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Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells., Competing Interests: C.T., K.V.K, B.V.B., F.S., J.D. and W.V.d.E. are employees of Janssen Research & Development, LLC, and some own stock/stock options in Johnson & Johnson. M.M. and P.-Y.S were employees of Janssen Research & Development, LLC, at the time of the study., (© 2023 The Author(s).)

The imbalance between production and clearance of amyloid β (Aβ) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aβ is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aβ concentrations include prevention of de novo production of Aβ through inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aβ deposits via passive Aβ immunotherapy. We have developed a novel, high affinity antibody against Aβ peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aβ species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multiple animal models have been created to gain insight into Alzheimer's disease (AD) pathology. Among the most commonly used models are transgenic mice overexpressing human amyloid precursor protein (APP) with mutations linked to familial AD, resulting in the formation of amyloid β plaques, one of the pathological hallmarks observed in AD patients. However, recent evidence suggests that the overexpression of APP by itself can confound some of the reported observations. Therefore, we investigated in the present study the App NL-G-F model, an App knock-in (App-KI) mouse model that develops amyloidosis in the absence of APP-overexpression. Our findings at the behavioral, electrophysiological, and histopathological level confirmed an age-dependent increase in Aβ1-42 levels and plaque deposition in these mice in accordance with previous reports. This had apparently no consequences on cognitive performance in a visual discrimination (VD) task, which was largely unaffected in App NL-G-F mice at the ages tested. Additionally, we investigated neurophysiological functioning of several brain areas by phase-amplitude coupling (PAC) analysis, a measure associated with adequate cognitive functioning, during the VD task (starting at 4.5 months) and the exploration of home environment (at 5 and 8 months of age). While we did not detect age-dependent changes in PAC during home environment exploration for both the wild-type and the App NL-G-F mice, we did observe subtle changes in PAC in the wild-type mice that were not present in the App NL-G-F mice.

Rationale: Immuno-PET imaging may prove to be a diagnostic and progression/intervention biomarker for Alzheimer's disease (AD) with improved sensitivity and specificity. Immuno-PET imaging is based on the coupling of an antibody with a chelator that captures a radioisotope thus serving as an in-vivo PET ligand. A robust and quality controlled process for linking the chelator to the-antibody is fundamental for the success of this approach., Methods: The structural integrities of two monoclonal antibodies (trastuzumab and JRF/AβN/25) and the quantity of desferal-based chelator attached following modification of the antibodies were assessed by online desalting and intact mass analysis. Enzymatic steps for the deglycosylation and removal of C-terminal lysine was performed sequentially and in a single tube to improve intact mass data., Results: Intact mass analysis demonstrated that inclusion of enzymatic processing was critical to correctly derive the quantity of chelator linked to the monoclonal antibodies. For trastuzumab, enzymatic cleaving of the glycans was sufficient, whilst additional removal of the C-terminal lysine was necessary for JRF/AβN/25 to ensure reproducible assessment of the relatively low amount of attached chelator., Conclusions: An efficient intact mass analysis-based process was developed to reproducibly determine the integrity of monoclonal antibodies and the quantity of attached chelator. This technique could serve as an essential quality control approach for the development and production of immuno-PET tracers., (This article is protected by copyright. All rights reserved.)

