Decoding distinctive features of plasma extracellular vesicles in amyotrophic lateral sclerosis

Abstract

Abstract Background Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem motor neuron disease for which currently there is no effective treatment. There is an urgent need to identify biomarkers to tackle the disease’s complexity and help in early diagnosis, prognosis, and therapy. Extracellular vesicles (EVs) are nanostructures released by any cell type into body fluids. Their biophysical and biochemical characteristics vary with the parent cell’s physiological and pathological state and make them an attractive source of multidimensional data for patient classification and stratification. Methods We analyzed plasma-derived EVs of ALS patients (n = 106) and controls (n = 96), and SOD1G93A and TDP-43Q331K mouse models of ALS. We purified plasma EVs by nickel-based isolation, characterized their EV size distribution and morphology respectively by nanotracking analysis and transmission electron microscopy, and analyzed EV markers and protein cargos by Western blot and proteomics. We used machine learning techniques to predict diagnosis and prognosis. Results Our procedure resulted in high-yield isolation of intact and polydisperse plasma EVs, with minimal lipoprotein contamination. EVs in the plasma of ALS patients and the two mouse models of ALS had a distinctive size distribution and lower HSP90 levels compared to the controls. In terms of disease progression, the levels of cyclophilin A with the EV size distribution distinguished fast and slow disease progressors, a possibly new means for patient stratification. Immuno-electron microscopy also suggested that phosphorylated TDP-43 is not an intravesicular cargo of plasma-derived EVs. Conclusions Our analysis unmasked features in plasma EVs of ALS patients with potential straightforward clinical application. We conceived an innovative mathematical model based on machine learning which, by integrating EV size distribution data with protein cargoes, gave very high prediction rates for disease diagnosis and prognosis.

Keywords

• Extracellular vesicles
• HSP90
• PPIA
• Phosphorylated TDP-43
• Biomarkers
• Machine learning