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Purpose: To assess the impact of glaucoma-related vision loss on measures of out-loud reading, including time to say individual words, interval time between consecutive words, lexical errors, skipped words, and repetitions., Methods: Glaucoma subjects (n = 63) with bilateral visual field loss and glaucoma suspect controls (n = 57) were recorded while reading a standardized passage out loud. A masked evaluator determined the start and end of each recorded word and identified reading errors., Results: Glaucoma subjects demonstrated longer durations to recite individual words (265 vs. 243 ms, P < 0.001), longer intervals between words (154 vs. 124 ms, P < 0.001), and longer word/post-word interval complexes (the time spanned by the word and the interval following the word; 419 vs. 367 ms, P < 0.001) than controls. In multivariable analyses, each 0.1 decrement in log contrast sensitivity (logCS) was associated with a 15.0 ms longer word/post-interval complex (95% confidence interval [CI] = 9.6-20.4; P < 0.001). Contrast sensitivity was found to significantly interact with word length, word frequency, and word location at the end of a line with regards to word/post-word interval complex duration (P < 0.05 for all). Glaucoma severity was also associated with more lexical errors (Odds ratio = 1.20 for every 0.1 logCS decrement; 95% CI = 1.02-1.39, P < 0.05), but not with more skipped or repeated words., Conclusions: Glaucoma patients with greater vision loss make more lexical errors, are slower in reciting longer and less frequently used words, and more slowly transition to new lines of text. These problem areas may require special attention when designing methods to rehabilitate reading in patients with glaucoma., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Research indicates that female risk of developing Alzheimer's disease (AD) is greater than that of males. Moderate reduction of calorie intake, known as calorie restriction (CR), reduces pathology in AD mouse models and is a potentially translatable prevention measure for individuals at-risk for AD, as well as an important tool for understanding how the brain endogenously attenuates age-related pathology. Whether sex influences the response to CR remains unknown. In this study, we assessed the effect of CR on beta-amyloid peptide (Aβ) pathology and hippocampal CA1 neuron specific gene expression in the Tg2576 mouse model of cerebral amyloidosis. Relative to ad libitum (AL) feeding, CR feeding significantly reduced hippocampal Aβ burden in 15-month-old female, but not age-matched male, Tg2576 mice. Sustained CR also significantly reduced expression of presenilin enhancer 2 (Psenen) and presenilin 1, components of the γ-secretase complex, in Tg2576 females. These results indicate that long-term CR significantly reduces age-dependent female Tg2576 Aβ pathology, which is likely to involve CR-mediated reductions in γ-secretase-dependent amyloid precursor protein (APP) metabolism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The entorhinal cortex (EC) is one of the first brain areas to display neuropathology in Alzheimer's disease. A mouse model which simulates amyloid-β (Aβ) neuropathology, the Tg2576 mouse, was used to address these early changes. Here, we show EC abnormalities occur in 2- to 4-month-old Tg2576 mice, an age before Aβ deposition and where previous studies suggest that there are few behavioral impairments. First we show, using a sandwich enzyme-linked immunosorbent assay, that soluble human Aβ40 and Aβ42 are detectable in the EC of 2-month-old Tg2576 mice before Aβ deposition. We then demonstrate that 2- to 4-month-old Tg2576 mice are impaired at object placement, an EC-dependent cognitive task. Next, we show that defects in neuronal nuclear antigen expression and myelin uptake occur in the superficial layers of the EC in 2- to 4-month-old Tg2576 mice. In slices from Tg2576 mice that contained the EC, there were repetitive field potentials evoked by a single stimulus to the underlying white matter, and a greater response to reduced extracellular magnesium ([Mg(2+)]o), suggesting increased excitability. However, deep layer neurons in Tg2576 mice had longer latencies to antidromic activation than wild type mice. The results show changes in the EC at early ages and suggest that altered excitability occurs before extensive plaque pathology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the subjective and objective measures of ocular surface disease in patients with glaucoma., Design: Cross-sectional study., Participants: Sixty-four glaucoma subjects with bilateral visual field (VF) loss and 59 glaucoma suspects with normal VFs., Methods: Consecutive patients were recruited prospectively from the Wilmer Eye Institute Glaucoma Clinic., Main Outcome Measures: Tear film breakup time (TBUT), corneal staining score (0-15), and Schirmer's test results were included as objective metrics, whereas the Ocular Surface Disease Index (OSDI) questionnaire was administered to assess symptoms. Total OSDI score, vision-related subscore (derived from questions about vision and task performance), and discomfort-related subscore (derived from questions about ocular surface discomfort) were calculated for each subject., Results: Seventy-five percent (48/64) of glaucoma subjects and 41% (24/59) of glaucoma suspects were receiving topical medications. The corneal staining grade was greater in glaucoma subjects than in glaucoma suspects (6.4 vs. 4.1; P<0.001), but groups did not differ with regard to TBUT or Schirmer's results (P>0.20 for both). Multivariate regression models showed that topical glaucoma therapy burden was associated with a significantly higher total corneal staining grade (β, +0.9 for each additional glaucoma drop; 95% confidence interval [CI], 0.5-1.3; P<0.001), but not with TBUT or Schirmer's results (P>0.20 for both). Glaucoma subjects had significantly higher total OSDI scores than glaucoma suspects (16.7 vs. 7.9; P<0.001). This largely was the result of higher vision-related subscores in the glaucoma group (11.1 vs. 3.3; P<0.001). Ocular discomfort-related subscores, however, were similar in both groups (5.7 vs. 4.6; P = 0.30). In multivariate analyses, each 5-decibel decrement in better-eye VF mean deviation was associated with a 4.7-point increase in total OSDI score (95% CI, 1.9-7.5; P = 0.001) and a 3.7-point increase in the vision-related subscore (95% CI, 1.7-5.6; P<0.001) but did not predict a higher discomfort-related subscore (β, 1.1 point; P = 0.07). Topical glaucoma therapy burden was not associated with higher total OSDI score or vision- or discomfort-related subscore (P>0.20 for all)., Conclusions: Glaucoma is associated with significant ocular surface disease, and topical glaucoma therapy burden seems predictive of corneal staining severity. However, OSDI is a poor metric for capturing ocular surface disease in glaucoma because symptoms seem to be related largely to VF loss., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptides (Aβ) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H(+)-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aβ production.