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in the elderly population. In this study, we used the APP/PS1 transgenic mouse model to explore the feasibility of using diffusion kurtosis imaging (DKI) as a tool for the early detection of microstructural changes in the brain due to amyloid-β (Aβ) plaque deposition., Methods: We longitudinally acquired DKI data of wild-type (WT) and APP/PS1 mice at 2, 4, 6 and 8 months of age, after which these mice were sacrificed for histological examination. Three additional cohorts of mice were also included at 2, 4 and 6 months of age to allow voxel-based co-registration between diffusion tensor and diffusion kurtosis  metrics and immunohistochemistry., Results: Changes were observed in diffusion tensor (DT) and diffusion kurtosis (DK) metrics in many of the 23 regions of interest that were analysed. Mean and axial kurtosis were greatly increased owing to Aβ-induced pathological changes in the motor cortex of APP/PS1 mice at 4, 6 and 8 months of age. Additionally, fractional anisotropy (FA) was decreased in APP/PS1 mice at these respective ages. Linear discriminant analysis of the motor cortex data indicated that combining diffusion tensor and diffusion kurtosis metrics permits improved separation of WT from APP/PS1 mice compared with either diffusion tensor or diffusion kurtosis metrics alone. We observed that mean kurtosis and FA are the critical metrics for a correct genotype classification. Furthermore, using a newly developed platform to co-register the in vivo diffusion-weighted magnetic resonance imaging with multiple 3D histological stacks, we found high correlations between DK metrics and anti-Aβ (clone 4G8) antibody, glial fibrillary acidic protein, ionised calcium-binding adapter molecule 1 and myelin basic protein immunohistochemistry. Finally, we observed reduced FA in the septal nuclei of APP/PS1 mice at all ages investigated. The latter was at least partially also observed by voxel-based statistical parametric mapping, which showed significantly reduced FA in the septal nuclei, as well as in the corpus callosum, of 8-month-old APP/PS1 mice compared with WT mice., Conclusions: Our results indicate that DKI metrics hold tremendous potential for the early detection and longitudinal follow-up of Aβ-induced pathology.

In this study, we investigated the effects of chronic administration of an inhibitor of the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) on Alzheimer-related pathology by multitracer PET imaging in transgenic APPPS1-21 (TG) mice. Methods: Wild-type (WT) and TG mice received vehicle or BACE inhibitor (60 mg/kg) starting at 7 wk of age. Outcome measures of brain metabolism, neuroinflammation, and amyloid-β pathology were obtained through small-animal PET imaging with 18 F-FDG, 18 F-peripheral benzodiazepine receptor ( 18 F-PBR), and 18 F-florbetapir ( 18 F-AV45), respectively. Baseline scans were acquired at 6-7 wk of age and follow-up scans at 4, 7, and 12 mo. 18 F-AV45 uptake was measured at 8 and 13 mo of age. After the final scans, histologic measures of amyloid-β (4G8), microglia (ionized calcium binding adaptor molecule 1), astrocytes (glial fibrillary acidic protein), and neuronal nuclei were performed. Results: TG mice demonstrated significant age-associated increases in 18 F-AV45 uptake. An effect of treatment was observed in the cortex ( P = 0.0014), hippocampus ( P = 0.0005), and thalamus ( P < 0.0001). Histology confirmed reduction of amyloid-β pathology in TG-BACE mice. Regardless of treatment, TG mice demonstrated significantly lower 18 F-FDG uptake than WT mice in the thalamus ( P = 0.0004) and hippocampus ( P = 0.0332). Neuronal nucleus staining was lower in both TG groups in the thalamus and cortex. 18 F-PBR111 detected a significant age-related increase in TG mice ( P < 0.0001) but did not detect the treatment-induced reduction in activated microglia as demonstrated by histology. Conclusion: Although 18 F-FDG, 18 F-PBR111, and 18 F-AV45 all detected pathologic alterations between TG and WT mice, only 18 F-AV45 could detect an effect of BACE inhibitor treatment. However, changes in WT binding of 18 F-AV45 undermine the specificity of this effect., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Understanding differences in Alzheimer's disease biomarkers before the pathology becomes evident can contribute to an improved understanding of disease pathogenesis and treatment. A decrease in amyloid-β (Aβ)42 in cerebrospinal fluid (CSF) is suggested to be a biomarker for Aβ deposition in brain. However, the relevance of CSF Aβ levels prior to deposition is not entirely known. Dogs are similar to man with respect to amyloid-β protein precursor (AβPP)-processing, age-related amyloid plaque deposition, and cognitive dysfunction. In the current study, we evaluated the relation between CSF Aβ42 levels and cognitive performance in young to middle-aged dogs (1.5-7 years old). Additionally, CSF sAβPPα and sAβPPβ were measured to evaluate AβPP processing, and CSF cytokines were measured to determine the immune status of the brain. We identified two groups of dogs showing consistently low or high CSF Aβ42 levels. Based on prior studies, it was assumed that at this age no cerebral amyloid plaques were likely to be present. The cognitive performance was evaluated in standard cognition tests. Low or high Aβ concentrations coincided with low or high sAβPPα, sAβPPβ, and CXCL-1 levels, respectively. Dogs with high Aβ concentrations showed significant learning impairments on delayed non-match to position (DNMP), object discrimination, and reversal learning compared to dogs with low Aβ concentrations. Our data support the hypothesis that high levels of CSF Aβ in dogs coincide with lower cognitive performance prior to amyloid deposition. Further experiments are needed to investigate this link, as well as the relevance with respect to Alzheimer's disease pathology progression.

