Clinical utility of an antibody-free LC-MS method to detect brain amyloid deposition in cognitively unimpaired individuals from the screening visit of the A4 Study.

INTRODUCTION: This study explored the ability of plasma amyloid beta (Aß)42/Aß40 to identify brain amyloid deposition in cognitively unimpaired (CU) individuals. METHODS: Plasma Aß was quantified with an antibody-free high-performance liquid chromatography tandem mass spectrometry method from Araclon Biotech (ABtest-MS) in a subset of 731 CU individuals from the screening visit of the Anti-Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study, to assess associations of Aß42/Aß40 with Aß positron emission tomography (PET). RESULTS: A model including Aß42/Aß40, age, apolipoprotein E ε4, and recruitment site identified Aß PET status with an area under the curve of 0.88 and an overall accuracy of 81%. A plasma-based pre-screening step could save up to 42% of the total number of Aß PET scans. DISCUSSION: ABtest-MS accurately identified brain amyloid deposition in a population of CU individuals, supporting its implementation in AD secondary prevention trials to reduce recruitment time and costs. Although a certain degree of heterogeneity is inherent to large and multicentric trials, ABtest-MS could be more robust to pre-analytical bias compared to other immunoprecipitation mass spectrometry methods. HIGHLIGHTS: Plasma amyloid beta (Aß)42/Aß40 accurately identified brain Aß deposition in cognitively unimpaired individuals from the Anti-Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study.The inclusion of the recruitment site in the predictive models has a non-negligible effect.A plasma biomarker-based model could reduce recruitment costs in Alzheimer's disease secondary prevention trials.Antibody-free liquid chromatography mass spectrometry methods may be more robust to pre-analytical variability than other platforms.

Clinical performance of an antibody-free assay for plasma Aß42/Aß40 to detect early alterations of Alzheimer's disease in individuals with subjective cognitive decline.

BACKGROUND: Accessible and cost-effective diagnostic tools are urgently needed to accurately quantify blood biomarkers to support early diagnosis of Alzheimer's disease (AD). In this study, we investigated the ability of plasma amyloid-beta (Aß)42/Aß40 ratio measured by an antibody-free mass-spectrometric (MS) method, ABtest-MS, to detect early pathological changes of AD. METHODS: This cohort study included data from the baseline and 2-year follow-up visits from the Fundació ACE Healthy Brain Initiative (FACEHBI) study. Plasma Aß42/Aß40 was measured with ABtest-MS and compared to 18F-Florbetaben PET as the reference standard (cutoff for early amyloid deposition of 13.5 centiloids). Cross-validation was performed in an independent DPUK-Korean cohort. Additionally, associations of plasma Aß42/Aß40 with episodic memory performance and brain atrophy were assessed. RESULTS: The FACEHBI cohort at baseline included 200 healthy individuals with subjective cognitive decline (SCD), of which 36 (18%) were Aß-PET positive. Plasma Aß42/Aß40 levels were significantly lower in Aß-PET positive individuals (median [interquartile range, IQR], 0.215 [0.203-0.236]) versus Aß-PET negative subjects (median [IQR], 0.261 [0.244-0.279]) (P < .001). Plasma Aß42/Aß40 was significantly correlated with Aß-PET levels (rho = -0.390; P < .001) and identified Aß-PET status with an area under the receiver operating characteristic curve (AUC) of 0.87 (95% confidence interval [CI], 0.80-0.93). A cutoff for the Aß42/Aß40 ratio of 0.241 (maximum Youden index) yielded a sensitivity of 86.1% and a specificity of 80.5%. These findings were cross-validated in an independent DPUK-Korean cohort (AUC 0.86 [95% CI 0.77-0.95]). Lower plasma Aß42/Aß40 ratio was associated with worse episodic memory performance and increased brain atrophy. Plasma Aß42/Aß40 at baseline predicted clinical conversion to mild cognitive impairment and longitudinal changes in amyloid deposition and brain atrophy at 2-year follow-up. CONCLUSIONS: This study suggests that plasma Aß42/Aß40, as determined by this MS-based assay, has potential value as an accurate and cost-effective tool to identify individuals in the earliest stages of AD, supporting its implementation in clinical trials, preventative strategies and clinical practice.

