Rufina Leung

Association of Plasma Clusterin Concentration With Severity, Pathology, and Progression in Alzheimer Disease

CONTEXT Blood-based analytes may be indicators of pathological processes in Alzheimer disease (AD). OBJECTIVE To identify plasma proteins associated with AD pathology using a combined proteomic and neuroimaging approach. DESIGN Discovery-phase proteomics to identify plasma proteins associated with correlates of AD pathology. Confirmation and validation using immunodetection in a replication set and an animal model. SETTING A multicenter European study (AddNeuroMed) and the Baltimore Longitudinal Study of Aging. PARTICIPANTS Patients with AD, subjects with mild cognitive impairment, and healthy controls with standardized clinical assessments and structural neuroimaging. MAIN OUTCOME MEASURES Association of plasma proteins with brain atrophy, disease severity, and rate of clinical progression. Extension studies in humans and transgenic mice tested the association between plasma proteins and brain amyloid. RESULTS Clusterin/apolipoprotein J was associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in AD. Increased plasma concentration of clusterin was predictive of greater fibrillar amyloid-beta burden in the medial temporal lobe. Subjects with AD had increased clusterin messenger RNA in blood, but there was no effect of single-nucleotide polymorphisms in the gene encoding clusterin with gene or protein expression. APP/PS1 transgenic mice showed increased plasma clusterin, age-dependent increase in brain clusterin, as well as amyloid and clusterin colocalization in plaques. CONCLUSIONS These results demonstrate an important role of clusterin in the pathogenesis of AD and suggest that alterations in amyloid chaperone proteins may be a biologically relevant peripheral signature of AD.

Plasma proteins predict conversion to dementia from prodromal disease

The study aimed to validate previously discovered plasma biomarkers associated with AD, using a design based on imaging measures as surrogate for disease severity and assess their prognostic value in predicting conversion to dementia.

Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease

Abberant lipid metabolism is implicated in Alzheimers disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases. In a subsequent validation study (n = 141), PC variation in a bigger sample set was investigated, and the same 3 PCs were found to be significantly lower in AD patients: PC 16:0/20:5 (p < 0.001), 16:0/22:6 (p < 0.05), and 18:0/22:6 (p < 0.01). A receiver operating characteristic (ROC) analysis of the PCs, combined with apolipoprotein E (ApoE) data, produced an area under the curve predictive value of 0.828. Confirmatory investigations into the background biochemistry indiciated no significant change in plasma levels of 3 additional PCs of similar structure, total choline containing compounds or total plasma omega fatty acids, adding to the evidence that specific PCs play a role in AD pathology.

Plasma lipidomics analysis finds long chain cholesteryl esters to be associated with Alzheimer's disease.

There is an urgent need for the identification of Alzheimer’s disease (AD) biomarkers. Studies have now suggested the promising use of associations with blood metabolites as functional intermediate phenotypes in biomedical and pharmaceutical research. The aim of this study was to use lipidomics to identify a battery of plasma metabolite molecules that could predict AD patients from controls. We performed a comprehensive untargeted lipidomic analysis, using ultra-performance liquid chromatography/mass spectrometry on plasma samples from 35 AD patients, 40 elderly controls and 48 individuals with mild cognitive impairment (MCI) and used multivariate analysis methods to identify metabolites associated with AD status. A combination of 10 metabolites could discriminate AD patients from controls with 79.2% accuracy (81.8% sensitivity, 76.9% specificity and an area under curve of 0.792) in a novel test set. Six of the metabolites were identified as long chain cholesteryl esters (ChEs) and were reduced in AD (ChE 32:0, odds ratio (OR)=0.237, 95% confidence interval (CI)=0.10–0.48, P=4.19E−04; ChE 34:0, OR=0.152, 95% CI=0.05–0.37, P=2.90E−04; ChE 34:6, OR=0.126, 95% CI=0.03–0.35, P=5.40E−04; ChE 32:4, OR=0.056, 95% CI=0.01–0.24, P=6.56E−04 and ChE 33:6, OR=0.205, 95% CI=0.06–0.50, P=2.21E−03, per (log2) metabolite unit). The levels of these metabolites followed the trend control>MCI>AD. We, additionally, found no association between cholesterol, the precursor of ChE and AD. This study identified new ChE molecules, involved in cholesterol metabolism, implicated in AD, which may help identify new therapeutic targets; although, these findings need to be replicated in larger well-phenotyped cohorts.

Plasma Transthyretin as a Candidate Marker for Alzheimer's Disease

Diagnosis of the progressive neurodegenerative disorder Alzheimers disease (AD) can only definitively be made postmortem. The most promising AD biomarkers identified to date are found in cerebrospinal fluid (CSF). Among these, one of the most interesting candidates is transthyretin (TTR), the carrier of thyroxine and retinol, which also binds with amyloid-β (Aβ), and it has been suggested that it protects against Aβ deposition. A biomarker detectable in plasma would have great diagnostic value and could be of use for determining disease progression and the monitoring of therapeutic efficacy due to its greater accessibility over CSF-based markers. We aimed to validate TTR as a prognostic marker in AD and to determine its relation with cognitive measures. We examined the plasma protein levels of TTR in 90 people with late-onset AD and 50 age-matched non-demented controls (NDC) by immunoblotting and found lower plasma TTR levels in AD compared to NDC (p = 0.004). We then quantified plasma TTR by enzyme-linked immunosorbent assays in a larger independent cohort (n = 270) including subjects with mild to severe AD. Plasma TTR levels were significantly lower in AD cases with rapid cognitive decline and with severe cognitive impairment. Regression analyses showed plasma TTR levels also predicted cognitive decline over the ensuing 6 months. These data indicate that plasma TTR is a strong candidate AD biomarker that should be included in the development of blood based biomarker panels for disease diagnosis and also suggests that plasma TTR is a marker of disease severity and progression.