The β-site amyloid-β protein precursor (AβPP) cleaving enzyme-1 (BACE1) is the rate limiting enzyme in the generation of amyloid-β peptide (Aβ) from AβPP, one of the major pathways in Alzheimer's disease (AD) pathology. Increased BACE1 levels and activity have been reported in the brain of patients with sporadic AD. Therefore, changes of BACE1 levels in the cerebrospinal fluid (CSF) have also been investigated as a possible biomarker of the disease. We analyzed BACE1 levels in CSF of elderly healthy participants before and after chronic treatment with a BACE inhibitor (BACEi) and evaluated the correlation between BACE1 levels and downstream AD markers. Overall, BACE1 CSF levels showed strong correlations to all downstream AD markers investigated. This is the first reported finding that shows BACE1 levels in CSF were well correlated to its end product Aβ1 - 42. As previously described, BACE1 levels were strongly correlated to total-tau and phosphorylated tau levels in CSF. Generally, chronic BACE inhibition did not influence BACE1 CSF protein levels. Follow-up studies including early-stage AD pathophysiology and prodromal AD patients will help to understand the importance of measuring BACE1 routinely in daily clinical practice and AD clinical trials.

Objectives: Safety, tolerability, pharmacokinetics, and pharmacodynamics of a novel β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor, JNJ-54861911, were assessed after single and multiple dosing in healthy participants., Methods: Two randomized, placebo-controlled, double-blind studies were performed using single and multiple ascending JNJ-54861911 doses (up to 14 days) in young and elderly healthy participants. Regular blood samples and frequent CSF samples, up to 36 hours after last dose, were collected to assess the pharmacokinetic and pharmacodynamic (Aβ, sAPPα,β,total levels) profiles of JNJ-54861911., Results: JNJ-54861911 was well-tolerated, adverse events were uncommon and unrelated to JNJ-54861911. JNJ-54861911 showed dose-proportional CSF and plasma pharmacokinetic profiles. Plasma- and CSF-Aβ and CSF-sAPPβ were reduced in a dose-dependent manner. Aβ reductions (up to 95%) outlasted exposure to JNJ-54861911. APOE ε4 carrier status and baseline Aβ levels did not influence Aβ/sAPPβ reductions., Conclusion: JNJ-54861911, a potent brain-penetrant BACE1 inhibitor, achieved high and stable Aβ reductions after single and multiple dosing in healthy participants.

Purpose: The aim of this study was to evaluate the in vitro and in vivo characteristics of [(89)Zr]JRF/AβN/25, a radiolabeled monoclonal antibody directed against amyloid-β (Aβ)., Procedures: JRF/AβN/25 was labeled with (89)Zr following modification with desferal. The affinity of the tracer for Aβ1-40 was determined in a saturation binding assay. In vitro stability was evaluated, and in vivo plasma stability and biodistribution of [(89)Zr]Df-Bz-JRF/AβN/25 were determined in wild-type mice. To evaluate whether the antibody can cross the blood-brain barrier, brain uptake in wild-type mice was additionally assessed by ex vivo autoradiography., Results: [(89)Zr]Df-Bz-JRF/AβN/25 was obtained in an average radiochemical yield of 50 % and a radiochemical purity of >97 %. A saturation binding assay demonstrated specific binding of [(89)Zr]Df-Bz-JRF/AβN/25 to Aβ1-40 with nanomolar affinity. The tracer was stable in buffer and proved to be stable in vivo with >92 % intact monoclonal antibody (mAb) remaining in the plasma at 48 h post injection. A biodistribution study showed a slow blood clearance with no significant accumulation of activity in any of the organs. Furthermore, [(89)Zr]Df-Bz-JRF/AβN/25 demonstrated modest brain penetration, which slowly decreased in time. This cerebral uptake was confirmed by ex vivo autoradiography., Conclusions: [(89)Zr]Df-Bz-JRF/AβN/25 binds with high affinity to Aβ1-40. The tracer displays an acceptable in vivo stability and is able to cross the blood-brain barrier. [(89)Zr]Df-Bz-JRF/AβN/25 might therefore be a potential candidate for in vivo imaging of Aβ deposition in the brain.