Longitudinal evaluation of the natural history of amyloid-ß in plasma and brain.

Plasma amyloid-ß peptide concentration has recently been shown to have high accuracy to predict amyloid-ß plaque burden in the brain. These amyloid-ß plasma markers will allow wider screening of the population and simplify and reduce screening costs for therapeutic trials in Alzheimer's disease. The aim of this study was to determine how longitudinal changes in blood amyloid-ß track with changes in brain amyloid-ß. Australian Imaging, Biomarker and Lifestyle study participants with a minimum of two assessments were evaluated (111 cognitively normal, 7 mild cognitively impaired, 15 participants with Alzheimer's disease). Amyloid-ß burden in the brain was evaluated through PET and was expressed in Centiloids. Total protein amyloid-ß 42/40 plasma ratios were determined using ABtest® assays. We applied our method for obtaining natural history trajectories from short term data to measures of total protein amyloid-ß 42/40 plasma ratios and PET amyloid-ß. The natural history trajectory of total protein amyloid-ß 42/40 plasma ratios appears to approximately mirror that of PET amyloid-ß, with both spanning decades. Rates of change of 7.9% and 8.8%, were observed for total protein amyloid-ß 42/40 plasma ratios and PET amyloid-ß, respectively. The trajectory of plasma amyloid-ß preceded that of brain amyloid-ß by a median value of 6 years (significant at 88% confidence interval). These findings, showing the tight association between changes in plasma and brain amyloid-ß, support the use of plasma total protein amyloid-ß 42/40 plasma ratios as a surrogate marker of brain amyloid-ß. Also, that plasma total protein amyloid-ß 42/40 plasma ratios has potential utility in monitoring trial participants, and as an outcome measure.

Total Aß42/Aß40 ratio in plasma predicts amyloid-PET status, independent of clinical AD diagnosis.

OBJECTIVE: To explore whether the plasma total ß-amyloid (Aß) Aß42/Aß40 ratio is a reliable predictor of the amyloid-PET status by exploring the association between these 2 variables in a subset of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging cohort. METHODS: Taking plasma samples at 3 separate time points, month 18 (n = 176), month 36 (n = 169), and month 54 (n = 135), we assessed the total Aß42/Aß40 ratio in plasma (TP42/40) with regard to neocortical Aß burden via PET standardized uptake value ratio (SUVR) and investigated both association with Aß-PET status and correlation (and agreement) with SUVR. RESULTS: The TP42/40 plasma ratio was significantly reduced in amyloid-PET-positive participants at all time points (p < 0.0001). Adjusting for covariates age, gender, APOE ε4 allele status, and clinical classification clearly affects the significance, with p values reduced and only comparisons at 54 months retaining significance (p = 0.006). Correlations with SUVR were similar across each time point, with Spearman ρ reaching -0.64 (p < 0.0001). Area under the curve values were highly reproducible over time points, with values ranging from 0.880 at 36 months to 0.913 at 54 months. In assessments of the healthy control group only, the same relationships were found. CONCLUSIONS: The current study demonstrates reproducibility of the plasma assay to discriminate between amyloid-PET positive and negative over 3 time points, which can help to substantially reducing the screening rate of failure for clinical trials targeting preclinical or prodromal disease. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that plasma total Aß42/Aß40 ratio is associated with neocortical amyloid burden as measured by PET SUVR.

Plasma Aß42/40 ratio alone or combined with FDG-PET can accurately predict amyloid-PET positivity: a cross-sectional analysis from the AB255 Study.

