Abstract
IntroductionPeripheral biomarkers to diagnose Alzheimer's disease (AD) have not been established. Given parallels between neuron and platelet biology, we hypothesized platelet membrane-associated protein changes may differentiate patients clinically defined with probable AD from noncognitive impaired controls.MethodsPurified platelets, confirmed by flow cytometry were obtained from individuals before fractionation by ultracentrifugation. Following a comparison of individual membrane fractions by SDS-PAGE for general proteome uniformity, equal protein weight from the membrane fractions for five representative samples from AD and five samples from controls were pooled. AD and control protein pools were further divided into molecular weight regions by one-dimensional SDS-PAGE, prior to digestion in gel. Tryptic peptides were analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Ionized peptide intensities were averaged for each identified protein in the two pools, thereby measuring relative protein abundance between the two membrane protein pools. Log2-transformed ratio (AD/control) of protein abundances fit a normal distribution, thereby permitting determination of significantly changed protein abundances in the AD pool.ResultsWe report a comparative analysis of the membrane-enriched platelet proteome between patients with mild to moderate AD and cognitively normal, healthy subjects. A total of 144 proteins were determined significantly altered in the platelet membrane proteome from patients with probable AD. In particular, secretory (alpha) granule proteins were dramatically reduced in AD. Of these, we confirmed significant reduction of thrombospondin-1 (THBS1) in the AD platelet membrane proteome by immunoblotting. There was a high protein-protein connectivity of proteins in other pathways implicated by proteomic changes to the proteins that define secretory granules.ConclusionsDepletion of secretory granule proteins is consistent with a preponderance of post-activated platelets in circulation in AD. Significantly changed pathways implicate additional AD-related defects in platelet glycoprotein synthesis, lipid homeostasis, amyloidogenic proteins, and regulators of protease activity, many of which may be useful plasma membrane-expressed markers for AD. This study highlights the utility of LC-MS/MS to quantify human platelet membrane proteins and suggests that platelets may serve as a source of blood-based biomarkers in neurodegenerative disease.
Highlights
Peripheral biomarkers to diagnose Alzheimer’s disease (AD) have not been established
The changes we describe for amyloidogenic proteins including THBS1, beta-2 microglobulin (B2M), and gelsolin in the platelet membrane proteome in AD are consistent with what is known to occur in individuals diagnosed with AD, it is apparent that alone, these protein changes are not markers with adequate specificity for AD obviating their inclusion into broader multianalyte profiles that consider a panel of changing proteins, be it on the membranes of platelets, or in cerebrospinal fluid (CSF)
In this study, we purified platelet membrane proteins for quantitative proteomics and identify potential biomarkers and pathways affected in patients with clinically diagnosed AD
Summary
Peripheral biomarkers to diagnose Alzheimer’s disease (AD) have not been established. Alzheimer’s disease (AD) is the most common form of dementia worldwide. It is characterized by the accumulation of extracellular beta amyloid (Ab) plaques and intracellular tau tangles as well as gliosis and neuronal cell death [1,2,3]. As the population ages and the number of people affected with AD increases, it is becoming increasingly important to identify biomarkers that can be used to diagnose the disease as early as possible. While significant progress has been made in brain imaging and characterizing fluid biomarkers of AD in cerebrospinal fluid (CSF) [6,7], peripheral biomarkers have not been well established for clinical use. Bloodbased biomarkers are especially attractive in a clinical setting compared to CSF, because blood samples are relatively easy to obtain
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