Network Analysis of the Cerebrospinal Fluid Proteome Reveals Shared and Unique Differences Between Sporadic and Familial Forms of Amyotrophic Lateral Sclerosis

Background: Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease involving loss of motor neurons, typically results in death within 3-5 years of disease onset. Although roughly 10 % of cases can be linked to a specific inherited mutation (e.g., C9orf72 hexanucleotide repeat expansion or SOD1 mutation), the cause of the majority of cases is unknown. Consequently, there is a critical need for biomarkers that reflect disease onset and progression across ALS subgroups. Methods: We employed tandem mass tag mass spectrometry (TMT-MS) based proteomics on cerebrospinal fluid (CSF) to identify and quantify 2105 proteins from ALS patients with sporadic disease (n=35), C9orf72 ALS (n=10), and SOD1 ALS (n=6), as well as age-matched healthy controls (n=44) and asymptomatic C9orf72 carriers (n=6). We used differential protein abundance and network analyses to determine how protein profiles vary across disease types in ALS CSF. Results: Integrated differential and co-expression network analysis identified proteomic differences between ALS and control, and differentially abundant proteins between sporadic, C9orf72 and SOD1 ALS. Groups of proteins also differentiated asymptomatic C9orf72 mutation carriers from those with C9orf72 ALS, marking a pre-symptomatic proteomic signature of C9orf72 ALS. Similarly, additional proteins differentiated asymptomatic from controls. Leveraging additional publicly available ALS and AD proteomic datasets, we validated our ALS CSF network and identified ALS-specific proteins within Module 5 (M5)-Extracellular matrix (e.g., IGF2, RARRES2, LGALS3, GALNT15, and LYZ) and shared biomarkers across neurodegenerative diseases linked to Module 10 (M10)-Ubiquitination/Gluconeogenesis (e.g., NEFL, NEFM, CHIT1, and CHI3L1). Conclusions: This study represents a comprehensive analysis of the CSF proteome across sporadic and genetic causes of ALS that resolves differences among these disease subgroups and points to varying pathogenic pathways that result in disease. ### Competing Interest Statement N.T.S, A.I.L and D.M.D. are co-founders and consultants of Emtherapro. D.M.D. and N.T.S are co-founders of Arc Proteomics.