Novel insights into RAGE signaling pathways during the progression of amyotrophic lateral sclerosis in RAGE-deficient SOD1 G93A mice

Abstract

Amyotrophic lateral sclerosis (ALS) is neurodegenerative disease characterized by a progressive loss of motor neurons resulting in paralysis and muscle atrophy. One of the most prospective hypothesis on the ALS pathogenesis suggests that excessive inflammation and advanced glycation end-products (AGEs) accumulation play a crucial role in the development of ALS in patients and SOD1 G93A mice. Hence, we may speculate that RAGE, receptor for advanced glycation end-products and its proinflammatory ligands such as: HMGB1, S100B and CML contribute to ALS pathogenesis. The aim of our studies was to decipher the role of RAGE as well as provide insight into RAGE signaling pathways during the progression of ALS in SOD1 G93A and RAGE-deficient SOD1 G93A mice. In our study, we observed alternations in molecular pattern of proinflammatory RAGE ligands during progression of disease in RAGE KO SOD1 G93A mice compared to SOD1 G93A mice. Moreover, we observed that the amount of beta actin (ACTB) as well as Glial fibrillary acidic protein (GFAP) was elevated in SOD1 G93A mice when compared to mice with deletion of RAGE. These data contributes to our understanding of implications of RAGE and its ligands in pathogenesis of ALS and highlight potential targeted therapeutic interventions at the early stage of this devastating disease. Moreover, inhibition of the molecular cross-talk between RAGE and its proinflammatory ligands may abolish neuroinflammation, gliosis and motor neuron damage in SOD1 G93A mice. Hence, we hypothesize that attenuated interaction of RAGE with its proinflammatory ligands may improve well-being and health status during ALS in SOD1 G93A mice. Therefore, we emphasize that the inhibition of RAGE signaling pathway may be a therapeutic target for neurodegenerative diseases.

Fig 1. Schematic diagram.

The diagram demonstrating subsequent stages of the experiment and showing the area of the spinal cord being examined; for the purpose of the study we examined lumbar motor spinal cord ventral horn lamina. The red line indicates the spinal cord. The figure is created by ourselves. Figure may be similar but not identical to the original image and is therefore for illustrative purposes only.

Fig 2

The relative expression of HMGB1 (A), S100B (B) and RHOJ (C) mRNAs in the lumbar spinal cord harvested from SOD1 transgenic mice with congenital, ALS RAGE knockout SOD1 transgenic mice with congenital ALS, and wild type mice at 90 days’ time points. Data are presented as means ± S.E.M. relative to the geometric mean of the expression level of IPO8 and 18SrRNA. Abbreviation: WT–wild type group, n = 6; SOD1 group, n = 6; RAGE KO SOD1, n = 5.

Fig 3

The relative amount of proteins: HMGB1 (A), S100B (B), CML (C), ACTB (D) in the lumbar spinal cord harvested from SOD1 transgenic mice with congenital ALS, RAGE knockout SOD1 transgenic mice with congenital ALS, and wild type mice at terminal time point quantified by western blot. Figure panels are indicated above the corresponding western blot set. Data are presented as means ± S.E.M. There are no statistical differences in relative amount of proteins. Abbreviation: WT–wild type group, n = 3; SOD1 transgenic mice with congenital ALS, n = 3; RAGE KO SOD1 –RAGE knockout SOD1 transgenic mice with congenital ALS, n = 4, at terminal endpoints.

Fig 4

The relative amount of proteins: HMGB1 (A), S100B(B), CML(C), ACTB (D) in the lumbar spinal cord harvested from SOD1 transgenic mice with congenital ALS, RAGE knockout SOD1 transgenic mice with congenital ALS, and wild type mice at 90 and terminal time points quantified by western blot. Figure panels are indicated above the corresponding western blot set. Data are presented as means ± S.E.M. Statistical differences in relative amount of protein respectively * 0.01 ≤ P ≤ 0.05; ** 0.001 ≤ P≤ 0.01. Abbreviation: RAGE KO SOD1 90 –RAGE knockout SOD1 transgenic mice with congenital ALS n = 4 at 90 time point, RAGE KO SOD1 T–RAGE knock out SOD1 transgenic mice with congenital ALS n = 4, at terminal endpoints.

Fig 5. Neuronal count and GFAP as well as NeuN expression in terminal-stage SOD1 transgenic mouse ventral horn of spinal cord.

A. DAPI positive nuclei (blue), GFAP protein showed (green), NeuN neurons showed (red). Co-localization (yellow staining) of GFAP-NeuN in mice spinal cord. Images were taken under 20× objective with 0.7 numerical aperture (20× /0.7). Scale bar  =  20 μm. For the purpose of the study we examined lumbar motor spinal cord ventral horn lamina. B. Effect of RAGE knockout on nuclear changes and assess apoptosis in ALS mice. Arrows indicated alternations in nuclei. C. Genetic deletion of RAGE effects on glial marker (GFAP protein, green colour) in the spinal cord of SOD1 mice. D. SOD1 mice lacking RAGE have the same level of NeuN in spinal cord (motor neurons–red). Statistical differences in relative amount of protein respectively * 0.01 ≤ P ≤ 0.05; ** 0.001 ≤ P≤ 0.01.

Fig 6. Summary of the study.

Genetic deletion of RAGE reduces glial marker and major inflammatory proteins in the spinal cord of SOD1 G93A mice. The figure is created by ourselves. Figure may be similar but not identical to the original image and is therefore for illustrative purposes only.