Background: Cerebrospinal fluid (CSF) amyloid-beta (Aβ) peptides are predictive biomarkers for Alzheimer's disease and are proposed as pharmacodynamic markers for amyloid-lowering therapies. However, frequent sampling results in fluctuating CSF Aβ levels that have a tendency to increase compared with baseline. The impact of sampling frequency, volume, catheterization procedure, and ibuprofen pretreatment on CSF Aβ levels using continuous sampling over 36 h was assessed., Methods: In this open-label biomarker study, healthy participants (n = 18; either sex, age 55-85 years) were randomized into one of three cohorts (n = 6/cohort; high-frequency sampling). In all cohorts except cohort 2 (sampling started 6 h post catheterization), sampling through lumbar catheterization started immediately post catheterization. Cohort 3 received ibuprofen (800 mg) before catheterization. Following interim data review, an additional cohort 4 (n = 6) with an optimized sampling scheme (low-frequency and lower volume) was included. CSF Aβ(1-37), Aβ(1-38), Aβ(1-40), and Aβ(1-42) levels were analyzed., Results: Increases and fluctuations in mean CSF Aβ levels occurred in cohorts 1-3 at times of high-frequency sampling. Some outliers were observed (cohorts 2 and 3) with an extreme pronunciation of this effect. Cohort 4 demonstrated minimal fluctuation of CSF Aβ both on a group and an individual level. Intersubject variability in CSF Aβ profiles over time was observed in all cohorts., Conclusions: CSF Aβ level fluctuation upon catheterization primarily depends on the sampling frequency and volume, but not on the catheterization procedure or inflammatory reaction. An optimized low-frequency sampling protocol minimizes or eliminates fluctuation of CSF Aβ levels, which will improve the capability of accurately measuring the pharmacodynamic read-out for amyloid-lowering therapies., Trial Registration: ClinicalTrials.gov NCT01436188 . Registered 15 September 2011.

Introduction: The accumulation of amyloid-β is a pathological hallmark of Alzheimer's disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-β antibody (JRF/AβN/25) to non-invasively assess amyloid-β burden in aged transgenic mice (APPPS1-21) with μPET imaging., Methods: We investigated the antibody JRF/AβN/25 that binds to full-length Aβ. JRF/AβN/25 was radiolabeled with a [(89)Zr]-desferal chelate and intravenously injected into 12-13 month aged APPPS1-21 mice and their wild-type (WT) controls. Mice underwent in vivo μPET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [(89)Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AβN/25 and finally we assessed the possible confounding effects of blood retention., Results: Voxel-wise analysis of μPET data demonstrated significant [(89)Zr]-Df-Bz-JRF/AβN/25 retention in APPPS1-21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [(89)Zr]-Df-Bz-JRF/AβN/25 was found at 4 and 7 days pi in APPPS1-21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AβN/25 only partially blocked [(89)Zr]-Df-Bz-JRF/AβN/25 uptake indicative of a high contribution of non-specific binding., Conclusion: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [(89)Zr]-Df-Bz-JRF/AβN/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