The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated τ protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects τ pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on τ phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display τ hyperphosphorylation. τ phosphorylation at τ-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, τ was hyperphosphorylated at the τ-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and τ hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated τ phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like τ hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology.

Olfaction is often impaired in Alzheimer's disease (AD) and is also dysfunctional in mouse models of the disease. We recently demonstrated that short-term passive anti-murine-Aβ immunization can rescue olfactory behavior in the Tg2576 mouse model overexpressing a human mutation of the amyloid precursor protein (APP) after β-amyloid deposition. Here we tested the ability to preserve normal olfactory behaviors by means of long-term passive anti-murine-Aβ immunization. Seven-month-old Tg2576 and non-transgenic littermate (NTg) mice were IP-injected biweekly with the m3.2 murine-Aβ-specific antibody until 16 mo of age when mice were tested in the odor habituation test. While Tg2576 mice treated with a control antibody showed elevations in odor investigation times and impaired odor habituation compared to NTg, olfactory behavior was preserved to NTg levels in m3.2-immunized Tg2576 mice. Immunized Tg2576 mice had significantly less β-amyloid immunolabeling in the olfactory bulb and entorhinal cortex, yet showed elevations in Thioflavin-S labeled plaques in the piriform cortex. No detectable changes in APP metabolite levels other than Aβ were found following m3.2 immunization. These results demonstrate efficacy of chronic, long-term anti-murine-Aβ m3.2 immunization in preserving normal odor-guided behaviors in a human APP Tg model. Further, these results provide mechanistic insights into olfactory dysfunction as a biomarker for AD by yielding evidence that focal reductions of Aβ may be sufficient to preserve olfaction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Although anti-human β-amyloid (Aβ) immunotherapy clears brain β-amyloid plaques in Alzheimer's disease (AD), targeting additional brain plaque constituents to promote clearance has not been attempted. Endogenous murine Aβ is a minor Aβ plaque component in amyloid precursor protein (APP) transgenic AD models, which we show is ∼3%-8% of the total accumulated Aβ in various human APP transgenic mice. Murine Aβ codeposits and colocalizes with human Aβ in amyloid plaques, and the two Aβ species coimmunoprecipitate together from brain extracts. In the human APP transgenic mouse model Tg2576, passive immunization for 8 weeks with a murine-Aβ-specific antibody reduced β-amyloid plaque pathology, robustly decreasing both murine and human Aβ levels. The immunized mice additionally showed improvements in two behavioral assays, odor habituation and nesting behavior. We conclude that passive anti-murine Aβ immunization clears Aβ plaque pathology--including the major human Aβ component--and decreases behavioral deficits, arguing that targeting minor endogenous brain plaque constituents can be beneficial, broadening the range of plaque-associated targets for AD therapeutics., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The paiche, Arapaima gigas represents a socio-economically important species in the Peruvian Amazon, and actually an intensive production for human consumption has emerged during the last years. Therefore, more studies are required in fish farming development, especially concerning populations of parasites that affect fish production yields. Eighty specimens of paiche collected between September and October of 2011 from semi-intensive fish farm in Loreto State, Peru, were examined for their helminthic parasites. Five species were recorded parasitizing A. gigas: Dawestrema cycloancistrium and Dawestrema cycloancistrioides (Monogenea) on gills, Trichodina sp. (Protozoa) on the skin, Caballerotrema arapaimense (Trematoda) in stomachs and Philometra senticosa (Nematoda) in the swim bladder. Highest prevalence was recorded for D. cycloancistrium (100.0%), D. cycloancistrioides (83.0%) and Trichodina sp. (50.0%) and highest values of mean intensity and mean abundance were recorded for D. cycloancistrium (260) parasites per individual. The results confirm the necessity of constant monitoring of fish, seeking the diagnosis and timely control of infestations with parasites, in order to eradicate the mortality of the host that leads unviable the fish farming intended for human consumption.

