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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 (Aβ)42/Aβ40 ratio measured by an antibody-free mass-spectrometric (MS) method, ABtest-MS, to detect early pathological changes of AD.This cohort study included data from the baseline and 2-year follow-up visits from the Fundació ACE Healthy Brain Initiative (FACEHBI) study. Plasma Aβ42/Aβ40 was measured with ABtest-MS and compared toThe FACEHBI cohort at baseline included 200 healthy individuals with subjective cognitive decline (SCD), of which 36 (18%) were Aβ-PET positive. Plasma Aβ42/Aβ40 levels were significantly lower in Aβ-PET positive individuals (median [interquartile range, IQR], 0.215 [0.203-0.236]) versus Aβ-PET negative subjects (median [IQR], 0.261 [0.244-0.279]) (P.001). Plasma Aβ42/Aβ40 was significantly correlated with Aβ-PET levels (rho = -0.390; P.001) and identified Aβ-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 Aβ42/Aβ40 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 Aβ42/Aβ40 ratio was associated with worse episodic memory performance and increased brain atrophy. Plasma Aβ42/Aβ40 at baseline predicted clinical conversion to mild cognitive impairment and longitudinal changes in amyloid deposition and brain atrophy at 2-year follow-up.This study suggests that plasma Aβ42/Aβ40, 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.

ObjectiveTo explore whether the plasma total β-amyloid (Aβ) Aβ42/Aβ40ratio 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.MethodsTaking plasma samples at 3 separate time points, month 18 (n = 176), month 36 (n = 169), and month 54 (n = 135), we assessed the total Aβ42/Aβ40ratio in plasma (TP42/40) with regard to neocortical Aβ burden via PET standardized uptake value ratio (SUVR) and investigated both association with Aβ-PET status and correlation (and agreement) with SUVR.ResultsThe TP42/40 plasma ratio was significantly reduced in amyloid-PET–positive participants at all time points (p< 0.0001). Adjusting for covariates age, gender,APOEε4 allele status, and clinical classification clearly affects the significance, withpvalues 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.ConclusionsThe 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 evidenceThis study provides Class II evidence that plasma total Aβ42/Aβ40ratio is associated with neocortical amyloid burden as measured by PET SUVR.

BackgroundTo 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 (Aβ) levels with the presence of pathological accumulation of Aβ in the brain measured by amyloid-PET. Both plasma Aβ42/40 ratio alone or combined with an FDG-PET-based biomarker of neurodegeneration were assessed as potential AD biomarkers.MethodsWe included 39 cognitively normal subjects and 20 patients with mild cognitive impairment from the AB255 Study who had undergone PiB-PET scans. Total Aβ40 and Aβ42 levels in plasma (TP42/40) were quantified using ABtest kits. Subjects were dichotomized as Aβ-PET positive or negative, and the ability of TP42/40 to detect Aβ-PET positivity was assessed by logistic regression and receiver operating characteristic analyses. Combination of plasma Aβ biomarkers and FDG-PET was further assessed as an improvement for brain amyloidosis detection and diagnosis classification.ResultsEighteen (30.5%) subjects were Aβ-PET positive. TP42/40 ratio alone identified Aβ-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 Aβ-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 Aβ-PET scans by 64%. Combination of both FDG-PET scores and plasma Aβ biomarkers was found to be the most accurate Aβ-PET predictor, with an AUC of 0.965 (95% CI = 0.913–0.100).ConclusionsPlasma TP42/40 ratio showed a relevant and significant potential as a screening tool to identify brain Aβ positivity in preclinical and prodromal stages of Alzheimer’s disease.

Plasma amyloid-β peptide concentration has recently been shown to have high accuracy to predict amyloid-β plaque burden in the brain. These amyloid-β 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-β track with changes in brain amyloid-β. 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-β burden in the brain was evaluated through PET and was expressed in Centiloids. Total protein amyloid-β 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-β 42/40 plasma ratios and PET amyloid-β. The natural history trajectory of total protein amyloid-β 42/40 plasma ratios appears to approximately mirror that of PET amyloid-β, with both spanning decades. Rates of change of 7.9% and 8.8%, were observed for total protein amyloid-β 42/40 plasma ratios and PET amyloid-β, respectively. The trajectory of plasma amyloid-β preceded that of brain amyloid-β 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-β, support the use of plasma total protein amyloid-β 42/40 plasma ratios as a surrogate marker of brain amyloid-β. Also, that plasma total protein amyloid-β 42/40 plasma ratios has potential utility in monitoring trial participants, and as an outcome measure., Total protein amyloid β42/40 plasma ratios determined by the ABtest® assay were able to effectively map and track longitudinal amyloid PET accumulation. This adds to the body of information that blood-based biomarkers hold utility for diagnosis, prognosis, monitoring and, thus, treatment in the field of Alzheimer’s., Graphical Abstract Graphical Abstract

Background Immunotherapy targeting the amyloid-β (Aβ) 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 Aβ40, 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-Aβ40 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-Aβ40 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.