BACKGROUND: To facilitate population screening and clinical trials of disease-modifying therapies for Alzheimer's disease, supportive biomarker information is necessary. This study was aimed to investigate the association of plasma amyloid-beta (Aß) levels with the presence of pathological accumulation of Aß in the brain measured by amyloid-PET. Both plasma Aß42/40 ratio alone or combined with an FDG-PET-based biomarker of neurodegeneration were assessed as potential AD biomarkers. METHODS: We included 39 cognitively normal subjects and 20 patients with mild cognitive impairment from the AB255 Study who had undergone PiB-PET scans. Total Aß40 and Aß42 levels in plasma (TP42/40) were quantified using ABtest kits. Subjects were dichotomized as Aß-PET positive or negative, and the ability of TP42/40 to detect Aß-PET positivity was assessed by logistic regression and receiver operating characteristic analyses. Combination of plasma Aß biomarkers and FDG-PET was further assessed as an improvement for brain amyloidosis detection and diagnosis classification. RESULTS: Eighteen (30.5%) subjects were Aß-PET positive. TP42/40 ratio alone identified Aß-PET status with an area under the curve (AUC) of 0.881 (95% confidence interval [CI] = 0.779-0.982). Discriminating performance of TP42/40 to detect Aß-PET-positive subjects yielded sensitivity and specificity values at Youden's cutoff of 77.8% and 87.5%, respectively, with a positive predictive value of 0.732 and negative predictive value of 0.900. All these parameters improved after adjusting the model for significant covariates. Applying TP42/40 as the first screening tool in a sequential diagnostic work-up would reduce the number of Aß-PET scans by 64%. Combination of both FDG-PET scores and plasma Aß biomarkers was found to be the most accurate Aß-PET predictor, with an AUC of 0.965 (95% CI = 0.913-0.100). CONCLUSIONS: Plasma TP42/40 ratio showed a relevant and significant potential as a screening tool to identify brain Aß positivity in preclinical and prodromal stages of Alzheimer's disease.

Blood Markers in Healthy-Aged Nonagenarians: A Combination of High Telomere Length and Low Amyloidß Are Strongly Associated With Healthy Aging in the Oldest Old.

Many factors may converge in healthy aging in the oldest old, but their association and predictive power on healthy or functionally impaired aging has yet to be demonstrated. By detecting healthy aging and in turn, poor aging, we could take action to prevent chronic diseases associated with age. We conducted a pilot study comparing results of a set of markers (peripheral blood mononuclear cell or PBMC telomere length, circulating Aß peptides, anti-Aß antibodies, and ApoE status) previously associated with poor aging or cognitive deterioration, and their combinations, in a cohort of "neurologically healthy" (both motor and cognitive) nonagenarians (n = 20) and functionally impaired, institutionalized nonagenarians (n = 38) recruited between 2014 and 2015. We recruited 58 nonagenarians (41 women, 70.7%; mean age: 92.37 years in the neurologically healthy group vs. 94.13 years in the functionally impaired group). Healthy nonagenarians had significantly higher mean PBMC telomere lengths (mean = 7, p = 0.001), this being inversely correlated with functional impairment, and lower circulating Aß40 (total in plasma fraction or TP and free in plasma fraction or FP), Aß42 (TP and FP) and Aß17 (FP) levels (FP40 131.35, p = 0.004; TP40 299.10, p = 0.007; FP42 6.29, p = 0.009; TP42 22.53, p = 0.019; FP17 1.32 p = 0.001; TP17 4.47, p = 0.3), after adjusting by age. Although healthy nonagenarians had higher anti-Aß40 antibody levels (net adsorbed signal or NAS ± SD: 0.211 ± 0.107), the number of participants that pass the threshold (NAS > 3) to be considered as positive did not show such a strong association. There was no association with ApoE status. Additionally, we propose a "Composite Neurologically Healthy Aging Score" combining TP40 and mean PBMC telomere length, the strongest correlation of measured biomarkers with neurologically healthy status in nonagenarians (AUC = 0.904).

Safety, tolerability and immunogenicity of an active anti-Aß40 vaccine (ABvac40) in patients with Alzheimer's disease: a randomised, double-blind, placebo-controlled, phase I trial.