Early endosomal changes, a prominent pathology in neurons early in Alzheimer's disease, also occur in neurons and peripheral tissues in Down syndrome. While in Down syndrome models increased amyloid-β protein precursor (AβPP) expression is known to be a necessary contributor on the trisomic background to this early endosomal pathology, increased AβPP alone has yet to be shown to be sufficient to drive early endosomal alterations in neurons. Comparing two AβPP transgenic mouse models, one that contains the AβPP Swedish K670N/M671L double mutation at the β-cleavage site (APP23) and one that has the AβPP London V717I mutation near the γ-cleavage site (APPLd2), we show significantly altered early endosome morphology in fronto-parietal neurons as well as enlargement of early endosomes in basal forebrain cholinergic neurons of the medial septal nucleus in the APP23 model, which has the higher levels of AβPP β-C-terminal fragment (βCTF) accumulation. Early endosomal changes correlate with a marked loss of the cholinergic population, which is consistent with the known dependence of the large projection cholinergic cells on endosome-mediated retrograde neurotrophic transport. Our findings support the idea that increased expression of AβPP and AβPP metabolites in neurons is sufficient to drive early endosomal abnormalities in vivo, and that disruption of the endocytic system is likely to contribute to basal forebrain cholinergic vulnerability.

Background: The management of health care, particularly for people with chronic conditions, combines the activities of health professionals, patients, informal carers and social networks that support them. Understanding the non-professional roles in health management requires information about the health related activities (HRA) that are undertaken by patients and informal carers. This understanding allows management planning that incorporates the capacity of patients and informal carers, as well as identifying the particular skills, knowledge and technical support that are necessary. This review was undertaken to identify how much time people with chronic illness and their informal carers spend on HRA., Methods: Literature searches of three electronic databases (CINAHL, Medline, and PubMed) and two journals (Time and Society, Sociology of Health and Illness) were carried out in 2011 using the following search terms (and derivatives): chronic illness AND time AND consumer OR carer. The search was aimed at finding studies of time spent on HRA. A scoping literature review method was utilised., Results: Twenty-two peer reviewed articles published between 1990 and 2010 were included for review. The review identified limited but specific studies about time use by people with a chronic illness and/or their carers. While illness work was seen as demanding, few studies combined inquiry about both defined tasks and defined time use. It also identified methodological issues such as consistency of definition and data collection methods, which remain unresolved., Conclusions: While HRA are seen as demanding by people doing them, few studies have measured actual time taken to carry out a comprehensive range of HRA. The results of this review suggest that both patients with chronic illness and informal carers may be spending 2 hours a day or more on HRA. Illnesses such as diabetes may be associated with higher time use. More empirical research is needed to understand the time demands of self-management, particularly for those affected by chronic illness.

We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) β-amyloidosis, the APP23 mouse, reduces β-amyloid (Aβ) pathology and Aβ levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Aβ plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Aβ plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Aβ pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Aβ pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Aβ pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Aβ pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Several studies found that FE65, a cytoplasmic adaptor protein, interacts with APP and LRP1, altering the trafficking and processing of APP. We have previously shown that FE65 interacts with the ApoE receptor, ApoER2, altering its trafficking and processing. Interestingly, it has been shown that FE65 can act as a linker between APP and LRP1 or ApoER2. In the present study, we tested whether FE65 can interact with another ApoE receptor, VLDLR, thereby altering its trafficking and processing, and whether FE65 can serve as a linker between APP and VLDLR., Results: We found that FE65 interacted with VLDLR using GST pull-down and co-immunoprecipitation assays in COS7 cells and in brain lysates. This interaction occurs via the PTB1 domain of FE65. Co-transfection with FE65 and full length VLDLR increased secreted VLDLR (sVLDLR); however, the levels of VLDLR C-terminal fragment (CTF) were undetectable as a result of proteasomal degradation. Additionally, FE65 increased cell surface levels of VLDLR. Moreover, we identified a novel complex between VLDLR and APP, which altered trafficking and processing of both proteins. Furthermore, immunoprecipitation results demonstrated that the presence of FE65 increased the interaction between APP and VLDLR in vitro and in vivo., Conclusions: These data suggest that FE65 can regulate VLDLR trafficking and processing. Additionally, the interaction between VLDLR and APP altered both protein's trafficking and processing. Finally, our data suggest that FE65 serves as a link between VLDLR and APP. This novel interaction adds to a growing body of literature indicating trimeric complexes with various ApoE Receptors and APP.

The neuritic plaque in the brain of Alzheimer's disease patients consists of an amyloid composed primarily of Aβ, an approximately 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Aβ plays a key role in the pathogenesis of the disease, and potential treatments that target Aβ production and/or Aβ accumulation in the brain as β-amyloid are being aggressively pursued. Methods to quantitate the Aβ peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Aβ in the brains of AD patients and β-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Aβ peptide. Here we describe methods for the recovery of both soluble and deposited Aβ from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.