The two pathognomonic lesions in the brain of AD patients are senile plaques and intraneuronal neurofibrillary tangles (NFT). Previous studies have demonstrated that amyloid-β (Aβ) is a component of both senile plaques and NFTs, and have showed that intracellular accumulation of Aβ 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 Aβx-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 Aβx-40 in AD.

Background: Long-term longitudinal studies with multimodal biomarkers are needed to delve into the knowledge of preclinical AD. Subjective cognitive decline has been proposed as a risk factor for the development of cognitive impairment. Thus, including individuals with SCD in observational studies may be a cost-effective strategy to increase the prevalence of preclinical AD in the sample. Objectives: To describe the rationale, research protocols and baseline characteristics of participants in the Fundació ACE Healthy Brain Initiative (FACEHBI). Design: FACEHBI is a clinical trial (EudraCT: 2014-000798-38) embedded within a long-term observational study of individuals with SCD. Setting: Participants have been recruited at the memory clinic of Fundació ACE (Barcelona) from two different sources: patients referred by a general practitioner and individuals from an Open House Initiative. Participants: 200 individuals diagnosed with SCD with a strictly normal performance in a comprehensive neuropsychological battery. Measurements: Individuals will undergo an extensive neuropsychological protocol, risk factor assessment and a set of multimodal biomarkers including florbetaben PET, structural and functional MRI, diffusion tensor imaging, determination of amyloid species in plasma and neurophthalmologic assessment with optical coherence tomography. Results: Two hundred individuals have been recruited in 15 months. Mean age was 65.9 years; mean MMSE was 29.2 with a mean of 14.8 years of education. Conclusions: FACEHBI is a long-term study of cognition, biomarkers and lifestyle that has been designed upon an innovative symptom-based approach using SCD as target population. It will shed light on the pathophysiology of preclinical AD and the role of SCD as a risk marker for the development of cognitive impairment.

Background Immunotherapy targeting the amyloid-β (Aβ) 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 Aβ40, 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-Aβ40 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-Aβ40 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. Electronic supplementary material The online version of this article (10.1186/s13195-018-0340-8) contains supplementary material, which is available to authorized users.

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-β (Aβ) 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 Aβ40 and Aβ42 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 Aβ 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

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-β (Aβ) isoforms present in both transgenic mouse models, we have developed two chromatographic methods, one acidic and the other basic, for the characterization of the Aβ 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 Aβ, 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 Aβ species in the brain of the APP/PS1 transgenic mouse, whereas, in contrast, a much lower number of Aβ 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.

The most common animal models currently used for Alzheimer disease (AD) research are transgenic mice that express a mutant form of human Abeta precursor protein (APP) and/or some of the enzymes implicated in their metabolic processing. However, these transgenic mice carry their own APP and APP-processing enzymes, which may interfere in the production of different amyloid-beta (Abeta) peptides encoded by the human transgenes. Additionally, the genetic backgrounds of the different transgenic mice are a possible confounding factor with regard to crucial aspects of AD that they may (or may not) reproduce. Thus, although the usefulness of transgenic mice is undisputed, we hypothesized that additional relevant information on the physiopathology of AD could be obtained from other natural non-transgenic models. We have analyzed the chick embryo and the dog, which may be better experimental models because their enzymatic machinery for processing APP is almost identical to that of humans. The chick embryo is extremely easy to access and manipulate. It could be an advantageous natural model in which to study the cell biology and developmental function of APP and a potential assay system for drugs that regulate APP processing. The dog suffers from an age-related syndrome of cognitive dysfunction that naturally reproduces key aspects of AD including Abeta cortical pathology, neuronal degeneration and learning and memory disabilities. However, dense core neuritic plaques and neurofibrillary tangles have not been consistently demonstrated in the dog. Thus, these species may be natural models with which to study the biology of AD, and could also serve as assay systems for Abeta-targeted drugs or new therapeutic strategies against this devastating disease.

