Platelet TLR4 activity in neurodegenerative diseases

Abstract

Purpose : Primary open-angle glaucoma (POAG) subtypes of high tension (HTG; historical IOP >21 mmHg), normal tension glaucoma (NTG), and Alzheimer’s disease (AD) are common ocular and neurodegenerative diseases that feature a proinflammatory phenotype. Previously, our laboratory found that HTG and AD but not NTG patients had increased levels of procoagulant platelets that were reduced by inhibitors of innate immune toll like-receptor 4 (TLR4). The purpose of this study was to isolate biologically active TLR4.

Methods : Whole blood was obtained from control (n=4), HTG (n=4) and AD (n=4) subjects after institutional review board approval and written consent. Platelets were used as surrogate tissue sources for the study. Platelets were isolated by centrifugation, lysed by freeze-thaw, pre-cleared with magnetic beads alone followed by magnetic beads with anti-tubulin and subjected to magnetic pull-down using beads conjugated with a TLR4 antibody (Abcam). Platelet samples were challenged with graded amounts of TLR4 agonists, e.g. β-amyloid 1-42 (Anaspec). Samples were separated by electrophoresis in Coomassie blue native 3-12 gradient polyacrylamide gels. TLR4 was visualized by western blot and pull-down proteins were visualized with silver staining. TLR4 content was determined by western blot densitometry using recombinant TLR4 (R&D) as a standard and by flow cytometry.

Results : An estimate of TLR4 in platelet lysate was 0.30 ng/μg protein (western blot) and 0.03 ng/μg protein (flow cytometry). Analysis of the pull-down assay of TLR4 positive protein was ~20 ng/µg protein. Silver staining of the TLR4 pull-down from control subjects revealed several bands (~4 major and 2 minor bands). Notably, TLR4 was dimerized in the presence of agonists and the degree of dimerization determined by densitometry was higher in patients with HTG and AD (p=0.03).

Conclusions : A partially purified TLR4 complex was visualized in the TLR4 pull-down assay and neurodegenerative diseases exhibited increased TLR4 dimerization. The strategy of using blue native gel electrophoresis is to retain the biological activity of ligands bound to the TLR4 complex. Future studies will focus on the identity of ligands within the TLR4 complex. The goal is to fingerprint TLR4 ligands and activation status in POAG and AD in comparison to normal subjects. This unique fingerprint may identify the putative proinflammatory factors which cause HTG, AD, and related neurodegenerative diseases.

This is a 2020 ARVO Annual Meeting abstract.