Proteomic analysis of human serum Extracellular Vesicles reveals early diagnostic markers for Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by the deposition of misfolded proteins leading to the death of motor neurons. Several ALS-associated proteins, including TAR DNA-binding protein 43 (TDP-43) and Superoxide dismutase-1 (SOD-1), have been linked to small extracellular vesicles (EVs). However, the role of these EVs and their cargo in ALS patients, prior to treatment intervention, has not been investigated. This study aims to identify the earliest protein changes facilitated by EVs in ALS by examining the serum of recently diagnosed ALS patients. EVs were isolated from the serum of ALS (n = 25) and healthy control (HC, n = 9) patients before undergoing proteomics analysis. This resulted in the identification of a panel of 9 significantly up-regulated proteins and included haptoglobin and hemoglobin subunits, complement, and afamin, which are involved in pathways including heme homeostasis and autophagy. The identification of haptoglobin in ALS serum EVs suggests it has potential as an early diagnostic biomarker whilst activation of autophagy pathways suggests early recruitment of clearance pathways in ALS. This study uncovers the processes and proteins facilitated through small EVs in the initial stages of ALS. Proteomics data are available via ProteomeXchange with identifier PXD036652. Statement of significance of the study The role of small EVs, which are involved in cell-to-cell communication, and their cargo in the initiation of ALS has not been investigated. This study is the first to identify the earliest protein changes occurring in ALS through small EV facilitation. This study examined serum from newly diagnosed ALS patients, prior to treatment intervention. Therefore, the EVs, isolated from ALS and healthy control patients, captured novel ALS associated changes without confoundment from medication, which could mask early changes. A panel of 9 statistically up-regulated proteins was identified after mass spectrometry analysis. These included: haptoglobin and hemoglobin subunits, complement, and afamin. The identification of up-regulated levels of these proteins in the ALS serum EVs suggests they have potential as diagnostic biomarkers whilst identifying pathways including chaperone mediated autophagy (CMA) and microautophagy suggests early recruitment of clearance pathways in ALS. Therefore, this study uncovered the proteins being facilitated through small EVs in the initial stages of ALS. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the National Health and Medical Research Council (GNT1041413 to A.F.H.) and the MND Foundation, Australia (A.F.H.). N.V. is supported by an Australian Postgraduate Scholarship. Motor Neuron Disease Foundation Australia and the National Health and Medical Research Council. Allocations from an Ian Potter Foundation grant (#31110299) and an ARC LIEF scheme grant (LE200100117) contributed to the purchase of the Thermo Eclipse mass spectrometer. P.L., E.B., A.F., and I.Q. were supported by the Association pour la Recherche sur la SLA (ARSLA). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Human ethics committee of Lyon University Hospital, Lyon , France gave ethical approval for this work (Human ethics: HCL.2016.710). Human ethics committee of La Trobe University, Melbourne, Australia gave ethical approval for this work (Human ethics: HEC17090). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Proteomics data are available via ProteomeXchange with identifier PXD036652. * (ALS) : Amyotrophic Lateral Sclerosis (EVs) : Extracellular Vesicles (TDP-43) : TAR DNA-binding protein 43 (SOD1) : Superoxide Dismutase 1 (HC) : Healthy control (MVB) : Multivesicular body (BCA) : Bicinchoninic acid (SDS) : Sodium dodecyl sulphate (TFA) : Trifluoroacetic acid (ACN) : Acetonitrile (GO) : Gene ontology (MAC) : Membrane attack complex (CMA) : Chaperone mediated autophagy (Lamp2A) : Lysosomal associated membrane protein 2A (FTD) : Frontotemporal Dementia