To date there are no conclusive reports on the usefulness of determining amyloid peptides in the serum of patients with Alzheimer's disease (AD). Only anecdotal works deal with the changes in the peptides produced by cholinesterase inhibitors. In this study, the authors investigated and studied the clinical significance of plasmatic Aβ-40 and Aβ-42 peptide levels in a series of 34 consecutive patients with AD. The baseline levels of the Aβ-40 peptide correlated negatively with the Mini Examen Cognoscitivo (Spanish version of the Mini-Mental test) score. Complete follow-up was possible in 22 patients. After 6 months of treatment with galantamine, the mean Aβ-40 peptide levels decreased from 31.86 to 24.22 pg/mL. The baseline levels of Aβ-40 were predictive of response to treatment in the Alzheimer's Disease Assessment Scale—Cognitive Subscale. The authors conclude that determining plasmatic Aβ-40 peptide levels could be useful in predicting and monitoring response to treatment in AD.

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 β-amyloid (Aβ) 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 Aβ levels. Pearson correlation analyses adjusted for the covariates age, sex, apolipoprotein E (ApoE), education and creatinine levels showed neuroimaging ROIs were associated with Aβ 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 Aβ 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 Aβ(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 Aβ(1–40) cell-bound fraction. AB128 and ADNI plasma Aβ measurements were not related to any brain morphometric measurement. Conclusions Association of cell-bound Aβ(1–40) in blood with left hippocampal volume was much stronger than previously observed in Aβ plasma fractions. If confirmed, this observation will require careful interpretation and must be taken into account for blood amyloid-based biomarker development.

Renal reabsorption is the main mechanism that controls mannose homeostasis. This takes place through a specific Na-coupled uphill transport system, the molecular identity of which is unknown. We prepared and screened a size-selected rat kidney cortex cDNA library through the expression of mannose transport in Xenopus laevis oocytes. We have identified a membrane protein that induces high-affinity and specific Na-dependent transport of d-mannose and d-glucose in X. laevis oocytes, most likely through stimulation of the capacity of an endogenous transport system of the oocyte. Sequencing has revealed that the cDNA encodes the counterpart of the human membrane-associated protein MAP17, previously known by its overexpression in renal, colon, lung, and breast carcinomas. We show that MAP17 is a 12.2-kDa nonglycosylated membrane protein that locates to the brush-border plasma membrane and the Golgi apparatus of transfected cells and that it is expressed in the proximal tubules of the kidney cortex and in the spermatids of the seminiferous tubules. It spans twice the cell membrane, with both termini inside the cell, and seems to form homodimers through intracellular Cys55, a residue also involved in transport expression. MAP17 is responsible for mannose transport expression in oocytes by rat kidney cortex mRNA. The induced transport has the functional characteristics of a Na-glucose cotransporter (SGLT), because d-glucose and α-methyl-d-glucopyranoside are also accepted substrates that are inhibited by phloridzin. The corresponding transporter from the proximal tubule remains to be identified, but it is different from the known mammalian SGLT-1, -2, and -3.

Plasma amyloid beta (Aβ) levels are being investigated as potential biomarkers for Alzheimer's disease. In AB128 cross-sectional study, a number of medical relevant correlates of blood Aβ40 or Aβ42 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 Aβ40 and Aβ42 levels measured in three different blood compartments called i) Aβ directly accessible (DA) in the plasma, ii) Aβ 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 Aβ40 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 Aβ40 levels/DBP correlation are in accordance with previous reports. Of note, DA Aβ40 and the RP Aβ40 were also strongly associated with creatinine levels (r=0.599, P<

Neprilysin (NEP) is the principal amyloidβ(Aβ) 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 Aβ-associated neurodegenerative diseases, such as AD.

The β-amyloid precursor proteins (βAPPs) are a family of glycosylated transmembrane proteins that include in their sequences the β-amyloid peptide, a major component of the characteristic amyloid deposits or senile plaques found in the brains of Alzheimer's disease patients and aged Down's syndrome subjects. Various βAPP isoforms, mainly βAPP-695, βAPP-714, βAPP-751 and βAPP-770, the number corresponding to the number of amino acids they encode, resulting from the alternative splicing of a single primary transcript have been described. Using oligonucleotides recognizing each of the four major Alzheimer's βAPP mRNAs, we have found that each βAPP mRNA displays a specific temporal and spatial pattern of expression. The prototype isoform βAPP-695 occurs early in cells actively implicated in morphogenetic events, as those mesodermal cells invaginating at the level of the primitive streak, and it is later restricted to the neurectodermal (neural tube, neural crest and neurogenic placode) derivatives. By contrast, the longest isoform βAPP-770 appears later and restricted to mesodermal and endodermal derivatives. The isoforms βAPP-714 and βAPP-751 are still expressed later than the other two isoforms and distributed ubiquitously, though βAPP-714 transcripts predominate typically within the neural tube. (C) 2000 Elsevier Science Ireland Ltd., This work was supported by grants SAF97-0238 and DGA P51/97 to M.S. from C.I.C.Y.T. and Aragon's Government, respectively