Blood-Based Biomarker Candidates of Cerebral Amyloid Using PiB PET in Non-Demented Elderly

Increasingly, clinical trials for Alzheimers disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p <  0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q <  0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.

Investigation of common, low-frequency and rare genome-wide variation in anorexia nervosa

Anorexia nervosa (AN) is a complex neuropsychiatric disorder presenting with dangerously low body weight, and a deep and persistent fear of gaining weight. To date, only one genome-wide significant locus associated with AN has been identified. We performed an exome-chip based genome-wide association studies (GWAS) in 2158 cases from nine populations of European origin and 15 485 ancestrally matched controls. Unlike previous studies, this GWAS also probed association in low-frequency and rare variants. Sixteen independent variants were taken forward for in silico and de novo replication (11 common and 5 rare). No findings reached genome-wide significance. Two notable common variants were identified: rs10791286, an intronic variant in OPCML (P=9.89 × 10−6), and rs7700147, an intergenic variant (P=2.93 × 10−5). No low-frequency variant associations were identified at genome-wide significance, although the study was well-powered to detect low-frequency variants with large effect sizes, suggesting that there may be no AN loci in this genomic search space with large effect sizes.

Blood-based biomarker discovery using aptamer capture (SOMAscan) platform technology

Background:The use of biomarkers to identify individuals at risk for developing late-onset Alzheimer’s disease (LOAD) is of interest for the design of therapeutic prevention or delay of onset clinical trials. A biomarker risk assignment algorithm (BRAA) based on APOE and TOMM40 ’523 genotypes and age is being used to enrich an international phase 3, double-blind, randomized, placebo-controlled clinical trial. This presentation reports preliminary data on the performance of the BRAA, specifically precision of the BRAA as a function of the experimental variation of the genotype assays, predictive characteristics of the algorithm to identify MCI due to AD, and comparative data for CSF and imaging (fMRI) based biomarkers.Methods: A simulation study was performed to determine how the experimental variation of the APOE and TOMM40 ’523 assays impacts the risk assignment by the BRAA. Performance of the BRAA (odds ratio, improvement in net reclassification rate vs. versions of the algorithm based only on age and/or APOE genotype) was calculated in a retrospective analysis of the Alzheimer’s Disease Neuroimaging Initiative data (n 1⁄4 660). Its performance (sensitivity and specificity) was compared to data from literature reports for proposed CSF and fMRI biomarkers. Results: The simulation study shows the expected precision of the BRAA to be>98%, based on the observed experimental variation of the TOMM40 ’523 and APOE assays. The odds ratio for using the algorithm to predict MCI or LOAD ranges from 3 to 5, and comparison of the full algorithm to a version based on APOE and age alone shows a significant (p < 0.0001) improvement in the net reclassification rate. The performance of this informative genotype BRAA compares favorably (PPV, NPV 70-80%) with CSF and imaging (fMRI) biomarkers. Conclusions: The performance characteristics of the biomarker risk algorithm support its use as a pharmacogenetic enrichment tool for stratification of individuals at high or low risk for developingMCI due to AD in a phase 3 clinical trial. The data from this prospective trial will be used to support qualification of the BRAA by regulatory agencies.

Small Chronic Alterations in Gene Expression and Circulating Blood Cells in Alzheimer's Disease

Background: There is evidence of modest benefits from “in-person” Brain Training using specific theoretical approaches. Millions of people worldwide use Brain Training games to try and improve cognitive performance. If effective this could provide a straightforward, cost-effective intervention and widely accessible intervention. There is however very limited empirical evidence examining the potential value of Brain Training Games.Methods: More than 13,500 participants, including 2500 people over the age of 60 were randomized to “Reasoning Brain training”, “General Brain Training” or a control internet search task and completed at least one training task. Participants were asked to train for at least 30 minutes 3 times a week. Cognition was evaluated at baseline and after 6 weeks, focussing upon reasoning (Grammatical Reasoning), memory (Hopkins Verbal Learning), spatial working memory, Paired Associate Learning and attention (digit span). Results:We recently reported a large randomized controlled trial of more than 11,000 people under the age of 60 in NatureMedicine. Across all age groups significant age related declines in reasoning and memory were identified from the baseline data, but there were no specific age related changes in other aspects of cognition. We recently reported a large randomized controlled trial of more than 11,000 people under the age of 60 in Nature Medicine, identifying no overall benefits over 6 weeks of training in a composite cognitive outcome. For the over 60 population, the mean age of participants was 65.4 sd 5.2, with an age range of 60-90. The mean number of training sessions completed was 28.1. In a preliminary analysis both active training groups experienced significant improvements in performance on the specific training games. In the outcome assessments, those receiving Reasoning Brain training had a significant improvement in Grammatical Reasoning in comparison to the control group. The participants receivingGeneral Brain Training experienced a significant but smaller benefit in Grammatical Reasoning. There was no significant benefit in memory or any of the other outcome measures. Conclusions: Preliminary results from the RCT in over 60’s suggest that reasoning based Brain Training games may confer some benefit in reasoning.