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Background: The accumulation of amyloid beta (Aβ) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aβ fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts., Methods: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aβ immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aβ42 flies, and Aβ42 seeded cultured neurons., Results: Amyloid fibrils are primarily composed by Aβ42 and unexpectedly harbor Aβ38 but generally lack Aβ40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aβ42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aβ42 toxicity in vivo., Conclusions: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology., (© 2023. Editorial Group and BioMed Central Ltd., part of Springer Nature.)

Background: Amyloid plaque deposition and axonal degeneration are early events in AD pathogenesis. Aβ disrupts microtubules in presynaptic dystrophic neurites, resulting in the accumulation of impaired endolysosomal and autophagic organelles transporting β-site amyloid precursor protein cleaving enzyme (BACE1). Consequently, dystrophic neurites generate Aβ42 and significantly contribute to plaque deposition. Farnesyltransferase inhibitors (FTIs) have recently been investigated for repositioning toward the treatment of neurodegenerative disorders and block the action of farnesyltransferase (FTase) to catalyze farnesylation, a post-translational modification that regulates proteins involved in lysosome function and microtubule stability. In postmortem AD brains, FTase and its downstream signaling are upregulated. However, the impact of FTIs on amyloid pathology and dystrophic neurites is unknown., Methods: We tested the effects of the FTIs LNK-754 and lonafarnib in the 5XFAD mouse model of amyloid pathology., Results: In 2-month-old 5XFAD mice treated chronically for 3 months, LNK-754 reduced amyloid plaque burden, tau hyperphosphorylation, and attenuated the accumulation of BACE1 and LAMP1 in dystrophic neurites. In 5-month-old 5XFAD mice treated acutely for 3 weeks, LNK-754 reduced dystrophic neurite size and LysoTracker-Green accumulation in the absence of effects on Aβ deposits. Acute treatment with LNK-754 improved memory and learning deficits in hAPP/PS1 amyloid mice. In contrast to LNK-754, lonafarnib treatment was less effective at reducing plaques, tau hyperphosphorylation and dystrophic neurites, which could have resulted from reduced potency against FTase compared to LNK-754. We investigated the effects of FTIs on axonal trafficking of endolysosomal organelles and found that lonafarnib and LNK-754 enhanced retrograde axonal transport in primary neurons, indicating FTIs could support the maturation of axonal late endosomes into lysosomes. Furthermore, FTI treatment increased levels of LAMP1 in mouse primary neurons and in the brains of 5XFAD mice, demonstrating that FTIs stimulated the biogenesis of endolysosomal organelles., Conclusions: We show new data to suggest that LNK-754 promoted the axonal trafficking and function of endolysosomal compartments, which we hypothesize decreased axonal dystrophy, reduced BACE1 accumulation and inhibited amyloid deposition in 5XFAD mice. Our results agree with previous work identifying FTase as a therapeutic target for treating proteinopathies and could have important therapeutic implications in treating AD., (© 2022. The Author(s).)

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer's disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