BACKGROUND: Immunotherapy targeting the amyloid-ß (Aß) peptide is a promising strategy for the treatment of Alzheimer's disease (AD); however, none of the active or passive vaccines tested have been demonstrated to be effective to date. We have developed the first active vaccine against the C-terminal end of Aß40, ABvac40, and assessed its safety and tolerability in a phase I clinical trial. METHODS: A randomised, double-blind, placebo-controlled, parallel-group, phase I study of ABvac40 was conducted with patients aged 50-85 years with mild to moderate AD. Participants were entered into three separate groups according to time of study entry and were randomly allocated to receive ABvac40 or placebo (overall ratio 2:1). The first group received two half-doses of ABvac40 or placebo, whereas the second and third groups received two and three full doses, respectively. All treatments were administered subcutaneously at 4-week intervals. Patients, carers and investigators were blind to treatment allocation throughout the study. The primary objective was to assess the safety and tolerability of ABvac40 by registering all adverse events (AEs). All patients who received at least one dose of treatment were included in the safety analysis. The secondary objective was to evaluate the immunogenicity of ABvac40 by titration of specific anti-Aß40 antibodies in plasma. RESULTS: Twenty-four patients were randomly allocated: 16 patients to the ABvac40 group and 8 patients to the placebo group. All randomised patients completed the study, therefore the intention-to-treat and safety populations were identical. Overall, 71 AEs affecting 18 patients were recorded: 11 (69%) in the ABvac40 group and 7 (88%) in the placebo group (p = 0.6214). Neither incident vasogenic oedema nor sulcal effusion (amyloid-related imaging abnormalities corresponding to vasogenic oedema and sulcal effusions) nor microhaemorrhages (amyloid-related imaging abnormalities corresponding to microhaemorrhages and hemosiderin deposits) were detected throughout the study period in the ABvac40-treated patients. Eleven of 12 (~92%) individuals receiving three injections of ABvac40 developed specific anti-Aß40 antibodies. CONCLUSIONS: ABvac40 showed a favourable safety and tolerability profile while eliciting a consistent and specific immune response. An ongoing phase II clinical trial is needed to confirm these results and to explore the clinical efficacy of ABvac40. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03113812 . Retrospectively registered on 14 April 2017.

Plasma amyloid ß 42/40 ratios as biomarkers for amyloid ß cerebral deposition in cognitively normal individuals.

INTRODUCTION: Plasma amyloid ß (Aß) peptides have been previously studied as candidate biomarkers to increase recruitment efficiency in secondary prevention clinical trials for Alzheimer's disease. METHODS: Free and total Aß42/40 plasma ratios (FP42/40 and TP42/40, respectively) were determined using ABtest assays in cognitively normal subjects from the Australian Imaging, Biomarker and Lifestyle Flagship Study. This population was followed-up for 72 months and their cortical Aß burden was assessed with positron emission tomography. RESULTS: Cross-sectional and longitudinal analyses showed an inverse association of Aß42/40 plasma ratios and cortical Aß burden. Optimized as a screening tool, TP42/40 reached 81% positive predictive value of high cortical Aß burden, which represents 110% increase over the population prevalence of cortical Aß positivity. DISCUSSION: These findings support the use of plasma Aß42/40 ratios as surrogate biomarkers of cortical Aß deposition and enrichment tools, reducing the number of subjects submitted to invasive tests and, consequently, recruitment costs in clinical trials targeting cognitively normal individuals.

Neurofibrillary Tangles of Aßx-40 in Alzheimer's Disease Brains.

The two pathognomonic lesions in the brain of AD patients are senile plaques and intraneuronal neurofibrillary tangles (NFT). Previous studies have demonstrated that amyloid-ß (Aß) is a component of both senile plaques and NFTs, and have showed that intracellular accumulation of Aß is toxic for cells and precedes the appearance of extracellular amyloid deposits. Here we report that there are numerous intraneuronal NFT and extraneuronal NFT immunoreactive for Aßx-40 in which there is no co-localization with tau staining suggesting the existence of two different neurodegenerating populations associated with the intracellular accumulation of either tau protein or Aßx-40 in AD.