Amyloid-containing tissue, whether from human patients or an animal model of a disease, is typically characterized by various biochemical and immunohistochemical techniques, many of which are described in detail in this volume. In this chapter, we describe a straightforward technique for the homogenization of tissue prior to these analyses. The technique is particularly well suited for performing a large number of different biochemical analyses on a single mouse brain hemisphere. Starting with this homogenate multiple characterizations can be done, including western blot analysis and isolation of membrane-associated proteins, both of which are described here. Additional analyses can readily be performed on the tissue homogenate, including the ELISA quantitation of Aβ in the brain of a transgenic mouse model of β-amyloid deposition. The ELISA technique is described in detail in Chapter 34.

The proteolytic machinery comprising metalloproteases and γ-secretase, an intramembrane aspartyl protease involved in Alzheimer's disease, cleaves several substrates in addition to the extensively studied amyloid precursor protein. Some of these substrates, such as N-cadherin, are synaptic proteins involved in synapse remodeling and maintenance. Here we show, in rats and mice, that metalloproteases and γ-secretase are physiologic regulators of synapses. Both proteases are synaptic, with γ-secretase tethered at the synapse by δ-catenin, a synaptic scaffolding protein that also binds to N-cadherin and, through scaffolds, to AMPA receptor and a metalloprotease. Activity-dependent proteolysis by metalloproteases and γ-secretase takes place at both sides of the synapse, with the metalloprotease cleavage being NMDA receptor-dependent. This proteolysis decreases levels of synaptic proteins and diminishes synaptic transmission. Our results suggest that activity-dependent substrate cleavage by synaptic metalloproteases and γ-secretase modifies synaptic transmission, providing a novel form of synaptic autoregulation.

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

Deposition of amyloid β peptides (Aβs) in extracellular amyloid plaques within the human brain is a hallmark of Alzheimer's disease (AD). Aβ derives from proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. The initial cleavage by β-secretase results in shedding of the APP ectodomain and generation of APP C-terminal fragments (APP-CTFs), which can then be further processed within the transmembrane domain by γ-secretase, resulting in release of Aβ. Here, we demonstrate that accumulation of sphingolipids (SLs), as occurs in lysosomal lipid storage disorders (LSDs), decreases the lysosome-dependent degradation of APP-CTFs and stimulates γ-secretase activity. Together, this results in increased generation of both intracellular and secreted Aβ. Notably, primary fibroblasts from patients with different SL storage diseases show strong accumulation of potentially amyloidogenic APP-CTFs. By using biochemical, cell biological, and genetic approaches, we demonstrate that SL accumulation affects autophagic flux and impairs the clearance of APP-CTFs. Thus, accumulation of SLs might not only underlie the pathogenesis of LSDs, but also trigger increased generation of Aβ and contribute to neurodegeneration in sporadic AD.

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-β peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-β peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-β peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-β peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-β peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.

Familial Danish dementia (FDD) is a progressive neurodegenerative disease with cerebral deposition of Dan-amyloid (ADan), neuroinflammation, and neurofibrillary tangles, hallmark characteristics remarkably similar to those in Alzheimer's disease (AD). We have generated transgenic (tg) mouse models of familial Danish dementia that exhibit the age-dependent deposition of ADan throughout the brain with associated amyloid angiopathy, microhemorrhage, neuritic dystrophy, and neuroinflammation. Tg mice are impaired in the Morris water maze and exhibit increased anxiety in the open field. When crossed with TauP301S tg mice, ADan accumulation promotes neurofibrillary lesions, in all aspects similar to the Tau lesions observed in crosses between beta-amyloid (Abeta)-depositing tg mice and TauP301S tg mice. Although these observations argue for shared mechanisms of downstream pathophysiology for the sequence-unrelated ADan and Abeta peptides, the lack of codeposition of the two peptides in crosses between ADan- and Abeta-depositing mice points also to distinguishing properties of the peptides. Our results support the concept of the amyloid hypothesis for AD and related dementias, and suggest that different proteins prone to amyloid formation can drive strikingly similar pathogenic pathways in the brain.

An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.

In Alzheimer's disease, microglia cluster around beta-amyloid deposits, suggesting that these cells are important for amyloid plaque formation, maintenance and/or clearance. We crossed two distinct APP transgenic mouse strains with CD11b-HSVTK mice, in which nearly complete ablation of microglia was achieved for up to 4 weeks after ganciclovir application. Neither amyloid plaque formation and maintenance nor amyloid-associated neuritic dystrophy depended on the presence of microglia.