Reduction of amyloid beta (Aβ) has been shown to be effective in treating Alzheimer's disease (AD), but the underlying assumption that neurons are the main source of pathogenic Aβ is untested. Here, we challenge this prevailing belief by demonstrating that oligodendrocytes are an important source of Aβ in the human brain and play a key role in promoting abnormal neuronal hyperactivity in an AD knock-in mouse model. We show that selectively suppressing oligodendrocyte Aβ production improves AD brain pathology and restores neuronal function in the mouse model in vivo. Our findings suggest that targeting oligodendrocyte Aβ production could be a promising therapeutic strategy for treating AD. Neurons are thought to be the primary source of pathogenic amyloid beta (Aβ) in Alzheimer's disease. This study identifies oligodendrocytes as significant contributors to Aβ production and shows that suppressing Aβ in oligodendrocytes rescues early neuronal dysfunction in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Dysregulation of calcium homeostasis has been hypothesized to play a role in Alzheimer's disease (AD) pathogenesis. Increased calcium levels can impair axonal transport, disrupt synaptic transmission, and ultimately lead to cell death. Given the potential role of calcium dyshomeostasis in AD, there is interest in testing the ability of already approved drugs targeting various calcium channels to affect amyloid pathology and other aspects of disease. The objective of this study was to test the effects of FDA-approved L-type calcium channel antagonist nimodipine on amyloid accumulation and dystrophic neurite formation in 5XFAD mice, a mouse model of amyloid pathology. 5XFAD transgenic mice and non-transgenic littermates were treated with vehicle or nimodipine-containing chow from two to eight months of age, then brains were harvested and amyloid pathology assessed by immunoblot and immunofluorescence microscopy analyses. Nimodipine was well tolerated and crossed the blood brain barrier, as expected, but there was no effect on Aβ accumulation or on the relative amount of neuritic dystrophy, as assessed by either immunoblot, dot blot or immunofluorescence imaging of Aβ42 and dystrophic neurite marker LAMP1. While we conclude that nimodipine treatment is not likely to improve amyloid pathology or decrease neuritic dystrophy in AD, it is worth noting that nimodipine did not worsen the phenotype suggesting its use is safe in AD patients. [ABSTRACT FROM AUTHOR]

There exist several dozen lines of transgenic mice that express human amyloid-β protein precursor (AβPP) with Alzheimer's disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. [ABSTRACT FROM AUTHOR]

Background The β-secretase, BACE1, cleaves APP to initiate generation of the β-amyloid peptide, Aβ, that comprises amyloid plaques in Alzheimer's disease (AD). Reducing BACE1 activity is an attractive therapeutic approach to AD, but complete inhibition of BACE1 could have mechanism-based side-effects as BACE1-/- mice show deficits in axon guidance, myelination, memory, and other neurological processes. Since BACE1+/- mice appear normal there is interest in determining whether 50% reduction in BACE1 is potentially effective in preventing or treating AD. APP transgenic mice heterozygous for BACE1 have decreased Aβ but the extent of reduction varies greatly from study to study. Here we assess the effects of 50% BACE1 reduction on the widely used 5XFAD mouse model of AD. Results 50% BACE1 reduction reduces Aβ42, plaques, and BACE1-cleaved APP fragments in female, but not in male, 5XFAD/BACE1+/- mice. 5XFAD/BACE1+/+ females have higher levels of Aβ42 and steady-state transgenic APP than males, likely caused by an estrogen response element in the transgene Thy-1 promoter. We hypothesize that higher transgenic APP level in female 5XFAD mice causes BACE1 to no longer be in excess over APP so that 50% BACE1 reduction has a significant Aβ42 lowering effect. In contrast, the lower APP level in 5XFAD males allows BACE1 to be in excess over APP even at 50% BACE1 reduction, preventing lowering of Aβ42 in 5XFAD/BACE1+/- males. We also developed and validated a dot blot assay with an Aβ42-selective antibody as an accurate and cost-effective alternative to ELISA for measuring cerebral Aβ42 levels. Conclusions 50% BACE1 reduction lowers Aβ42 in female 5XFAD mice only, potentially because BACE1 is not in excess over APP in 5XFAD females with higher transgene expression, while BACE1 is in excess over APP in 5XFAD males with lower transgene expression. Our results suggest that greater than 50% BACE1 inhibition might be necessary to significantly lower Aβ, given that BACE1 is likely to be in excess over APP in the human brain. Additionally, in experiments using the 5XFAD mouse model, or other Thy-1 promoter transgenic mice, equal numbers of male and female mice should be used, in order to avoid artifactual gender-related differences. [ABSTRACT FROM AUTHOR]