Cognitive Composites Domain Scores Related to Neuroimaging Biomarkers within Probable-Amnestic Mild Cognitive Impairment-Storage Subtype.

The probable-amnestic (Pr-a) mild cognitive impairment (MCI)-storage subtype is a phenotype with 8.5 times more risk of conversion to dementia, mainly Alzheimer's disease (AD), than the possible non-amnestic (Pss-na) MCI. The aim of this study was to find the optimized cognitive composites (CCs) domain scores most related to neuroimaging biomarkers within Pr-aMCI-storage subtype patients. The Fundació ACE (ACE) study with 20 Pr-aMCI-storage subtype subjects (MCI) were analyzed. All subjects underwent a neuropsychological assessment, a structural MRI, FDG-PET, and PIB-PET. The adjusted hippocampal volume (aHV) on MRI, the standard uptake value ratio (SUVR) on FDG-PET and PIB-PET SUVR measures were analyzed. The construction of the CCs domain scores, and the aHV on MRI and FDG-PET SUVR measures, were replicated in the parental AB255 study database (n = 133 MCI). Partial correlations adjusted by age, gender, and education were calculated with the associated p-value among every CC domain score and the neuroimaging biomarkers. The results were replicated in the "MCI due to AD" with memory storage impairments from ADNI. Delayed Recall CC domain score was significantly correlated with PIB-PET SUVR (ß= -0.61, p = 0.003) in the ACE study and also with aHV on MRI (ß= 0.27, p = 0.01) and FDG-PET SUVR (ß= 0.27, p = 0.01) in the AB255 study. After a median survival time of 20.6 months, 85% from the ACE MCI converted to AD. The replication of our results in the ADNI dataset also confirmed our findings. Delayed Recall is the CC domain score best correlated with neuroimaging biomarkers associated with prodromal AD diagnosis.

Validation of Immunoassay-Based Tools for the Comprehensive Quantification of Aß40 and Aß42 Peptides in Plasma.

Recent advances in neuroimaging and cerebrospinal fluid (CSF) biomarker assays have provided evidence of a long preclinical stage of Alzheimer's disease (AD). This period is being increasingly targeted for secondary prevention trials of new therapies. In this context, the interest of a noninvasive, cost-effective amyloid-ß (Aß) blood-based test does not need to be overstated. Nevertheless, a thorough validation of these bioanalytical methods should be performed as a prerequisite for confident interpretation of clinical results. The aim of this study was to validate ELISA sandwich colorimetric ABtest40 and ABtest42 for the quantification of Aß40 and Aß42 in human plasma. The validation parameters assessed included precision, accuracy, sensitivity, specificity, recovery, and dilution linearity. ABtest40 and ABtest42 proved to be specific for their target peptide using Aß peptides with sequence similar to the target. Mean relative error in the quantification was found to be below 7.5% for both assays, with high intra-assay, inter-assay, and inter-batch precision (CV <9.0% on average). Sensitivity was assessed by determination of the limit of quantification fulfilling precision and accuracy criteria; it was established at 7.60 pg/ml and 3.60 pg/ml for ABtest40 and ABtest42, respectively. Plasma dilution linearity was demonstrated in PBS; however, dilution in a proprietary formulated buffer significantly increased the recovery of both Aß40 and Aß42 masked by matrix interactions, allowing a more comprehensive assessment of the free and total peptide levels in the plasma. In conclusion, both assays were successfully validated as tools for the quantification Aß40 and Aß42 in plasma.

Outstanding Phenotypic Differences in the Profile of Amyloid-ß between Tg2576 and APPswe/PS1dE9 Transgenic Mouse Models of Alzheimer's Disease.

