Neuroinflammation-Related Proteins NOD2 and Spp1 Are Abnormally Upregulated in Amyotrophic Lateral Sclerosis

Abstract

Background and objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology and poorly understood pathophysiology. There is no specific biomarker either for diagnosis or prognosis. The aim of our study was to investigate differentially expressed proteins in the CSF and serum from patients with ALS to determine their role in the disease process and evaluate their utility as diagnostic or prognostic biomarkers.

Methods: We performed mass spectrometry in the CSF from 3 patients with ALS and 3 healthy controls (HCs). The results were compared with motor cortex dysregulated transcripts obtained from 11patients with sporadic ALS and 8 HCs. Candidate proteins were tested using ELISA in the serum of 123 patients with ALS, 30 patients with Alzheimer disease (AD), 28 patients with frontotemporal dementia (FTD), and 102 HCs. Patients with ALS, AD, and FTD were prospectively recruited from January 2003 to December 2020. A group of age-matched HCs was randomly selected from the Sant Pau Initiative on Neurodegeneration cohort of the Sant Pau Memory Unit.

Results: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and osteopontin (Spp1) were differentially expressed in the CSF and the motor cortex transcriptome of patients with ALS compared with that in HCs (p < 0.05). NOD2 and Spp1 levels were significantly higher in sera from patients with ALS than in HCs (p < 0.001). Receiver operating characteristic analysis showed an area under the curve of 0.63 for NOD2 and 0.81 for Spp1. NOD2 levels were significantly lower in patients with AD and FTD than in patients with ALS (p < 0.0001), but we found no significant differences in Spp1 levels between patients with ALS, AD (p = 0.51), and FTD (p = 0.42). We found a negative correlation between Spp1 levels and ALS functional rating scale (r = -0.24, p = 0.009).

Discussion: Our discovery-based approach identified NOD2 as a novel biomarker in ALS and adds evidence to the contribution of Spp1 in the disease process. Both proteins are involved in innate immunity and autophagy and are increased in the serum from patients with ALS. Our data support a relevant role of neuroinflammation in the pathophysiology of the disease and may identify targets for disease-modifying treatments in ALS. Further longitudinal studies should investigate the diagnostic and prognostic value of NOD2 and Spp1 in clinical practice.

Figure 1. Box Plot Showing the Normalized RNA Expression Values of the 4 Genes Encoding the Prioritized Proteins in the Motor Cortex of Patients With Sporadic ALS and Healthy Controls Obtained Through RNA Sequencing

*p < 0.05; **p < 0.01 ***p < 0.001. sALS = sporadic amyotrophic lateral sclerosis.

Figure 2. Analysis of Diagnostic Utility of Serum Oligomerization Domain–Containing Protein 2 (NOD2) and Osteopontin (Spp1) Through Receiver Operating Characteristic (ROC) Curves

Sensitivity and specificity of serum (A) oligomerization domain–containing protein 2 (NOD2) and (B) osteopontin (Spp1) levels in patients with ALS and healthy controls. ALS = amyotrophic lateral sclerosis; HC = healthy control.

Figure 3. Levels of Nucleotide-Binding Oligomerization Domain–Containing Protein 2 (NOD2) and Osteopontin (Spp1) Across Diagnostic Groups (Controls, n = 102; ALS, n = 123; AD, n = 30; and FTD, n = 28)

Group differences between serum biomarker raw levels of (A) NOD2 and (B) Spp1. Error bars represent the 10th to 90th percentile range of data, and circles represent outliers. *<0.001. AD = Alzheimer disease; ALS = amyotrophic lateral sclerosis; FTD = frontotemporal dementia; HC = healthy control.

Figure 4. Correlation Between the Raw Osteopontin (Spp1) Levels and the ALSFRS-R Score at the Moment of Sample Extraction in the ALS Group

ALS = amyotrophic lateral sclerosis; ALSFRS-R = Revised ALS functional rating scale.