Background The role of mitochondrial dysfunction has long been implicated in age-related brain pathology, including Alzheimer's disease (AD). However, the mechanism by which mitochondrial dysfunction may cause neurodegeneration in AD is unclear. To model mitochondrial dysfunction in vivo, we utilized mice that harbor a knockin mutation that inactivates the proofreading function of mitochondrial DNA polymerase γ (PolgA D257A), so that these mice accumulate mitochondrial DNA mutations with age. PolgA D257A mice develop a myriad of mitochondrial bioenergetic defects and physical phenotypes that mimic premature ageing, with subsequent death around one year of age. Results We crossed the D257A mice with a well-established transgenic AD mouse model (APP/Ld) that develops amyloid plaques. We hypothesized that mitochondrial dysfunction would affect Aβ synthesis and/or clearance, thus contributing to amyloidogenesis and triggering neurodegeneration. Initially, we discovered that Aβ42 levels along with Aβ42 plaque density were increased in D257A; APP/Ld bigenic mice compared to APP/Ld monogenic mice. Elevated Aβ production was not responsible for increased amyloid pathology, as levels of BACE1, PS1, C99, and C83 were unchanged in D257A; APP/Ld compared to APP/Ld mice. However, the levels of a major Aβ clearance enzyme, insulin degrading enzyme (IDE), were reduced in mice with the D257A mutation, suggesting this as mechanism for increased amyloid load. In the presence of the APP transgene, D257A mice also exhibited significant brain atrophy with apparent cortical thinning but no frank neuron loss. D257A; APP/Ld mice had increased levels of 17 kDa cleaved caspase-3 and p25, both indicative of neurodegeneration. Moreover, D257A; APP/Ld neurons appeared morphologically disrupted, with swollen and vacuolated nuclei. Conclusions Overall, our results implicate synergism between the effects of the PolgA D257A mutation and Aβ in causing neurodegeneration. These findings provide insight into mechanisms of mitochondrial dysfunction that may contribute to the pathogenesis of AD via decreased clearance of Aβ. [ABSTRACT FROM AUTHOR]

Summary: The β secretase, widely known as β-site amyloid precursor protein cleaving enzyme 1 (BACE1), initiates the production of the toxic amyloid β (Aβ) that plays a crucial early part in Alzheimer's disease pathogenesis. BACE1 is a prime therapeutic target for lowering cerebral Aβ concentrations in Alzheimer's disease, and clinical development of BACE1 inhibitors is being intensely pursued. Although BACE1 inhibitor drug development has proven challenging, several promising BACE1 inhibitors have recently entered human clinical trials. The safety and efficacy of these drugs are being tested at present in healthy individuals and patients with Alzheimer's disease, and will soon be tested in individuals with presymptomatic Alzheimer's disease. Although hopes are high that BACE1 inhibitors might be efficacious for the prevention or treatment of Alzheimer's disease, concerns have been raised about potential mechanism-based side-effects of these drugs. The potential of therapeutic BACE1 inhibition might prove to be a watershed in the treatment of Alzheimer's disease. [Copyright &y& Elsevier]

The β-amyloid (Aβ) peptide is the major constituent of amyloid plaques in Alzheimer's disease (AD) brain and is likely to play a central role in the pathogenesis of this devastating neurodegenerative disorder. The β-secretase, β-site amyloid precursor protein cleaving enzyme (BACE1; also called Asp2, memapsin 2), is the enzyme responsible for initiating Aβ generation. Thus, BACE is a prime drug target for the therapeutic inhibition of Aβ production in AD. Since its discovery 10 years ago, much has been learned about BACE. This review summarizes BACE properties, describes BACE translation dysregulation in AD, and discusses BACE physiological functions in sodium current, synaptic transmission, myelination, and schizophrenia. The therapeutic potential of BACE will also be considered. This is a summary of topics covered at a symposium held at the 39th annual meeting of the Society for Neuroscience and is not meant to be a comprehensive review of BACE. [ABSTRACT FROM AUTHOR]

