To investigate disease biomarkers in ALS.

ALS is a neurodegenerative disease in which progressive muscle weakness occurs through the loss of upper and lower motor neurons. Bulbar ALS with symptom onset in the head and neck and non-bulbar ALS with spinal or limb onset are the two most common subtypes of ALS. Biomarkers in ALS have been intensely researched, but focus has been on neuronal biomarkers rather than primary pathology related to ALS. Our aim was to discover other novel biomarkers which may reflect the most common pathology of ALS through an unbiased proteomic analysis.

A total of 401 plasma samples were used, which originated from two independent cohorts (commercial and baseline samples from Phase 2 ALS clinical trial- NCT05740813) to investigate protein expression differences between ALS patients (n = 321) and healthy controls (n = 80). We performed proteomic analysis using the Olink platform. Preranked gene set enrichment analysis (GSEA) was used to determine significantly enriched pathways. Protein-protein interaction (PPI) plots were created to represent high confidence interactions between proteins.

Muscle-related proteins, such as MYBPC1 and MYOM3 were significantly upregulated in ALS versus healthy controls. GSEA of the differentially abundant proteins showed an enrichment of several different muscle-related pathways in ALS. In addition, muscle-related pathways were significantly upregulated in non-bulbar compared to bulbar onset ALS. PPI of muscle proteins demonstrated a highly connected network of muscle proteins in which the top four functional enrichments for prominent clusters were in myofibril and contractile fibers which are responsible for muscle contraction and relaxation.

The identification of these novel muscle-related changes allows us to build a broader biomarker strategy in clinical trials in patients with ALS by highlighting biomarkers of muscle function in addition to neuronal. These novel muscle-related changes can potentially also inform other disease indications which involve muscle loss.