is associated with formation of fibrillar deposits of amyloid beta-protein (Abeta). While the direct involvement of Abeta in AD has been well documented, the relations between Abeta production, amyloid formation, and neurodegeneration remain unknown. We propose that AD is initiated by a protein aging-related structural transformation in soluble Abeta. We hypothesize that spontaneous chemical modification of aspartyl residues in Abeta to transient succinimide induces a non-native conformation in a fraction of soluble Abeta, rendering it amyloidogenic and neurotoxic. Conformationally altered Abeta is characterized by increased stability in solution and the presence of a non-native beta-turn that determines folding of Abeta in solution and the structure of Abeta subunits incorporated into amyloid fibrils. While the soluble 'non-native' Abeta is both the factor triggering the neurodegenerative cascade and the precursor of amyloid plaques, these two events result from interaction of Abeta with different sets of cellular components and need not coincide in space and time. Extensive literature data and experimental evidence are provided in support of this hypothesis.     

PAR-4 is involved in regulation of beta-secretase cleavage of the Alzheimer amyloid precursor protein

Mounting evidence indicates that aberrant production and aggregation of amyloid beta-peptide (Abeta)-(1-42) play a central role in the pathogenesis of Alzheimer disease (AD). Abeta is produced when amyloid precursor protein (APP) is cleaved by beta- and gamma-secretases at the N and C termini of the Abeta domain, respectively. The beta-secretase is membrane-bound aspartyl protease, most commonly known as BACE1. Because BACE1 cleaves APP at the N terminus of the Abeta domain, it catalyzes the first step in Abeta generation. PAR-4 (prostate apoptosis response-4) is a leucine zipper protein that was initially identified to be associated with neuronal degeneration and aberrant Abeta production in models of AD. We now report that the C-terminal domain of PAR-4 is necessary for forming a complex with the cytosolic tail of BACE1 in co-immunoprecipitation assays and in vitro pull-down experiments. Overexpression of PAR-4 significantly increased, whereas silencing of PAR-4 expression by RNA interference significantly decreased, beta-secretase cleavage of APP. These results suggest that PAR-4 may be directly involved in regulating the APP cleavage activity of BACE1. Because the increased BACE1 activity observed in AD patients does not seem to arise from genetic mutations or polymorphisms in BACE1, the identification of PAR-4 as an endogenous regulator of BACE1 activity may have significant implications for developing novel therapeutic strategies for AD.