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) (β-secretase) initiates generation of β-amyloid (Aβ), which plays an early role in Alzheimer's disease (AD). BACE1 levels are increased in postmortem AD brain, suggesting BACE1 elevation promotes Aβ production and AD. Alternatively, the BACE1 increase may be an epiphenomenon of late-stage AD. To distinguish between these possibilities, we analyzed BACE1 elevation using a highly specific BACE1 antibody, BACE-Cat1, made in BACE1-/- mice, which mount a robust anti-BACE1 immune response. Previous BACE1 immunohistochemical studies lack consistent results because typical BACE1 antibodies produce nonspecific background, but BACE-Cat1 immunolabels BACE1 only. BACE1 elevation was recapitulated in two amyloid precursor protein (APP) transgenic mouse lines. 5XFAD mice form amyloid plaques at young ages and exhibit neuron loss. In contrast, Tg2576 form plaques at a more advanced age and do not show cell death. These two mouse lines allow differentiation between early Aβ-induced events and late phenomena related to neuron death. BACE1 levels became elevated in parallel with amyloid burden in each APP transgenic, starting early in 5XFAD and late in Tg2576. The increase in BACE1 protein occurred without any change in BACE1 mRNA level, indicating a posttranscriptional mechanism. In APP transgenic and AD brains, high BACE1 levels were observed in an annulus around Aβ42-positive plaque cores and colocalized with neuronal proteins. These results demonstrate that amyloid plaques induce BACE1 in surrounding neurons at early stages of pathology before neuron death occurs. We conclude that BACE1 elevation is most likely triggered by the amyloid pathway and may drive a positive-feedback loop in AD. [ABSTRACT FROM AUTHOR]

The pathogenesis of Alzheimer's disease is highly complex. While several pathologies characterize this disease, amyloid plaques, composed of the β-amyloid peptide are hallmark neuropathological lesions in Alzheimer's disease brain. Indeed, a wealth of evidence suggests that β-amyloid is central to the pathophysiology of AD and is likely to play an early role in this intractable neuro-degenerative disorder. The BACE1 enzyme is essential for the generation of β-amyloid. BACE1 knockout mice do not produce β-amyloid and are free from Alzheimer's associated pathologies including neuronal loss and certain memory deficits. The fact that BACE1 initiates the formation of β-amyloid, and the observation that BACE1 levels are elevated in this disease provide direct and compelling reasons to develop therapies directed at BACE1 inhibition thus reducing β-amyloid and its associated toxicities. However, new data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-deficient mice. Here we review BACE1 biology, covering aspects ranging from the initial identification and characterization of this enzyme to recent data detailing the apparent dysregulation of BACE1 in Alzheimer's disease. We pay special attention to the putative function of BACE1 during healthy conditions and discuss in detail the relationship that exists between key risk factors for AD, such as vascular disease (and downstream cellular consequences), and the pathogenic alterations in BACE1 that are observed in the diseased state. [ABSTRACT FROM AUTHOR]

ß-Secretase [ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1)] is the key rate-limiting enzyme for the production of the ß-amyloid (Aß) peptide involved in the pathogenesis of Alzheimer's disease (AD). BACE1 levels and activity are increased in AD brain and are likely to drive Aß overproduction, but the cause of BACE1 elevation in AD is unknown. Interestingly, cerebral glucose metabolism and blood flow are both reduced in preclinical AD, suggesting that impaired energy production may be an early pathologic event in AD. To determine whether reduced energy metabolism would cause BACE1 elevation, we used pharmacological agents (insulin, 2-deoxyglucose, 3-nitropropionic acid, and kainic acid) to induce acute energy inhibition in C57/B6 wild-type and amyloid precursor protein (APP) transgenic (Tg2576) mice. Four hours after treatment, we observed that reduced energy production caused a ˜150% increase of cerebral BACE1 levels compared with control. Although this was a modest increase, the effect was long-lasting, because levels of the BACE1 enzyme remained elevated for at least 7 d after a single dose of energy inhibitor. In Tg2576 mice, levels of the BACE1-cleaved APP ectodomain APPsß were also elevated and paralleled the BACE1 increase in both relative amount and duration. Importantly, cerebral Aß40 levels in Tg2576 were increased to ˜200% of control at 7 d after injection, demonstrating that energy inhibition was potentially amyloidogenic. These results support the hypothesis that impaired energy production in the brain may drive AD pathogenesis by elevating BACE1 levels and activity, which, in turn, lead to Aß overproduction. This process may represent one of the earliest pathogenic events in AD. [ABSTRACT FROM AUTHOR]