APPswe/PS1dE9 and Tg2576 are very common transgenic mouse models of Alzheimer's disease (AD), used in many laboratories as tools to research the mechanistic process leading to the disease. In order to augment our knowledge about the amyloid-ß (Aß) isoforms present in both transgenic mouse models, we have developed two chromatographic methods, one acidic and the other basic, for the characterization of the Aß species produced in the brains of the two transgenic mouse models. After immunoprecipitation and micro-liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry, 10 species of Aß, surprisingly all of human origin, were detected in the brain of Tg2576 mouse, whereas 39 species, of both murine and human origin, were detected in the brain of the APP/PS1 mouse. To the best of our knowledge, this is the first study showing the identification of such a high number of Aß species in the brain of the APP/PS1 transgenic mouse, whereas, in contrast, a much lower number of Aß species were identified in the Tg2576 mouse. Therefore, this study brings to light a relevant phenotypic difference between these two popular mice models of AD.

Aß1-17 is a major amyloid-ß fragment isoform in cerebrospinal fluid and blood with possible diagnostic value in Alzheimer's disease.

This work was prompted by the finding that Aß1-17 (Aß17) appeared to be the second-most abundant cerebrospinal fluid (CSF) Aß fragment, after Aß40. We developed an ELISA to quantify levels of Aß17 directly accessible in plasma (DA17), recovered from the proteomic plasma matrix (RP17) and associated with the cellular pellet (CP17) that remained after plasma collection. Then, we used a sample of 19 healthy control (HC), 27 mild cognitive impairment (MCI), and 17 mild Alzheimer's disease (AD) patients to explore the association of the diagnostic groups with those direct markers, their ratios or the ratios with their Aß40 or Aß42 counterparts. After dichotomization (d) for the median of the sample population, logistic regression analysis showed that in the AD versus HC subgroup, subjects with a dDA/CP17 higher than the median had a significantly greater risk of being AD than those with marker levels equal to or below the median (odds ratio OR; 95% confidence interval; 17.21; 1.42-208.81). Subjects with dRP17/42 below the median had an increased likelihood of being MCI (20.00; 1.17-333.33) or AD (40.00; 1.87-1000) versus being HC, than those with dRP17/42 higher than the median. Although the confidence intervals are wide, these findings suggest that assessment of Aß17 may increase the diagnostic performance of blood-based Aß tests which might be developed into minimally invasive first-step screening tests for people with increased risk for AD.

Association between cell-bound blood amyloid-ß(1-40) levels and hippocampus volume.

INTRODUCTION: The identification of early, preferably presymptomatic, biomarkers and true etiologic factors for Alzheimer's disease (AD) is the first step toward establishing effective primary and secondary prevention programs. Consequently, the search for a relatively inexpensive and harmless biomarker for AD continues. Despite intensive research worldwide, to date there is no definitive plasma or blood biomarker indicating high or low risk of conversion to AD. METHODS: Magnetic resonance imaging and ß-amyloid (Aß) levels in three blood compartments (diluted in plasma, undiluted in plasma and cell-bound) were measured in 96 subjects (33 with mild cognitive impairment, 14 with AD and 49 healthy controls). Pearson correlations were completed between 113 regions of interest (ROIs) (45 subcortical and 68 cortical) and Aß levels. Pearson correlation analyses adjusted for the covariates age, sex, apolipoprotein E (ApoE), education and creatinine levels showed neuroimaging ROIs were associated with Aß levels. Two statistical methods were applied to study the major relationships identified: (1) Pearson correlation with phenotype added as a covariate and (2) a meta-analysis stratified by phenotype. Neuroimaging data and plasma Aß measurements were taken from 630 Alzheimer's Disease Neuroimaging Initiative (ADNI) subjects to be compared with our results. RESULTS: The left hippocampus was the brain region most correlated with Aß(1-40) bound to blood cell pellets (partial correlation (pcor) = -0.37, P = 0.0007) after adjustment for the covariates age, gender and education, ApoE and creatinine levels. The correlation remained almost the same (pcor = -0.35, P = 0.002) if phenotype is also added as a covariate. The association between both measurements was independent of cognitive status. The left hemisphere entorhinal cortex also correlated with Aß(1-40) cell-bound fraction. AB128 and ADNI plasma Aß measurements were not related to any brain morphometric measurement. CONCLUSIONS: Association of cell-bound Aß(1-40) in blood with left hippocampal volume was much stronger than previously observed in Aß plasma fractions. If confirmed, this observation will require careful interpretation and must be taken into account for blood amyloid-based biomarker development.