Gangliosides have been shown to be necessary for beta-amyloid (Abeta) binding and aggregation. GD3 synthase (GD3S) is responsible for biosynthesis of the b- and c-series gangliosides, including two of the four major brain gangliosides. We examined Abeta-ganglioside interactions in neural tissue from mice lacking the gene coding for GD3S (St8sia1), and in a double-transgenic (APP/PSEN1) mouse model of Alzheimer's disease cross-bred with GD3S-/- mice. In primary neurons and astrocytes lacking GD3S, Abeta-induced cell death and Abeta aggregation were inhibited. Like GD3S-/- and APP/PSEN1 double-transgenic mice, APP/PSEN1/GD3S-/- "triple-mutant" mice are indistinguishable from wild-type mice on casual examination. APP/PSEN1 double-transgenics exhibit robust impairments on a number of reference-memory tasks. In contrast, APP/PSEN1/GD3S-/- triple-mutant mice performed as well as wild-type control and GD3S-/- mice. Consistent with the behavioral improvements, both aggregated and unaggregated Abeta and associated neuropathology were almost completely eliminated in triple-mutant mice. These results suggest that GD3 synthase may be a novel therapeutic target to combat the cognitive deficits, amyloid plaque formation, and neurodegeneration that afflict Alzheimer's patients.

The metabolism of the amyloid precursor protein (APP) and tau are central to the pathobiology of Alzheimer's disease (AD). We have examined the in vivo turnover of APP, secreted APP (sAPP), Abeta and tau in the wild-type and Tg2576 mouse brain using cycloheximide to block protein synthesis. In spite of overexpression of APP in the Tg2576 mouse, APP is rapidly degraded, similar to the rapid turnover of the endogenous protein in the wild-type mouse. sAPP is cleared from the brain more slowly, particularly in the Tg2576 model where the half-life of both the endogenous murine and transgene-derived human sAPP is nearly doubled compared to wild-type mice. The important Abeta degrading enzymes neprilysin and IDE were found to be highly stable in the brain, and soluble Abeta40 and Abeta42 levels in both wild-type and Tg2576 mice rapidly declined following the depletion of APP. The cytoskeletal-associated protein tau was found to be highly stable in both wild-type and Tg2576 mice. Our findings unexpectedly show that of these various AD-relevant protein metabolites, sAPP turnover in the brain is the most different when comparing a wild-type mouse and a beta-amyloid depositing, APP overexpressing transgenic model. Given the neurotrophic roles attributed to sAPP, the enhanced stability of sAPP in the beta-amyloid depositing Tg2576 mice may represent a neuroprotective response.

Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain.

Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains.

Calpains are calcium-dependent enzymes that determine the fate of proteins through regulated proteolytic activity. Calpains have been linked to the modulation of memory and are key to the pathogenesis of Alzheimer disease (AD). When abnormally activated, calpains can also initiate degradation of proteins essential for neuronal survival. Here we show that calpain inhibition through E64, a cysteine protease inhibitor, and the highly specific calpain inhibitor BDA-410 restored normal synaptic function both in hippocampal cultures and in hippocampal slices from the APP/PS1 mouse, an animal model of AD. Calpain inhibition also improved spatial-working memory and associative fear memory in APP/PS1 mice. These beneficial effects of the calpain inhibitors were associated with restoration of normal phosphorylation levels of the transcription factor CREB and involved redistribution of the synaptic protein synapsin I. Thus, calpain inhibition may prove useful in the alleviation of memory loss in AD.

Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75.

Using transgenic mice expressing human cystatin C (encoded by CST3), we show that cystatin C binds soluble amyloid-beta peptide and inhibits cerebral amyloid deposition in amyloid-beta precursor protein (APP) transgenic mice. Cystatin C expression twice that of the endogenous mouse cystatin C was sufficient to substantially diminish amyloid-beta deposition. Thus, cystatin C has a protective role in Alzheimer's disease pathogenesis, and modulation of cystatin C concentrations may have therapeutic implications for the disease.

Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42.

Epidemiological studies support an association between vascular risk factors, including hypercholesterolemia, and Alzheimer's disease (AD). Recently, there has been much interest in the possibility that hypercholesterolemia might directly promote beta-amyloid (Abeta) production. Indeed, in vitro studies have shown that increasing cellular cholesterol levels enhances Abeta production. However, studies in AD transgenic mouse models have not consistently found that elevated plasma cholesterol leads to increased Abeta production or deposition in vivo. In this study, we determined whether elevated peripheral cholesterol influences Abeta production in mice with a null mutation of the low-density lipoprotein receptor (LDLR). We show that dramatically elevated plasma cholesterol levels, whether induced by high cholesterol, high fat, or high fat/high cholesterol diets, did not affect either levels of brain Abeta40, Abeta42, or APP, or the Abeta42/40 or APP-CTF/APP ratios, nor substantially alter brain cholesterol levels. ApoE protein levels in brain were, however, elevated, in LDLR-/- mice by post-transcriptional mechanisms. Collectively, these studies argue that plasma cholesterol levels do not normally regulate production of brain Abeta.