Synaptic dysfunction is one of the earliest events in the pathogenesis of Alzheimer disease (AD). However, the molecular mechanisms underlying synaptic defects in AD are largely unknown. We report here that B-amyloid (Aβ), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that is essential for synaptic vesicle recycling and, hence, for memory formation and information processing. The Aβ-induced dynamin 1 decrease occurred in the absence of overt synaptic loss and was also observed in the Tg2576 mouse model of AD. In addition, our results provided evidence that the Aβ-induced decrease in dynamin 1 was likely the result of a calpain-mediated cleavage of dynamin 1 protein and possibly the down-regulation of dynamin 1 gene expression. These data suggest a mechanism to explain the early cognitive loss without a major decline in synapse number observed in AD and propose a novel therapeutic target for AD intervention. [ABSTRACT FROM AUTHOR]

Focuses on a study which tested the suppression of amyloid pathology development and inflammatory responses in the brains of amyloid precursor protein-expressing transgenic mice. Characteristics of Alzheimer's disease; Materials and methods; Results; Discussion.

BACE initiates the production of β-amyloid (Aβ), the likely cause of Alzheimer''s disease (AD). In this issue of Neuron, Tesco et al. show that during apoptosis caspase-3 cleaves the adaptor protein GGA3, which is required for BACE lysosomal degradation, consequently stabilizing BACE and elevating Aβ generation. [Copyright &y& Elsevier]

A correction to the article "The Alzheimer's β-secretase BACE1 localizes to normal presynaptic terminals and to dystrophic presynaptic terminals surrounding amyloid plaques" that was published in the previous issue is presented.

X11 and X11-like proteins (X11L) are neuronal adaptor proteins whose association to the cytoplasmic domain of amyloid β-protein precursor (APP) suppresses the generation of amyloid β-protein (Aβ) implicated in Alzheimer disease pathogenesis. The amyloidogenic, but not amyloidolytic, metabolism of APP was selectively increased in the brain of mutant mice lacking X11L (Sano, Y., Syuzo-Takabatake, A., Nakaya, T., Saito, Y., Tomita, S., Itohara, S., and Suzuki, T. (2006) J. Biol. Chem. 281, 37853-37860). To reveal the actual role of X11 proteins (X11s) in suppressing amyloidogenic cleavage of APP in vivo, we generated X11 and X11L double knock-out mice and analyzed the metabolism of APP. The mutant mice showed enhanced β-site cleavage of APP along with increased accumulation of Aβ in brain and increased colocalization of APP with β-site APP-cleaving enzyme (BACE). In the brains of mice deficient in both X11 and X11L, the apparent relative subcellular distributions of both mature APP and its β-C-terminal fragment were shifted toward the detergent-resistant membrane (DRM) fraction, an organelle in which BACE is active and both X11s are not nearly found. These results indicate that X11s associate primarily with APP molecules that are outside of DRM, that the dissociation of APP-X11/X11L complexes leads to entry of APP into DRM, and that cleavage of uncomplexed APP by BACE within DRM is enhanced by X11s deficiency. Present results lead to an idea that the dysfunction of X11L in the interaction with APP may recruit more APP into DRM and increase the generation of even if BACE activity did not increase in brain. [ABSTRACT FROM AUTHOR]

