Leukocyte Derived Microvesicles as Disease Progression Biomarkers in Slow Progressing Amyotrophic Lateral Sclerosis Patients

Abstract

The lack of biomarkers in Amyotrophic Lateral Sclerosis (ALS) makes it difficult to determine the stage of the disease in patients and, therefore, it delays therapeutic trials. Microvesicles (MVs) are possible biomarkers implicated in physiological and pathological functions, however, their role in ALS remains unclear. We investigated whether plasma derived microvesicles could be overrepresented in a group of 40 patients affected by ALS compared to 28 Alzheimer’s Disease (AD) patients and 36 healthy volunteers. Leukocyte derived MVs (LMVs) compared to endothelial, platelet, erythrocyte derived MVs, were mostly present in ALS patients compared to AD patients and healthy donors. Correlation analysis corrected for the presence of confounding variables (riluzole, age at onset, site of onset, gender) was tested between PRL (Progression Rate at the Last visit) and LMVs, and a statistically significant value was found (Pearson partial correlation r = 0.407, p = 0.006). We also investigated SOD1, TDP-43 intravesicular protein level in LMVs. Misfolded SOD1 was selectively transported by LMVs and its protein level was associated with the percentage of LMVs in slow progressing patients ( r = 0.545, p = 0.033). Our preliminary findings suggest that LMVs are upregulated in ALS patients and they can be considered possible markers of disease progression.

Different RNA profiles in plasma derived small and large extracellular vesicles of Neurodegenerative diseases patients

“…We previously demonstrated significant differences between LEVs and SEVs derived from plasma for dimension, markers, protein loading (see figure S1a, b, c, d) [35,40,55]. Cellular miRNAs and RNAs can selectively traffick into LEVs and SEVs, so we first identified differentially expressed miRNAs (DE miRNAs) and mRNAs (DE mRNA) in SEVs and LEVs among the four groups of patients (AD, PD, ALS, FTD) and the healthy controls (CTRs) (Table 3, Table 4, Table S1, Table S2).…”

“…Venous blood (15 ml) was collected in sodium citrate tubes from all patients and controls and processed as previously described [35,40]. Briefly, platelet-free plasma was centrifuged at 20,000xg for 1 hour.…”

“…The supernatant of LEVs was filtered through a 0.2 μm filter and spun in an Optima MAX-TL Ultracentrifuge at 100,000xg for 1 hour at 4°C and SEVS pellet was washed with 1 ml of filtered 1X PBS. Western Blot analysis for LEVs markers (Annexin V-Abcam, Inc., United States) and for SEVs markers (Alix-Abcam, Inc., United States), Transmission Electron Microscopy (TEM) and Nanoparticle-tracking analysis (NTA) were run to confirm LEVs and SEVs purity as we previously described [35,40].…”

Molecular and Cellular Mechanisms Affected in ALS

“…Extracellular vesicles encompass different types such as apoptotic vesicles, microvesicles, and exosomes, all of which can affect the functionality of the recipient cells [ 174 , 175 , 176 , 177 ]. The last decade has seen several investigations into the relevance of exosomes to ALS, either in propagating the disease or as biomarkers [ 6 , 178 , 179 , 180 , 181 ]. In the ALS context, exosomes secreted by astrocytes, neurons, or microglia are suspected to carry neurotoxic elements such as mutated SOD1 or C9orf72-derived DPR and to be responsible for motor neuron death [ 178 , 179 , 182 ].…”

Extracellular vesicles and amyotrophic lateral sclerosis: from misfolded protein vehicles to promising clinical biomarkers

“…Similarly, TDP-43 has been retrieved in brain-derived exosomes from CSF of ALS patients, possibly reflecting in vivo the underlying neuropathological findings [ 54 ]. Moreover, leukocyte-derived MVs have been found at elevated levels in ALS patients and have shown a slight correlation with progression rate at the last visit, possibly representing biomarkers of disease progression [ 83 ].…”