Neprilysin is poorly expressed in the prefrontal cortex of aged dogs with cognitive dysfunction syndrome.

Neprilysin (NEP) is the principal amyloid ß (A ß ) degrading peptidase; this activity may protect against Alzheimer's disease (AD), the most important age-related neurodegenerative process. The aim of this work was to analyze NEP mRNA expression in the frontal cortex of dogs with and without canine cognitive dysfunction syndrome (CDS), which is considered a natural model for AD. Expression of canine cerebral NEP mRNA was assessed by RT-PCR followed by qPCR in young, aged-cognitively unimpaired (CU), and aged-cognitively impaired (CI) dogs. On average, aged-CI dogs showed 80% (P < 0.01) lower expression levels of NEP mRNA than their aged-CU counterparts. Furthermore, the standard deviation of the qPCR measurements was more than 6 times higher in the cognitively healthy animals (young and aged-CU) than in the aged-CI group. Another interesting find is the determination of a positive correlation between NEP expression and the number of cholinergic neurons in basal telencephalon, indicating a probable connection between both events in these types of neurodegeneration processes. These results suggest that high expression levels of NEP might be a protective factor for canine CDS and, most likely, for other A ß -associated neurodegenerative diseases, such as AD.

Blood amyloid beta levels in healthy, mild cognitive impairment and Alzheimer's disease individuals: replication of diastolic blood pressure correlations and analysis of critical covariates.

Plasma amyloid beta (Aß) levels are being investigated as potential biomarkers for Alzheimer's disease. In AB128 cross-sectional study, a number of medical relevant correlates of blood Aß40 or Aß42 were analyzed in 140 subjects (51 Alzheimer's disease patients, 53 healthy controls and 36 individuals diagnosed with mild cognitive impairment). We determined the association between multiple variables with Aß40 and Aß42 levels measured in three different blood compartments called i) Aß directly accessible (DA) in the plasma, ii) Aß recovered from the plasma matrix (RP) after diluting the plasma sample in a formulated buffer, and iii) associated with the remaining cellular pellet (CP). We confirmed that diastolic blood pressure (DBP) is consistently correlated with blood DA Aß40 levels (r=-0.19, P=0.032). These results were consistent in the three phenotypic groups studied. Importantly, the observation resisted covariation with age, gender or creatinine levels. Observed effect size and direction of Aß40 levels/DBP correlation are in accordance with previous reports. Of note, DA Aß40 and the RP Aß40 were also strongly associated with creatinine levels (r=0.599, P<<0.001) and to a lesser extent to urea, age, hematocrit, uric acid and homocysteine (p<0.001). DBP and the rest of statistical significant correlates identified should be considered as potential confounder factors in studies investigating blood Aß levels as potential AD biomarker. Remarkably, the factors affecting Aß levels in plasma (DA, RP) and blood cell compartments (CP) seem completely different.

Several direct and calculated biomarkers from the amyloid-ß pool in blood are associated with an increased likelihood of suffering from mild cognitive impairment.