Accumulation of β-amyloid (Aβ) deposits is a primary pathological feature of Alzheimer disease that is correlated with neurotoxicity and cognitive decline. The role of glycogen synthase kinase-3 (GSK-3) in Alzheimer disease pathogenesis has been debated. To study the role of GSK-3 in Aβ pathology, we used 5XFAD mice co-expressing mutated amyloid precursor protein and presenilin-1 that develop massive cerebral Aβ loads. Both GSK-3 isozymes (α/β) were hyperactive in this model. Nasal treatment of 5XFAD mice with a novel substrate competitive GSK-3 inhibitor, L803-mts, reduced Aβ deposits and ameliorated cognitive deficits. Analyses of 5XFAD hemi-brain samples indicated that L803-mts restored the activity of mammalian target of rapamycin (mTOR) and inhibited autophagy. Lysosomal acidification was impaired in the 5XFAD brains as indicated by reduced cathepsin D activity and decreased N-glycoyslation of the vacuolar ATPase subunit V0a1, a modification required for lysosomal acidification. Treatment with L803-mts restored lysosomal acidification in 5XFAD brains. Studies in SH-SY5Y cells confirmed that GSK-3α and GSK-3β impair lysosomal acidification and that treatment with L803-mts enhanced the acidic lysosomal pool as demonstrated in LysoTracker Red-stained cells. Furthermore, L803-mts restored impaired lysosomal acidification caused by dysfunctional presenilin-1. We provide evidence thatmTORis a target activated by GSK-3 but inhibited by impaired lysosomal acidification and elevation in amyloid precursor protein/Aβ loads. Taken together, our data indicate that GSK-3 is a player in Aβ pathology. Inhibition of GSK-3 restores lysosomal acidification that in turn enables clearance ofAβ burdens and reactivation of mTOR. These changes facilitate amelioration in cognitive function. [ABSTRACT FROM AUTHOR]

Abstract: Amyloid plaques composed of the 42 amino acid form of amyloid-β peptide (Aβ42) are a pathological hallmark of Alzheimer''s disease (AD), but soluble and intraneuronal Aβ42 are the more proximal causes of synaptic dysfunction and neurotoxicity. Apolipoprotein E (apoE) modulates this disease process, as inheritance of the ɛ4 allele of the apoE gene is the primary genetic risk factor for AD. To address the solubility of Aβ42 and apoE, the 5xFAD-specific extraction profile for Aβ42 was optimized, a protein extraction protocol was optimized in the presence of minimal to extensive Aβ42 pathology. Sequential extractions with TBS, TBS+Triton X-100 (TBSX), and guanidine-HCl (GuHCl) or formic acid (FA) were used with tissue from young and old wild type or mice expressing 5 familial AD mutations (5xFAD), in disease-susceptible or -resistant brain regions. In older 5xFAD mice, the extraction of insoluble Aβ42 and m-apoE protein was increased with FA compared to GuHCl. The 5 FAD mutations significantly increase production of Aβ42, recapitulating AD-like pathology at a greatly accelerated rate. Consistent protein extraction and the specificity of extractions for soluble or membrane-associated proteins were demonstrated. Age-dependent increases in Aβ42 were observed in all extraction fractions, particularly in the cortex and hippocampus. In both young and old 5xFAD mice, Aβ42 is TBS- or GuHCl-soluble. While in WT mice m-apoE is TBSX-soluble, in 5xFAD mice m-apoE is TBS- or GuHCl-soluble. Thus, the 5xFAD-specific extraction profile of Aβ42 paralleled that of m-apoE. As now characterized, this method identifies the extraction profile for disease relevant apoE and Aβ in the brain, both normal or modified due to neuropathological processes. [Copyright &y& Elsevier]

Abstract: Alzheimer''s disease (AD) is characterized by depositions of β-amyloid (Aβ) aggregates as amyloid in the brain. To facilitate diagnosis of AD by radioligand imaging, several highly specific small-molecule amyloid ligands have been developed. Because amyloid ligands display excellent pharmacokinetics properties and brain bioavailability, and because we have previously shown that some amyloid ligands bind the highly neurotoxic Aβ oligomers (AβO) with high affinities, they may also be valuable candidates for anti-Aβ therapies. Here we identified two fluorene compounds from libraries of amyloid ligands, initially based on their ability to block cell death secondary to intracellular AβO. We found that the lead fluorenes were able to reduce the amyloid burden including the levels of AβO in cultured neurons and in 5xFAD mice. To explain these in vitro and in vivo effects, we found that the lead fluorenes bind and destabilize AβO as shown by electron paramagnetic resonance spectroscopy studies, and block the harmful AβO-synapse interaction. These fluorenes and future derivatives, therefore, have a potential use in AD therapy and research. [Copyright &y& Elsevier]