Background: Overlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimer's disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-β (Aβ) isoforms might improve the AD versus non-AD differential diagnosis., Objective: To determine the added diagnostic value of Aβ isoforms, Aβ(1-37), Aβ(1-38), and Aβ(1-40), as compared to the AD CSF biomarkers Aβ(1-42), T-tau, and P-tau(181P)., Methods: CSF from patients with dementia due to AD (n = 50), non-AD dementias (n = 50), mild cognitive impairment due to AD (n = 50) and non-demented controls (n = 50) was analyzed with a prototype multiplex assay using MSD detection technology. The non-AD group consisted of frontotemporal dementia (FTD; n = 17), dementia with Lewy bodies (DLB; n = 17), and vascular dementia (n = 16)., Results: Aβ(1-37) and Aβ(1-38) increased accuracy to differentiate AD from FTD or DLB. Aβ(1-37), Aβ(1-38), and Aβ(1-40) levels correlated with Mini-Mental State Examination scores and disease duration in dementia due to AD. The Aβ(1-42)/Aβ(1-40) ratio improved diagnostic performance of Aβ(1-42) in most differential diagnostic situations. Aβ(1-42) levels were lower in APOE ε4 carriers compared to non-carriers., Conclusions: Aβ isoforms help to differentiate AD from FTD and DLB. Aβ isoforms increase diagnostic performance of Aβ(1-42). In contrast to Aβ1-42, Aβ isoforms seem to be correlated with disease severity in AD. Adding the Aβ isoforms to the current biomarker panel could enhance diagnostic accuracy.

Beta-secretase is the first cleavage enzyme of amyloid-β protein precursor (AβPP) in the amyloidogenic pathway, leading to the formation of the plaque forming Amyloid-β (Aβ)1-42 peptide. BACE (beta-site AβPP cleaving enzyme) 1 inhibition is therefore considered to be a promising disease modifying therapy for Alzheimer's disease. An early assessment of the in vivo activity of BACE inhibitors was done in dogs since AβPP processing is the same as in humans and this species easily enables longitudinal cerebrospinal fluid (CSF) sampling. Aβ changes in CSF compared to baseline are used to evaluate target engagement of the compounds. Levels of Aβ1-37, Aβ1-38, Aβ1-40, and Aβ1-42 in CSF are measured with immunoassay (Mesoscale electrochemiluminescence technology) and with an ultra high-performance liquid chromatography mass spectrometry (UPLC-MS/MS). Two experimental BACE inhibitors were evaluated. With the immunoassay, a dose dependent decrease is observed for all four Aβ peptides. Measurements with the UPLC-MS/MS are in line with the immunoassay for Aβ1-37, Aβ1-38, and Aβ1-40, however, for Aβ1-42, differences are sometimes observed when comparing to changes seen in the other peptides with UPLC-MS/MS and with immunoassay results. Generally lower concentrations are measured with immunoassay. The reason for these differences is still unknown. Aβ1-42 is more prone to form aggregates compared to the other peptides. One hypothesis could be that while the immunoassay only measures free Aβ, bound and aggregated Aβ peptides are at least partially dissolved with the UPLC-MS/MS method, since acetonitrile is added to the CSF samples. This increases variability in the concentration of Aβ peptide measured with UPLC-MS/MS, especially for Aβ1-42, potentially masking the compound effect on Aβ1-42 levels.

Cramer et al. (Reports, 23 March 2012, p. 1503; published online 9 February 2012) tested bexarotene as a potential β-amyloid-lowering drug for Alzheimer's disease (AD). We were not able to reproduce the described effects in several animal models. Drug formulation appears very critical. Our data call for extreme caution when considering this compound for use in AD patients.

Gamma-secretase, a membrane bound protease which cleaves the transmembrane protein amyloid-β protein precursor (AβPP), is a therapeutic target for Alzheimer's disease. Gamma-secretase inhibitors (GSIs) and modulators (GSMs) are being investigated as potential disease-modifying agents. Preclinical in vivo models to monitor the activity on gamma-secretase are described in different species such as mouse, rat, and guinea pigs. All these models have their value in testing compounds with amyloid lowering properties, however, compound characteristics and pharmacokinetic properties, as well as other species characteristics such as limited sampling volumes of cerebrospinal fluid (CSF), recommended the use of a larger, non-rodent animal species. For this purpose, a screening model in dogs was developed for testing GSIs and GSMs. We showed that GSIs and GSMs had a dose- and time-dependent effect on Aβ(37), Aβ(38), Aβ(40), and Aβ(42) in CSF. Changes in liver function were evidenced by a transient increase in bilirubin with the GSMs and incidental increases in alanine aminotransferase for GSMs as well as GSIs. Microarray analysis of liver biopsies enabled to elucidate potential mechanisms behind the liver function changes. The relevance of the liver findings should be further evaluated in chronic pre-clinical safety studies and in humans. Based on our data, we can conclude that the dog is a very appropriate species to assess efficacy and safety of compounds which have an effect on AβPP processing such as GSMs, GSIs, and BACE-inhibitors.