In the present study, we have examined the cellular mechanisms mediating the regulation of renal proximal tubular sodium-coupled inorganic phosphate (Na/Pi) transport by thyroid hormone (T3) in young and aged rats. Young hypothyroid rats showed a marked decrease in Na/Pi cotransport activity, which was associated with parallel decreases in type II Na/Pi cotransporter (NaPi-2) protein and messenger RNA (mRNA) abundance. In contrast, administration of long-term physiological and supraphysiological doses of T3 resulted in significant increases in Na/Pi cotransport activity, protein, and mRNA levels. Nuclear run-on experiments indicated that thyroid hormone regulates NaPi-2 mRNA levels by a transcriptional mechanism. In aged rats, although there were no changes in T3 serum levels (when compared with young animals), there were significant decreases in serum Pi concentration, renal Na/Pi cotransport activity, and NaPi-2 protein and mRNA abundance. These effects were mediated, at least in part, by a reduction in...

Calcitonin mRNA and calcitonin gene-related peptide (CGRP) mRNA both are generated from the calcitonin gene because of tissue-specific alternative splicing of the primary transcript. It is currently established that, of the two mature transcripts, calcitonin mRNA is far the predominant transcript produced in thyroid C-cells whereas only CGRP mRNA is produced in the nervous system. However, here we provide evidence that the two splicing forms of the chick calcitonin primary transcript are found within the developing central nervous system, although displaying specific patterns of expression. While CGRP mRNA is first expressed in motor neurons at rather advanced stages of embryogenesis, calcitonin mRNA is expressed in the floor plate and dorsal rhombencephalon from earliest stages.

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-β (Aβ)1-40 and Aβ1-42: 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 Aβ 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 Aβ levels may be associated with the initial stages of AD. Thus, these Aβ blood biomarkers might be useful tools for screening for those at increased risk of developing AD.

Brain levels of amyloid-β (Aβ) 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 Aβ present in the samples. Here we present data suggesting that standard protocols can lead to a dramatic underestimation of the Aβ 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 Aβ content in brain tissues.

Elevated levels of amyloid beta (Aβ) 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 Aβ deposition; (3) enhanced astrocyte-mediated Aβ 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. © 2013 Elsevier Inc.

The present study was aimed at assessing the capability of Aβ1-40 and Aβ1-42 levels in undiluted plasma (UP), diluted plasma (DP), and cell bound (CB) to distinguish between early stages of Alzheimer's disease (AD), amnesic mild cognitive impairment (MCI), and healthy control (HC). Four blood samples from each participant were collected during one month and the levels of Aβ1-40 and Aβ1-42 were determined by a blinded proprietary ELISA sandwich (Araclon Biotech. Zaragoza, Spain). First striking result was that the amount of Aβ1-40 and Aβ1-42 in UP represented only a small proportion (~15%) of the total beta-amyloid pool in blood (βAPB) described here as the sum of Aβ1-40 and Aβ1-42 in blood where they are free in plasma, bound to plasma proteins, and bound to blood cells. Furthermore, we found that levels of Aβ1-40 and Aβ1-42 in UP, DP, and CB were significantly higher in MCI when compared to HC. On average, the totalβAPB was 1.8 times higher in MCI than in HC (P=0.03) and allowed to discriminate between MCI and HC with a sensitivity and specificity over 80%. Thus, quantification of several markers of theβAPB could be useful and reliable in the discrimination between MCI and HC.