The sequential processing of the APP (amyloid precursor protein) by the beta- and gamma-secretase and generation of the Abeta (amyloid-beta) peptide is a primary pathological factor in AD (Alzheimer's disease). Regulation of the processing or turnover of these proteins represents potential targets for the development of AD therapies. Sumoylation is a process by which SUMOs (small ubiquitin-like modifiers) are covalently conjugated to target proteins, resulting in a number of functional consequences. These include regulation of protein-protein interactions, intracellular trafficking and protein stability, which all have the potential to impact on several aspects of the amyloidogenic pathway. The present study examines the effects of overexpression and knockdown of the major SUMO isoforms (SUMO1, 2 and 3) on APP processing and the production of Abeta peptides. SUMO3 overexpression significantly increased Abeta40 and Abeta42 secretion, which was accompanied by an increase in full-length APP and its C-terminal fragments. These effects of SUMO3 were independent of its covalent attachment or chain formation, as mutants lacking the motifs responsible for SUMO chain formation or SUMO conjugation led to similar changes in Abeta. SUMO3 overexpression also up-regulated the expression of the transmembrane protease BACE (beta-amyloid-cleaving enzyme), but failed to affect levels of several other unrelated proteins. Suppression of SUMO1 or combined SUMO2+3 by RNA interference did not affect APP levels or Abeta production. These findings confirm a specific effect of SUMO3 overexpression on APP processing and the production of Abeta peptides but also suggest that endogenous sumoylation is not essential and likely plays an indirect role in modulating the amyloid processing pathway.

Background: Amyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study., Methodology: In order to evaluate whether the phosphorylation state of Thr668 controlled brain Abeta levels, we studied the levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in brains from "knock-in" mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein., Conclusions: The levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Abeta40 or Abeta42 in vivo.

Protein aggregation is an established pathogenic mechanism in Alzheimer's disease, but little is known about the initiation of this process in vivo. Intracerebral injection of dilute, amyloid-beta (Abeta)-containing brain extracts from humans with Alzheimer's disease or beta-amyloid precursor protein (APP) transgenic mice induced cerebral beta-amyloidosis and associated pathology in APP transgenic mice in a time- and concentration-dependent manner. The seeding activity of brain extracts was reduced or abolished by Abeta immunodepletion, protein denaturation, or by Abeta immunization of the host. The phenotype of the exogenously induced amyloidosis depended on both the host and the source of the agent, suggesting the existence of polymorphic Abeta strains with varying biological activities reminiscent of prion strains.

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models.

Membrane-bound endosomal vesicles play an integral role in multiple cellular events, including protein processing and turnover, and often critically regulate the cell-surface availability of receptors and other plasma membrane proteins in many different cell types. Neurons are no exception, being dependent on endosomal function for housekeeping and synaptic events. Growing evidence suggests a link between neuronal endosomal function and Alzheimer's disease (AD) pathophysiology. Endosomal abnormalities invariably occur within neurons in AD brains, and endocytic compartments are one likely site for the production of the pathogenic beta-amyloid peptide (Abeta), which accumulates within the brain during the disease and is generated by proteolytic processing of the amyloid precursor protein (APP). The enzymes and events involved in APP processing are appealing targets for therapeutic agents aimed at slowing or reversing the pathogenesis of AD. The neuronal endosome may well prove to be the intracellular site of action for inhibitors of beta-amyloidogenic APP processing. We present here the view that knowledge of the endosomal system in the disease can guide drug discovery of AD therapeutic agents.

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Gamma-secretase depends on presence of presenilins (PS), Nct, Aph-1, and PEN-2 within a core complex. This endoproteolytic activity cleaves within transmembrane domains of amyloid-beta precursor protein (APP) and Notch, and familial Alzheimer's disease (FAD) mutations in PS1 or PS2 genes shift APP cleavage from production of amyloid-beta (Abeta) 40 peptide to greater production of Abeta42. Although studies in PS1/PS2-deficient embryonic cells define overlapping activities for these proteins, in vivo complementation of PS1-deficient animals described here reveals an unexpected spectrum of activities dictated by PS1 and PS2 alleles. Unlike PS1 transgenes, wild-type PS2 transgenes expressed in the mouse CNS support little Abeta40 or Abeta42 production, and FAD PS2 alleles support robust production of only Abeta42. Although wild-type PS2 transgenes failed to rescue Notch-associated skeletal defects in PS1 hypomorphs, a "gained" competence in this regard was apparent for FAD alleles of PS2. The range of discrete and divergent processing activities in mice reconstituted with different PS genes and alleles argues against gamma-secretase being a single enzyme with intrinsically relaxed substrate and cleavage site specificities. Instead, our studies define functionally distinct gamma-secretase variants. We speculate that extrinsic components, in combination with core complexes, may tailor functional variants of this enzyme to their preferred substrates.

The neuritic plaque in the brain of Alzheimer's disease (AD) patients consists of an amyloid composed primarily of Abeta, an approx 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Abeta plays a key role in the pathogenesis of the disease, and potential treatments that target Abeta production and/or Abeta accumulation in the brain as beta-amyloid are being aggressively pursued. Methods to quantitate the Abeta peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Abeta in the brains of AD patients and beta-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Abeta peptide. Here we describe methods for the recovery of both soluble and deposited Abeta from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.

