Different approaches to biomarker research for ALS: in search of novel neuronal markers associated with extracellular vesicles

Amyotrophic lateral sclerosis (ALS) is a fatal, complex, neurodegenerative disease characterized by the progressive loss of motor neurons. Its highly heterogeneous clinical presentation and underlying molecular biology make it difficult to diagnose the disease and manage patients effectively. The development of new drugs is hindered by the lack of preclinical models representing sporadic forms of ALS, which account for 90% of total cases. Currently, biomarker research for ALS is actively seeking new indicators to aid in diagnosis and prognosis, study target engagement, and facilitate participation in clinical research. Of all the proposed biomarkers, neurofilament light chain (NfL) is the most promising, having been demonstrated to be useful as both a diagnostic and a prognostic and pharmacodynamic biomarker. In this thesis, various strategies were implemented to study biofluids collected from ALS patients, aiming to identify protein-level alterations that may suggest pathways deregulated in disease conditions. First, plasma samples from ALS patients and controls were analyzed using the Olink platform. Four proteins were found to be differentially upregulated. Aside from the well-known neuronal damage marker NfL, the other three proteins are involved in inflammatory processes. This emphasizes the presence of inflammation in ALS. Next, to identify a signature related to changes in the CNS, we analyzed CSF samples from ALS patients at diagnosis, as well as from patients with Alzheimer’s disease and controls. This aimed to identify early indicators of disease. We employed MACSPlex technology to examine the alterations in markers present on the surface of extracellular vesicles (EVs). EVs are emerging as a powerful tool for studying neurodegenerative diseases. These naturally released membrane-bound nanoparticles consist of proteins, lipids, and nucleic acids and are produced by all cell types. Their main role is to mediate intercellular communication; they are released into the extracellular space and are found in all types of biofluids. We proposed that ALS is associated with a significant reduction in the oligodendrocytic marker O4 and an increase in the glycoprotein CD47. Furthermore, we conducted an in-depth characterization of novel markers linked to neuron-derived EVs. Neuronal EV markers in circulation may reflect initial damage to the blood-brain barrier. Moreover, they can help isolate neuronal-specific EVs for cargo analysis or further study. We isolated EVs through differential ultracentrifugation from primary cultures of cortical neurons and analyzed their proteomic content, specifically searching for transmembrane proteins enriched in central nervous system tissues. After having identified two candidates, we validated their neuronal specificity in mouse tissues, including brain and spinal cord EVs, and in iPSC-derived neurons. To enhance detection sensitivity, we used a novel single-molecule array (SiMoA) for EV analysis. We confirmed that SiMoA technology is feasible for detecting our candidate proteins in iPSC-derived EVs and verified their presence in biofluids. Preliminary results showed an increase in neuron-derived vesicles in the CSF of ALS patients, suggesting its potential as a new biomarker for future validation.