Alzheimer disease β-amyloid (An) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and γ-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Aβ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Aβ. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1. [ABSTRACT FROM AUTHOR]

Summary: β-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for β-amyloid (Aβ) production, is elevated in Alzheimer''s disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2α (eIF2α-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2α-P phosphatase PP1c, directly increases BACE1 and elevates Aβ production in primary neurons. Preventing eIF2α phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2α kinase PERK, or PERK inhibitor P58IPK blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2α-P, BACE1, Aβ, and amyloid plaques. Importantly, eIF2α-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2α-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2α phosphorylation increases BACE1 levels and causes Aβ overproduction, which could be an early, initiating molecular mechanism in sporadic AD. [Copyright &y& Elsevier]

The use of statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that block the synthesis of mevalonate (and downstream products such as cholesterol and nonsterol isoprenoids), as a therapy for Alzheimer disease is currently the subject of intense debate. It has been reported that statins reduce the risk of developing the disorder, and a link between cholesterol and Alzheimer disease pathophysiology has been proposed. Moreover, experimental studies focusing on the cholesterol-dependent effects of statins have demonstrated a close association between cellular cholesterol levels and amyloid production. However, evidence suggests that statins are pleiotropic, and the potential cholesterol-independent effects of statins on amyloid precursor protein (APP) metabolism and amyloid β-peptide (Aβ) genesis are unknown. In this study, we developed a novel in vitro system that enabled the discrete analysis of cholesterol-dependent and -independent (i.e. isoprenoid-dependent) statin effects on APP cleavage and Aβ formation. Given the recent interest in the role that intracellular Aβ may play in Alzheimer disease, we analyzed statin effects on both secreted and cell-associated Aβ. As reported previously, low cellular cholesterol levels favored the α-secretase pathway and decreased Aβ secretion presumably within the endocytic pathway. In contrast, low isoprenoid levels resulted in the accumulation of APP, amyloidogenic fragments, and Aβ likely within biosynthetic compartments. Importantly, low cholesterol and low isoprenoid levels appeared to have completely independent effects on APP metabolism and Aβ formation. Although the implications of these effects for Alzheimer disease pathophysiology have yet to be investigated, to our knowledge, these results provide the first evidence that isoprenylation is involved in determining levels of intracellular Aβ. [ABSTRACT FROM AUTHOR]

β-site APP cleaving enzyme 1 (BACE1) is the β-secretase enzyme required for generating pathogenic β-amyloid (Aβ) peptides in Alzheimer''s disease (AD). BACE1 knockout mice lack Aβ 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 Aβ-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 Aβ40 and Aβ42 levels and occurs before amyloid deposition in Tg2576 mice. Our gene-based approach demonstrates that lower Aβ levels are beneficial for AD-associated memory impairments, validating BACE1 as a therapeutic target for AD. [Copyright &y& Elsevier]

The formation of Alzheimer''s Aβ peptide is initiated when the amyloid precursor protein (APP) is cleaved by the enzyme β-secretase (BACE1); inhibition of this cleavage has been proposed as a means of treating Alzheimer''s disease. (AD) We have previously shown that young BACE1 knockout mice (BACE1 KO) do not generate Aβ but in other respects appear normal. Here we have extended this analysis to include both gene expression profiling and phenotypic assessment of older BACE1 KO animals to evaluate the impact of chronic Aβ deficiency. We did not detect global compensatory changes in neural gene expression in young BACE1 KO mice. In particular, expression of the β-secretase homolog BACE2 was not upregulated. Furthermore, we found no structural alterations in any organ, including all central and peripheral neural tissues, of BACE1 KO mice up to 14 months of age. Aged BACE1 KO mice engineered to overexpress human APP (BACE1 KO/APPtg) did not develop amyloid plaques. These data provide evidence that neither β-secretase nor Aβ plays a vital role in mouse physiology and that chronic β-secretase inhibition could be a useful approach in treating AD. [Copyright &y& Elsevier]