Amyloid-containing tissue, whether from human patients or an animal model of a disease, is typically characterized by various biochemical and immunohistochemical techniques, many of which are described in detail in this volume. In this chapter, we describe a straightforward technique for the homogenization of tissue prior to these analyses. The technique is particularly well-suited for performing a large number of different biochemical analyses on a single mouse brain hemisphere. Starting with this homogenate, multiple characterizations can be done, including Western blot analysis and isolation of membrane-associated proteins, both of which are described here. Additional analyses can readily be performed on the tissue homogenate, including the ELISA quantitation of Abeta in the brain of a transgenic mouse model of beta-amyloid deposition. The ELISA technique is described in detail in the following chapter.

Presenilin 1 or presenilin 2, nicastrin, APH-1, and PEN-2 form high molecular weight complexes that play a pivotal role in the cleavage of various Type I transmembrane proteins, including the beta-amyloid precursor protein. The specific function of PEN-2 is unclear. To explore its function and intermolecular interactions, we conducted deletion and mutagenesis studies on a series of conserved residues at the C terminus of PEN-2. These studies suggest that: 1) both the presence and amino acid sequence of the conserved DYLSF domain at the C terminus of PEN-2 (residues 90-94) is critical for binding PEN-2 to other components in the presenilin complex and 2) the overall length of the exposed C terminus is critical for functional gamma-secretase activity.

The colocalization of cystatin C, an inhibitor of cysteine proteases, with amyloid beta (Abeta) in parenchymal and vascular amyloid deposits in brains of Alzheimer's disease (AD) patients may reflect cystatin C involvement in amyloidogenesis. We therefore sought to determine the association of cystatin C with Abeta. Immunofluorescence analysis of transfected cultured cells demonstrated colocalization of cystatin C and beta amyloid precursor protein (betaAPP) intracellularly and on the cell surface. Western blot analysis of immunoprecipitated cell lysate or medium proteins revealed binding of cystatin C to full-length betaAPP and to secreted betaAPP (sbetaAPP). Deletion mutants of betaAPP localized the cystatin C binding site within betaAPP to the Abeta region. Cystatin C association with betaAPP resulted in increased sbetaAPP but did not affect levels of secreted Abeta. Analysis of the association of cystatin C and Abeta demonstrated a specific, saturable and high affinity binding between cystatin C and both Abeta(1-42) and Abeta(1-40). Notably, cystatin C association with Abeta results in a concentration-dependent inhibition of Abeta fibril formation.

Alzheimer's disease (AD) is associated with accumulation of beta-amyloid (Abeta). A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele. ApoE can act as a pathological chaperone of Abeta, promoting its conformational transformation from soluble Abeta into toxic aggregates. We determined if blocking the apoE/Abeta interaction reduces Abeta load in transgenic (Tg) AD mice. The binding site of apoE on Abeta corresponds to residues 12 to 28. To block binding, we synthesized a peptide containing these residues, but substituted valine at position 18 to proline (Abeta12-28P). This changed the peptide's properties, making it non-fibrillogenic and non-toxic. Abeta12-28P competitively blocks binding of full-length Abeta to apoE (IC50 = 36.7 nmol). Furthermore, Abeta12-28P reduces Abeta fibrillogenesis in the presence of apoE, and Abeta/apoE toxicity in cell culture. Abeta12-28P is blood-brain barrier-permeable and in AD Tg mice inhibits Abeta deposition. Tg mice treated with Abeta12-28P for 1 month had a 63.3% reduction in Abeta load in the cortex (P = 0.0043) and a 59.5% (P = 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice. Antibodies against Abeta were not detected in sera of treated mice; therefore the observed therapeutic effect of Abeta12-28P cannot be attributed to an antibody clearance response. Our experiments demonstrate that compounds blocking the interaction between Abeta and its pathological chaperones may be beneficial for treatment of beta-amyloid deposition in AD.

The E693Q mutation in the amyloid beta precursor protein (APP) leads to cerebral amyloid angiopathy (CAA), with recurrent cerebral hemorrhagic strokes and dementia. In contrast to Alzheimer disease (AD), the brains of those affected by hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) show few parenchymal amyloid plaques. We found that neuronal overexpression of human E693Q APP in mice (APPDutch mice) caused extensive CAA, smooth muscle cell degeneration, hemorrhages and neuroinflammation. In contrast, overexpression of human wild-type APP (APPwt mice) resulted in predominantly parenchymal amyloidosis, similar to that seen in AD. In APPDutch mice and HCHWA-D human brain, the ratio of the amyloid-beta40 peptide (Abeta40) to Abeta42 was significantly higher than that seen in APPwt mice or AD human brain. Genetically shifting the ratio of AbetaDutch40/AbetaDutch42 toward AbetaDutch42 by crossing APPDutch mice with transgenic mice producing mutated presenilin-1 redistributed the amyloid pathology from the vasculature to the parenchyma. The understanding that different Abeta species can drive amyloid pathology in different cerebral compartments has implications for current anti-amyloid therapeutic strategies. This HCHWA-D mouse model is the first to develop robust CAA in the absence of parenchymal amyloid, highlighting the key role of neuronally produced Abeta to vascular amyloid pathology and emphasizing the differing roles of Abeta40 and Abeta42 in vascular and parenchymal amyloid pathology.

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.