Background: The cerebrospinal fluid (CSF) biomarkers amyloid β (Aβ)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program., Methods: The program is open for laboratories using commercially available kits for Aβ, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Mölndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples., Results: Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aβ-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aβ triplex (AβN-42, AβN-40, and AβN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories., Conclusions: Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers., (Copyright © 2011 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Amyloid-beta (Abeta) plaques are pathological hallmarks of Alzheimer disease (AD). In addition, innate inflammatory responses, such as those mediated by microglia, are integral to the pathogenesis of AD. Interestingly, only dense-core plaques and not diffuse plaques are associated with neuritic and inflammatory pathology in AD patients as well as in mouse AD models. However, the precise neuropathological changes that occur in the brain in response to amyloid deposition are largely unknown. To study the molecular mechanism(s) responsible for Abeta-mediated neuropathology, we performed a gene expression analysis on laser-microdissected brain tissue of Tg2576 and APPPS1 mice that are characterized by different types of amyloid plaques and genetic backgrounds. Data were validated by image and biochemical analyses on different ages of Tg2576, APPPS1, and Abeta42-depositing BRI-Abeta42 mice. Consistent with an important role of inflammatory responses in AD, we identified progranulin (mouse Grn; human GRN) as one of the top ten up-regulated molecules in Tg2576 ( approximately 8-fold increased) and APPPS1 ( approximately 2-fold increased) mice compared to littermate controls, and among the eight significantly up-regulated molecules common to both mouse models. In addition, Grn levels correlated significantly with amyloid load, especially the dense-core plaque pathology (p < 0.001). We further showed that Grn is up-regulated in microglia and neurons and neurites around dense-core plaques, but not in astrocytes or oligodendrocytes, as has been shown in AD patients. Our data therefore support the ongoing use of these mouse models in drug trials, especially those with anti-inflammatory compounds. Moreover, the correlation of Grn with increasing disease severity in AD mouse models prompts human studies exploring the viability of GRN as a disease biomarker. Because loss of GRN has recently been shown to cause frontotemporal dementia and serves as a risk factor for AD, the strong GRN reactivity around dense-core plaques is consistent with an important role of this factor in AD pathogenesis., (2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

We previously described two transgenic mouse lines expressing sub-endogenous levels of the 'Austrian' APP-T714I mutation (driven by the prenatally active PDGF-beta promoter; APP-Au mice) and showing intraneuronal Abeta pathology and reduced brain volumes on MRI at 12 and 20 months of age. To further investigate whether reduced brain sizes were caused by neurodegeneration or a neurodevelopmental defect, we now measured brain volumes as early as postnatal day 10. At this age, a distinguishable reduction in brain volumes was absent, indicating that brain volume deficits in APP-Au mice are not caused by a neurodevelopmental defect. To further study the association between intraneuronal Abeta and reduced brain volumes, we further generated and analyzed an APP transgenic mouse model expressing both Austrian and Swedish (K670N/M671L) mutations (APP-SwAu mice). APP-Swedish mutation is known to lead to altered APP processing in the secretory pathway, precluding its later processing in endosomal-lysosomal compartments, the site of intraneuronal Abeta accumulation. Also, to have higher levels of transgene expression only after birth, a murine Thy-1 promoter was utilized for APP-SwAu mouse lines. Despite having five times higher transgene APP levels compared to APP-Au mice, APP-SwAu mice showed significantly lower intraneuronal Abeta levels in the absence of reduced brain volumes, suggesting that intraneuronal Abeta accumulation is related to reduced brain volumes in APP-Au mice. These data also provide a first in vivo indication of altered processing of APP-Swedish at sub-endogenous levels, an effect not observed in mouse models expressing the APP-Swedish mutation in high amounts.