Validation of cost-effective, non-invasive methods to identify early (pre-clinical) Alzheimer's disease (AD) is increasingly becoming a key research challenge. We have developed two ELISA sandwich colorimetric tests for the accurate detection of amyloid-ß (Aß)1-40 and Aß1-42: i) directly accessible (DA) in the plasma, ii) recovered from the plasma sample (RP) after diluting the plasma sample in a formulated buffer, and iii) associated with the remaining cellular pellet (CP). These tests were carried out on samples from healthy controls (n = 19) and individuals with mild cognitive impairment (MCI; n = 27) with amnestic-hippocampal syndrome to investigate whether this comprehensive approach may help to explain the association between blood Aß levels and MCI. A logistic regression analysis detected seven direct or calculated markers (CP 40, DA 42, RP 42, DA/CP 40, DA/RP 42, DA/CP 42, and DA 42/40) with significant odds ratios (OR) after they were dichotomized with regard to the median of the pooled population. In particular, the likelihood [OR (95% CI)] of having MCI for patients with catCP 40, catDA/RP 42, catDA/CP 42, or catDA 42/40 below the corresponding population median ("positive test") was 11.48 (1.87-70.52), 22.09 (3.19-152.61), 11.48 (1.87-70.50), and 9.54 (1.77-51.38)-fold higher, respectively, than in those with a "negative test" after adjusting for the effect of the ApoE genotype. These results are congruent with the hypothesis that changes in blood Aß levels may be associated with the initial stages of AD. Thus, these Aß blood biomarkers might be useful tools for screening for those at increased risk of developing AD.

Identification of ß-amyloid species in canine cerebrospinal fluid by mass spectrometry.

It is well known that several Aß species, including Aß40 and Aß42, are present in cerebrospinal fluid (CSF). Experimental results suggest that these species could play a role in Alzheimer's disease and might also have diagnostic significance. In the present work, the canine CSF ß-amyloid species profile has been identified by matrix-assisted laser desorption and ionization-time-of-flight/time of flight mass spectrometry (MALDI-TOF/TOF) analysis after immunoprecipitation with different Aß-specific antibodies. The results show that species arising from combined ß- and γ-secretase activities in humans, such as Aß1-33, Aß1-34, Aß1-37, Aß1-38, Aß1-39, Aß1-40, and Aß1-42, are also present in dogs. Species arising from combined α- and ß-secretase activities, as well as other Aß-degrading enzymes, are also present in both human and canine CSF, with the exception of Aß1-13, Aß1-14, and Aß1-18, which are not detected in dogs. A large number of species truncated at Glu-3 and Glu-11 have also been detected. To our knowledge, this work describes a most complete Aß species profile from canine CSF. The similarities between the canine and human CSF Aß profile reinforce the dog as a highly appropriate animal model for research in Alzheimer's disease.

Phosphodiesterase 7 inhibitor reduced cognitive impairment and pathological hallmarks in a mouse model of Alzheimer's disease.

Elevated levels of amyloid beta (Aß) peptide, hyperphosphorylation of tau protein, and inflammation are pathological hallmarks in Alzheimer's disease (AD). Phosphodiesterase 7 (PDE7) regulates the inflammatory response through the cyclic adenosine monophosphate signaling cascade, and thus plays a central role in AD. The aim of this study was to evaluate the efficacy of an inhibitor of PDE7, named S14, in a mouse model of AD. We report that APP/Ps1 mice treated daily for 4 weeks with S14 show: (1) significant attenuation in behavioral impairment; (2) decreased brain Aß deposition; (3) enhanced astrocyte-mediated Aß degradation; and (4) decreased tau phosphorylation. These effects are mediated via the cyclic adenosine monophosphate/cyclic adenosine monophosphate response element-binding protein signaling pathway, and inactivation of glycogen synthase kinase (GSK)3. Our data support the use of PDE7 inhibitors, and specifically S14, as effective therapeutic agents for the prevention and treatment of AD.

Optimized protocol for amyloid-ß extraction from the brain.

Brain levels of amyloid-ß (Aß) are frequently assessed in transgenic mice models of Alzheimer's disease. The procedure involves tissue sample homogenization using different buffers in a sequential process. No attempt has been made to assess if these procedures are able to extract the total amount of Aß present in the samples. Here we present data suggesting that standard protocols can lead to a dramatic underestimation of the Aß content. Results show that higher extraction buffer volumes and at least two repetitions of the soluble and membrane-bound extraction steps are necessary for a more accurate estimation of the Aß content in brain tissues.