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BACKGROUND: The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer's disease. METHODS: The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. RESULTS: We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. CONCLUSIONS: This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.
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INTRODUCTION: Early intervention in Alzheimer's disease (AD) requires the development of an easily administered test that is able to identify those at risk. Focusing on microRNA robustly detected in plasma and standardizing the analysis strategy, we sought to identify disease-stage specific biomarkers. METHODS: Using TaqMan microfluidics arrays and a statistical consensus approach, we assessed plasma levels of 185 neurodegeneration-related microRNA, in cohorts of cognitively normal amyloid ß-positive (CN-Aß+), mild cognitive impairment (MCI), and Alzheimer's disease (AD) participants, relative to their respective controls. RESULTS: Distinct disease stage microRNA biomarkers were identified, shown to predict membership of the groups (area under the curve [AUC] >0.8) and were altered dynamically with AD progression in a longitudinal study. Bioinformatics demonstrated that these microRNA target known AD-related pathways, such as the Phosphoinositide 3-kinase (PI3K-Akt) signalling pathway. Furthermore, a significant correlation was found between miR-27a-3p, miR-27b-3p, and miR-324-5p and amyloid beta load. DISCUSSION: Our results show that microRNA signatures alter throughout the progression of AD, reflect the underlying disease pathology, and may prove to be useful diagnostic markers.
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There is some debate regarding the differing levels of plasma homocysteine, vitamin B12 and serum folate between healthy controls (HC), mild cognitive impairment (MCI), and Alzheimer's disease (AD). As part of the Australian Imaging Biomarker Lifestyle (AIBL) study of aging cohort, consisting of 1,112 participants (768 HC, 133 MCI patients, and 211 AD patients), plasma homocysteine, vitamin B12, and serum and red cell folate were measured at baseline to investigate their levels, their inter-associations, and their relationships with cognition. The results of this cross-sectional study showed that homocysteine levels were increased in female AD patients compared to female HC subjects (+16%, p-value < 0.001), but not in males. Red cell folate, but not serum folate, was decreased in AD patients compared to HC (-10%, p-value = 0.004). Composite z-scores of short- and long-term episodic memory, total episodic memory, and global cognition all showed significant negative correlations with homocysteine, in all clinical categories. Increasing red cell folate had a U-shaped association with homocysteine, so that high red cell folate levels were associated with worse long-term episodic memory, total episodic memory, and global cognition. These findings underscore the association of plasma homocysteine with cognitive deterioration, although not unique to AD, and identified an unexpected abnormality of red cell folate.