Background Fibromyalgia (FM) is characterized by chronic, widespread muscular pain and tenderness and is generally associated with other somatic and psychological symptoms. Further, circulatory levels of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) may be altered in FM patients, possibly in association with their symptoms. Recently, rises in BMI have been suggested to contribute to increased circulating levels of proinflammatory cytokines in FM patients. Our aim was to measure the circulatory levels of proinflammatory cytokines to determine the influence of BMI on these levels in FM patients and healthy volunteers (HVs). In Spanish FM patients (n = 64) and HVs (n = 25), we measured BMI and serum concentrations of proinflammatory cytokines by capture ELISA. Findings There were significant differences in BMI levels between FM patients (26.40 ± 4.46) and HVs (23.64 ± 3.45) and significant increase in IL-6 in FM patients (16.28 ± 8.13 vs 0.92 ± 0.32 pg/ml) (P < 0.001). IL-1β and TNF-α decreased in FM patients compared with HVs. By ANCOVA, there was no significant association between BMI and TNF-α (F = 0.098, p = 0.75) or IL-6 (F = 0.221, p = 0.63) levels in FM patients. Conclusions Our analysis in FM patients of BMI as a covariate of proinflammatory cytokines levels showed that serum TNF-α and IL-6 levels are independent of BMI. Further studies are necessary to dissect these findings and their implication in future therapeutic approaches for FM patients.

Gentamicin causes endocytosis of Na/Pi cotransporter protein (NaPi-2).BackgroundRenal toxicity is a major side-effect of aminoglycoside antibiotics and is characterized by an early impairment in proximal tubular function. In a previous study, we have shown that gentamicin administration to the rat causes an early impairment in sodium gradient-dependent phosphate (Na/Pi) cotransport activity. The purpose of our current study was to determine the molecular mechanisms of the impairment in Na/Pi cotransport activity, specifically the role of the proximal tubular type II Na/Pi cotransporter.MethodsRats were treated for one, two, and three days with two daily injections of 30 mg/kg body weight gentamicin or the vehicle.ResultsGentamicin caused a progressive decrease in superficial cortical apical brush-border membrane (SC-BBM) Na/Pi cotransporter activity (856 ± 93 in control vs. 545 ± 87 pmol/mg BBM protein in 3-day gentamicin, P < 0.01). Western blot analysis showed a parallel and progressive decrease in SC-BBM Na/Pi cotransporter protein abundance, a 50% decrease after one day of treatment, a 63% decrease after two days of treatment, and an 83% decrease after three days treatment with gentamicin. In contrast, gentamicin treatment had no effect on Na/Pi cotransport activity or Na/Pi cotransporter protein abundance in BBM isolated from the juxtamedullary cortex (JMC-BBM). Immunofluorescence microscopy showed a major decrease in the expression of Na/Pi cotransporter protein in the apical membrane of the proximal convoluted tubule, with progressive intracellular accumulation of Na/Pi protein. Colocalization studies showed that in gentamicin-treated rats, Na/Pi protein was colocalized in the early endosomes and especially in the lysosomes. Northern blot analysis of cortical RNA interestingly showed no reduction in Na/Pi cotransporter mRNA abundance even after three days of gentamicin treatment.ConclusionWe conclude that gentamicin inhibits Na/Pi cotransport activity by causing a decrease in the expression of the type II Na/Pi cotransport protein at the level of the proximal tubular apical BBM and that inhibition of Na/Pi cotransport activity is most likely mediated by post-transcriptional mechanisms.

In this study we have analysed, by in situ hybridization, the expression of the genes for both α-CGRP and β-CGRP in hypoglossal motor nuclei following transection of the left hypoglossal nerve. Our results show that the gene for α-CGRP displays a peculiar sequence of regulation (a successive up-down-up-recovery sequence) within ipsilateral hypoglossal motoneurons in response to axotomy. It is initially up-regulated, then down-regulated (displaying mRNA levels below basal), and later again up-regulated before recovery. By contrast, the gene for β-CGRP displays a successive and distinct up-down-recovery sequence of regulation (it does not display a second increase in mRNA production). The first up-regulation of the α-CGRP gene occurs just during the early period of perineuronal glial reaction and the second up-regulation just during the period of delayed astrocyte reaction and muscle reinnervation. Because α-CGRP is a neuron-derived factor for many types of cells, including astrocytes and skeletal myocytes, our results suggest that the pleiotropic α-CGRP may be a motoneuron-derived trophic signal for both glial and skeletal muscle cells in order to maintain the motoneuron itself and, in consequence, might be of therapeutic interest in treating degenerative diseases of motoneurons. β-CGRP might be redundant within the hypoglossal motoneurons., M.S. was supported by a Juan March Foundation Fellowship. Part of the study was supported by a grant (PB91-0691) from the Spanish Ministry of Education and Science