Transgenic mouse models of Alzheimer's disease (AD) expressing high levels of amyloid precursor protein (APP) with familial AD (FAD) mutations have proven to be extremely useful in understanding pathogenic processes of AD especially those that involve amyloidogenesis. We earlier described Austrian APP T714I pathology that leads to one of the earliest AD age-at-onsets with abundant intracellular and extracellular amyloid deposits in brain. The latter strikingly was non-fibrillar diffuse amyloid, composed of N-truncated A beta 42 in absence of A beta 40. In vitro, this mutation leads to one of the highest A beta 42/A beta 40 ratios among all FAD mutations. We generated an APP T714I transgenic mouse model that despite having 10 times lower transgene than endogenous murine APP deposited intraneuronal A beta in brain by 6 months of age. Accumulations increased with age, and this was paralleled by decreased brain sizes on volumetric MRI, compared to age-matched and similar transgene-expressing APP wild-type mice, although, with these levels of transgenic expression we did not detect neuronal loss or significant memory impairment. Immunohistochemical studies revealed that the majority of the intraneuronal A beta deposits colocalized with late endosomal markers, although some A beta inclusions were also positive for lysosomal and Golgi markers. These data support earlier observations of A beta accumulation in the endosomal-lysosomal pathway and the hypothesis that intraneuronal accumulation of A beta could be an important factor in the AD pathogenesis.

During the last 10 years, a lot of progress has been made in unraveling the pathogenic cascade leading to Alzheimer disease (AD). According to the most widely accepted hypothesis, production and aggregation of the amyloid beta (Abeta) peptide plays a key role in AD, and thus therapeutic interference with these processes is the subject of intense research. However, some important aspects of the disease mechanism are not yet fully understood. There is no consensus as yet on whether the disease acts through a loss- (LOF) or a gain-of-function (GOF) mechanism. While for many years, an increased production of Abeta42 was considered to be the prime culprit for the initiation of the disease process, and accordingly Abeta42 is elevated by AD-related presenilin(PS) mutations, recent data strongly suggest that PS mutations also lead to a LOF of PS towards a plethora of its substrates including amyloid precursor protein. How this PS LOF, especially decreased Abeta40 secretion due to mutant PS, impacts on the disease pathogenesis is yet to be elucidated. Secondly, vascular abnormalities--frequently observed to co-occur with AD--might also play a critical role in the initiation and aggravation of AD pathology given that the elimination of Abeta through a vascular route is an important brain Abeta clearance mechanism and its failure leads to formation of vascular amyloidosis and dense-core plaques. In this review, we will first focus on the important issue of a LOF versus a GOF mechanism for AD due to mutant PS, as well as on the possible role of vascular damage and reduced perfusion in AD. Special emphasis will be given to some of the AD mouse models that have helped to gain insights into the disease mechanism. Secondly, considering these mechanistic insights, we will discuss some therapeutic strategies which are currently in clinical or preclinical trials for AD., (Copyright (c) 2007 S. Karger AG, Basel.)

The varied ways in which mutations in presenilins (PSEN1 and PSEN2) affect amyloid b precursor protein (APP) processing in causing early-onset familial Alzheimer disease (FAD) are complex and not yet properly understood. Nonetheless, one useful diagnostic marker is an increased ratio of Ab42 to Ab40 (Ab42/Ab40) in patients' brain and biological fluids as well as in transgenic mice and cells. We studied Ab and APP processing for a set of nine clinical PSEN mutations on a novel and highly reproducible enzyme-linked immunosorbent assay (ELISA)-based in vitro method and also sought correlation with brain Ab analyzed by image densitometry and mass spectrometry. All mutations significantly increased Ab42/Ab40 in vitro by significantly decreasing Ab40 with accumulation of APP C-terminal fragments, a sign of decreased PSEN activity. A significant increase in absolute levels of Ab42 was observed for only half of the mutations tested. We also showed that age-of-onset of PSEN1-linked FAD correlated inversely with Ab42/Ab40 (r = -0.89; P = 0.001) and absolute levels of Ab42 (r = -0.83; P = 0.006), but directly with Ab40 levels (r = 0.69; P = 0.035). These changes also partly correlated with brain Ab42 and Ab40 levels. Together, our data suggested that Ab40 might be protective by perhaps sequestering the more toxic Ab42 and facilitating its clearance. Also, the in vitro method we describe here is a valid tool for assaying the pathogenic potential of clinical PSEN mutations in a molecular diagnostic setting.