Platelet Phosphorylated TDP-43: An Exploratory Study for a Peripheral Surrogate Biomarker Development for Alzheimer’s Disease

Figures

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  Figure 1.. Prediction analysis of TDP-43 for natural disordered regions. (A) PONDR® analysis of TDP-43 for potential phosphorylation sites. Majority of the phosphorylation events were predicted at Serine (Ser) amino acid sites (359–410). Most of the Ser amino acids are located at C-terminus region (20 out of 41; 48.7%). (B) PONDR® VL3-BA analysis of TDP-43 for identifying the disordered sites. The VL3-BA predictor is a feedforward neural network that was trained on regions of 152 long regions of disorder that were characterized by various methods. The region close to C-terminus was identified as disordered sites for TDP-43 which is also most of the Serine amino acids are located. Data taken from the Molecular Kinetics website, under license from the WSU Research Foundation. PONDR is © 1999 by the WSU Research Foundation, all rights reserved.

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  Figure 2.. (A) TDP-43 distribution in Alzheimer's disease (AD) patient brain regions. The tissue from three different regions of the brain was used in this study. The tissue homogenates were analyzed by immunoblotting method. The protein band intensities were normalized to actin. Three postmortem AD patients and age-matched healthy human brain samples were utilized in this study (n = 3). The difference between control and AD in hippocampus region was found statistically significant (p ≤ 0.015) according to Mann–Whitney U rank sum test. (B) TDP-43 protein aggregation in the brain region of an Alzheimer's disease patient. The homogenates from different regions of the brain were resolved in nonreducing SDS-PAGE condition and immunoprobed with anti-TDP-43 (pan) antibody (1:1000 dilutions). The TDP-43 protein aggregation is relatively more prominent in hippocampus region. (C) Platelet lysate TDP-43 profile. This figure represents the early TDP-43 studies on platelets obtained from AD patients (n = 3) and age-matched healthy subjects (n = 3). The platelet lysates were analyzed by a classical immunoblotting assay using anti-TDP-43 (pan antibody [1:1000 dilutions]).

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  Figure 3.. (A) Phosphorylated TDP-43 profile in platelet lysates. An anti-phospho (Ser409/410–2) TDP-43 antibody was used as an immunoprobing agent. The signals from AD platelet lysates were more pronounced as compared with ALS (negative disease control) and healthy subjects (control). Inset figure shows a computer-generated pseudo band that marks prominent phosphorylated TDP-43 at about 62 kDa. (B) Platelet lysate phosphorylated TDP-43 protein profile. The platelet lysates obtained from local bio-repository (n = 10 in each group) and analyzed by capillary electrophoresis based gel-less and membrane-less system western assay developed by proteinsimple (WES). The electropherogram peaks indicate the noticeable difference at about 62 kDa protein that represents phosphorylated TDP-43 protein. Box-whiskers plot represents statistical values. Redline within the boxes mark the median. Mann–Whitney U rank sum test was employed for statistical analysis. Difference between AD and control platelet phosphorylated TDP-43 was found statistically significant (p ≤ 0.010).