In this study, we present evidence that neurogenic cells inhibit the differentiation of cardiogenic cells. When cells of the entire area pellucida at stage 5 were dissociated and reaggregated, the aggregates differentiated into neural tissues and other structures of any germ layer origin, except for heart tissues, despite the fact that the cardiogenic cells are already committed to differentiate. The phenomenon also occurs from stages 6 to 8, during which cells are in a higher state of commitment. Using combinations of different regions of the area pellucida, it was confirmed that neurogenic cells are responsible for the inhibition of cardiogenic cell differentiation. The inhibition is not species-specific because quail and chick neurogenic cells inhibit each other's cardiogenic cell differentiation. Direct contact between cardiogenic and neurogenic cells seems to be necessary for inhibition because cardiogenic cell differentiation takes place if cardiogenic and neurogenic cells are separated by a porous membrane. Dissociated neural tubes from early stages, but not dissociated telencephalon from 2-day-old chicks, also inhibit cardiogenic cell differentiation. These results suggest that neurogenic cells may sequester molecules necessary for the differentiation of cardiogenic cells rather than produce inhibitory molecules.

In order to investigate the modulation of the synthesis and the subcellular localization of the growth associated protein GAP-43 in neuronal cell bodies we have taken advantage of the well known regenerative properties of axotomized motor neurons of the facial and hypoglossal nuclei. Alterations in the levels of GAP-43 mRNA containing cells were studied by in situ hybridization histochemistry. The protein localization was examined using immunohistochemistry at the light and electron microscopic levels. Neurons from the control side showed undetectable levels of both GAP-43-like immunoreactivity and GAP-43 mRNA levels. Whereas axotomized neurons exhibited a marked increase in GAP-43 mRNA levels and in GAP-43-like immunoreactivity. Three to 50 days after axotomy, motor neurons ipsilateral to the lesion displayed a dense reticular or filamentous perinuclear distribution of the immunoreactivity in somata and proximal dendritic processes, corresponding to the location of the Golgi apparatus in these neurons. At the electron microscopic level the immunoreactivity was located in the cisternae of the Golgi complex and found to be associated with trans-side vesicles of these complexes. The myelinated fibers of the transectomized facial nerve also presented an intense GAP-43-like immunoreactivity. Twenty-one days after the axotomy a decay in the number of immunostained neurons and in the intensity of immunolabeled somata was observed. Our study reveals a rapid induction of GAP-43 mRNA and protein after axotomy. The localization of the newly synthesized GAP-43-like immunoreactivity to the Golgi apparatus seen in the present work suggests an early association of this protein with newly formed membranes prior to transport toward the terminals through the axons.

Background Mild cognitive impairment (MCI) of an amnestic type is a common condition in older people and highly predictive of Alzheimer's disease (AD). To date, there is no clear consensus regarding the best antecedent biomarker to predict early conversion to AD. Objective The aim of the study is to demonstrate that 1H magnetic resonance spectroscopy (MRS) of the brain in MCI patients may predict early conversion to dementia within the 2-year period after baseline assessment. Methods A cohort of patients fulfilling the criteria of amnestic MCI were enrolled consecutively. At baseline the patients underwent neuropsychological examination, standard blood tests and APOE genotype. 1H-MRS (1.5 T) of the brain was carried out by exploring two areas: the posteromedial bilateral parietal lobe and left medial occipital lobe. The patients were followed up to detect conversion to probable AD according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association group criteria. Results After a 2-year follow-up, 27 (38%) patients converted to AD. The mean N-acetyl-aspartate/creatine (NAA/Cr) ratio in the posteromedial bilateral parietal cortex was 1.38 in converters versus 1.49 in non-converters (p, Article summary Article focus Amnestic mild cognitive impairment (MCI) is a common condition at increased risk of conversion to Alzheimer's disease (AD). There is no clear consensus regarding the ideal antecedent biomarker to predict early conversion. There are a few longitudinal studies using brain magnetic resonance spectroscopy (MRS) as a predictor of conversion to dementia. We hypothesise that MRS in MCI may identify patients at risk of early conversion to dementia. Key messages MRS is a reliable biomarker of AD and predicts early conversion to dementia in MCI. Both posteromedial parietal and occipital regions were predictive of conversion, but the parietal region was better in terms of accuracy of classification. Neither the APOE4 genotype nor leuco-araiosis was predictive of conversion. Strengths and limitations This is a longitudinal study, which is a non-invasive, reproducible and widely available tool. However, this technique is not free of artefacts limiting the accuracy of metabolite levels. This study is based on early predictions (2 years from baseline). In the longer term, it is likely that all patients with objective memory impairment will convert to dementia.