The neuronal lysosomal system is a major degradative pathway, induced by cell stress and closely linked to Alzheimer disease (AD) and other neurodegenerative diseases. Here, we show that mutations of presenilin (PS) 1 and 2, which cause familial early-onset AD (FAD), induce more severe lysosomal system neuropathology in humans than does sporadic AD (SAD). Cathepsin D and B levels were higher in PS-FAD neocortex than in SAD and, unlike neurons in SAD, expressed higher levels of the cation-independent mannose-6-phosphate receptor. Lysosomal pathology was also evident in more populations of neurons in PS-FAD brains, including the less vulnerable neurons in laminae II and IV and affected neurons contained high numbers of hydrolase-positive vesicular compartments with a broader range of abnormal morphology. In transgenic mice expressing mutant amyloid precursor protein (APPswe), introducing mutant PSI significantly upregulated the lysosomal system in neocortical and hippocampal neurons. This upregulation, though milder in severity, resembled that seen in human PS-FAD. Accumulation of hydrolases in dystrophic neurites in senile plaques was particularly strong, suggesting that amyloid deposition may be a stimulus for local mobilization of the lysosomal system. PS1 mice lacking the APPswe transgene also had a mild lysosomal response in some neuronal populations, which was not seen in the APPswe mice. Our findings suggest that presenilin mutations have amyloid-independent effects on the lysosomal system, which are synergistic with the lysosomal system pathology that is associated with beta-amyloid.

BACKGROUND: The over-expression of transforming growth factor beta-1(TGF-beta1) has been reported to cause hydrocephalus, glia activation, and vascular amyloidbeta (Abeta) deposition in mouse brains. Since these phenomena partially mimic the cerebral amyloid angiopathy (CAA) concomitant to Alzheimer's disease, the findings in TGF-beta1 over-expressing mice prompted the hypothesis that CAA could be caused or enhanced by the abnormal production of TGF-beta1. This idea was in accordance with the view that chronic inflammation contributes to Alzheimer's disease, and drew attention to the therapeutic potential of anti-inflammatory drugs for the treatment of Abeta-elicited CAA. We thus studied the effect of anti-inflammatory drug administration in TGF-beta1-induced pathology. METHODS: Two-month-old TGF-beta1 mice and littermate controls were orally administered pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, or ibuprofen, a non steroidal anti-inflammatory agent, for two months. Glia activation was assessed by immunohistochemistry and western blot analysis; Abeta precursor protein (APP) by western blot analysis; Abeta deposition by immunohistochemistry, thioflavin-S staining and ELISA; and hydrocephalus by measurements of ventricle size on autoradiographies of brain sections. Results are expressed as means +/- SD. Data comparisons were carried with the Student's T test when two groups were compared, or ANOVA analysis when more than three groups were analyzed. RESULTS: Animals displayed glia activation, hydrocephalus and a robust thioflavin-S-positive vascular deposition. Unexpectedly, these deposits contained no Abeta or serum amyloid P component, a common constituent of amyloid deposits. The thioflavin-S-positive material thus remains to be identified. Pioglitazone decreased glia activation and basal levels of Abeta42- with no change in APP contents - while it increased hydrocephalus, and had no effect on the thioflavin-S deposits. Ibuprofen mimicked the reduction of glia activation caused by pioglitazone and the lack of effect on the thioflavin-S-labeled deposits. CONCLUSIONS: i) TGF-beta1 over-expressing mice may not be an appropriate model of Abeta-elicited CAA; and ii) pioglitazone has paradoxical effects on TGF-beta1-induced pathology suggesting that anti-inflammatory therapy may reduce the damage resulting from active glia, but not from vascular alterations or hydrocephalus. Identification of the thioflavin-S-positive material will facilitate the full appraisal of the clinical implication of the effects of anti-inflammatory drugs, and provide a more thorough understanding of TGF-beta1 actions in brain.

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity.

Increasing evidence points to synaptic plasticity impairment as one of the first events in Alzheimer's disease (AD). However, studies on synaptic dysfunction in different transgenic AD models that overexpress familial AD mutant forms of amyloid precursor protein (APP) and/or presenilin (PS) have provided conflicting results. Both long-term potentiation (LTP) and basal synaptic transmission (BST) have been found to be both unchanged and altered in different models and under differing experimental conditions. Because of their more robust amyloid-beta (Abeta) deposition, double transgenic mice currently are used by several laboratories as an AD model. Here, we report that mice overexpressing APP (K670N:M671L) together with PS1 (M146L) have abnormal LTP as early as 3 months of age. Interestingly, reduced LTP paralleled plaque appearance and increased Abeta levels and abnormal short-term memory (working memory). BST and long-term memory (reference memory) are impaired only later (approximately 6 months) as amyloid burden increases. Abeta pathology across different ages did not correlate with synaptic and cognitive deficits, suggesting that Abeta levels are not a marker of memory decline. In contrast, progression of LTP impairment correlated with the deterioration of working memory, suggesting that percentage of potentiation might be an indicator of the cognitive decline and disease progression in the APP/PS1 mice.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts.