Receptor for Advanced Glycation End Products and its Inflammatory Ligands are Upregulated in Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder of largely unknown pathogenesis. Recent studies suggest that enhanced oxidative stress and neuroinflammation contribute to the progression of the disease. Mounting evidence implicates the receptor for advanced glycation end-products (RAGE) as a significant contributor to the pathogenesis of certain neurodegenerative diseases and chronic conditions. It is hypothesized that detrimental actions of RAGE are triggered upon binding to its ligands, such as AGEs (advanced glycation end products), S100/calgranulin family members, and High Mobility Group Box-1 (HMGB1) proteins. Here, we examined the expression of RAGE and its ligands in human ALS spinal cord. Tissue samples from age-matched human control and ALS spinal cords were tested for the expression of RAGE, carboxymethyllysine (CML) AGE, S100B, and HMGB1, and intensity of the immunofluorescent and immunoblotting signals was assessed. We found that the expression of both RAGE and its ligands was significantly increased in the spinal cords of ALS patients versus age-matched control subjects. Our study is the first report describing co-expression of both RAGE and its ligands in human ALS spinal cords. These findings suggest that further probing of RAGE as a mechanism of neurodegeneration in human ALS is rational.

Keywords: CML; HMGB1; RAGE; S100B; amyotrophic lateral sclerosis; spinal cord.

FIGURE 1

RAGE expression is increased in human ALS spinal cord. (A) Thoracic spinal cord sections were immunostained for RAGE expression from control (left) or ALS (right) subjects. (B) Quantification of RAGE immunofluorescent staining intensity in spinal cord tissue. Representative images from each group are shown; control (n = 6) vs. ALS samples (n = 5). (C) mRNA expression levels for AGER were determined by quantitative RT-PCR (n = 3 samples/group). (D) Magnified images of RAGE immunostaining in ALS and control thoracic spinal cord, motor ventral horn. Clear staining of motor neuron and surrounding area is visible in the ALS sample as compared to control. Error bars represent mean ± SEM, ^∗p < 0.05. Scale bar: 100 μm.

FIGURE 2

Expression of RAGE and its ligands in control and ALS thoracic spinal cord tissue. (A) RAGE expression in control (A, top), and in ALS tissue (A, bottom). (B) S100B immunostaining in control tissue (B, top) and in ALS tissue (B, bottom). (C) HMGB1 immunostaining in the control tissue (C, top) and in ALS tissue (C, bottom). (D) CML immunostaining in control spinal cord (D, top) and in ALS spinal cord (D, bottom). (E–G) Quantification of immunostaining intensity revealed that expression of all studied proteins was significantly increased in ALS thoracic spinal cord tissue compared to controls. S100B (E) was increased about 70%, HMGB1 (F) displayed almost threefold increase and CML (G) showed almost double level of increase in immunostaining between ALS and control subjects. Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Error bars represent mean ± SEM, ^∗p < 0.05. Scale bar: 50 μm.

FIGURE 3

High magnification images of immunostaining for RAGE and its ligands in the thoracic spinal cord. Increased immunostaining pattern on the border of gray (lamina IX) and white matter was observed for (A) RAGE, (B) S100B, (C) HMGB1 and (D) CML in ALS versus control samples. Scale bar: 50 μm.

FIGURE 4

Co-expression of RAGE and RAGE ligands S100B, CML, and HMGB1 is higher in human ALS spinal cord. (A) Triple staining for RAGE (red), S100B (green), CML (blue) revealed increased immunoexpression of these proteins in the ALS spinal cord (A, right) as compared to controls (A, left) and a high degree of RAGE/ligand overlapping was observed in ALS samples (merged images). (B) Expression of RAGE (red) and its ligands, S100B (green) and HMGB1 (blue) was highly increased in the ALS (B, right) spinal cord as compared to controls (B, left) and a high degree of RAGE/ligand co-expression observed in ALS samples (merged images); control (n = 6) vs. ALS samples (n = 5). Scale bar: 100 μm. (C) A schematic diagram showing different regions of spinal cord; for the purpose of the study we examined thoracic motor spinal cord ventral horn lamina IX and surrounding white matter.

FIGURE 5

High magnification images of white/gray matter showing triple staining for RAGE and its ligands S100B, CML, and HMGB1. Immunostaining for RAGE (red) and its ligands S100B (green) and CML or HMGB1 (blue) revealed low immunoexpression in control tissue (A and C) and high immunoexpression in ALS tissue (B and D). Sections are representative of n = 6 control and n = 5 ALS tissue samples per condition. Scale bar: 100 μm.

FIGURE 6

Protein levels of RAGE and its ligands S100B and HMGB1 are higher in human ALS spinal cord. Western blot analysis of RAGE (A), RAGE ligands S100B (B) and HMGB1 (C) in control and ALS spinal cord tissue. The original blots were stripped followed by incubation with the other antigens under study. Signal for test antigen was then normalized to β-actin and the relative band densities were reported. n = 3 subjects/group. Error bars represent mean ± SEM, ^∗p < 0.05.

FIGURE 7

A proposed mechanism of RAGE action in ALS spinal cord. We propose that during pathological processes in ALS, neuronal and microglial RAGE becomes activated by RAGE ligands such as AGEs, S100B, and HMGB1. Once activated, RAGE triggers a cascade of metabolic changes, contributing to the release of reactive oxygen species (ROS) and inflammatory cytokines, subsequently resulting in altered protein structures and misfolded protein accumulation, impaired mitochondrial function and growing energy deficits ultimately leading to neuronal dysfunction and apoptosis.