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.

Keywords: SOD1; TDP-43; amyotrophic lateral sclerosis; biomarkers; disease progression; microvesicles.

FIGURE 1

LMVs overrepresentation in plasma from ALS patients. Flow cytometry dot plots of MVs isolated from plasma of a representative healthy control, an ALS patient and AD patient labeled with Annexin V and CD45 (A–C). While healthy control (A) and AD patient (C) presented few events similar to isotype control, ALS patient (B) had higher % CD45+/Annexin V+ MVs. Dot plots of MVs isolated from plasma of ALS patients (red), healthy controls (green), and AD patients (blue) labeled with Annexin V and CD45 (D), CD31 (E), CD235A (F), CD61 (G) markers. Results were referred as the percentage of Annexin V⁺ MVs expressing the cell lineage marker. While Annexin V+ MVs derived from endothelium (%CD31 MVs) (E), erythrocytes (%CD235a MVs) (F), platelets (%CD61 MVs) (G) were not different from the healthy control subjects and AD patients (ANOVA test, p > 0.05), the percentage of Annexin V+ MVs derived from leukocytes (%CD45 MVs) (D) was significantly enhanced in the plasma of ALS patients compared to the control group (ANOVA test, ^∗∗∗p < 0.001) and the AD patients (ANOVA test, ^∗∗p < 0.01). n CTRL = 36; n ALS = 40; n AD = 28.

FIGURE 2

CD45+/Annexin V+ MVs is the most informative feature to discriminate ALS group from AD patients and healthy matched control groups. Boxplot of percentage of marker positive MVs are shown for ALS, AD, and control samples: (A) CD45+/Annexin V+; (B) CD235a+/Annexin V+; (C) CD31+/Annexin V+; (D) CD61+/Annexin V+; (E) ROC curve for logistic regression with an AUC of 0.717 and an accuracy of 0.653.

FIGURE 3

Misfolded SOD1 enrichment in LMVs (CD45+ MVs) from ALS patients and controls. CD45+ MVs of patients (ALS 1 and ALS 2) and of CTRLs (CTRL1) were enriched with misfolded SOD1. Misfolded SOD1 was only found in LMVs. The cropped blots are used in the figure and full length blots are presented in Supplementary Figure S2. The immunodepleted (I-) fraction, which include all CD45 negative MVs from other cell origin, showed no or very little level of misfolded SOD1. TDP-43 was transported in its phosphorylated form by MVs of the IP and immunodepleted (I-) (A). ALS 1 and ALS 2 are referred to two slow progressing patients. Input = MVs whole lysate-20% of the IP; IP = CD45 immunoprecipitated MVs (LMVs); I- = immunodepleted. (B) Densitometric analysis of misfolded SOD1, normalized to CD45 band, revealed slight increase of misfolded SOD1 in all ALS patients compared to controls (0.8431 ± 0.1236, CTRL n = 10; 1.008 ± 0.1274, ALS n = 18).

FIGURE 4

LMVs percentage in ALS patients is correlated to progression rate at last visit and is correlated to misfolded SOD1 protein level in slow progressing ALS patients. The log of PRL and of LMVs was plotted and the line was drawn from the least squares regression. A significant correlation was found between LMVs and PRL (Pearson partial correlation r = 0.409, p = 0.006) (A). The PRL of slow progressing patients was positively correlated to LMVs levels (% CD45 MVs) (Pearson partial correlation r = 0.432, p = 0.022) (B). On the other hand, there was no correlation between LMVs levels and disease progression in fast progressing patients (Pearson partial correlation, r = –0.0037, p = 0.496) (C). CD45+ MVs percentage (%) was directly correlated with misfolded SOD1. Densitometric analysis of misfolded SOD1 was correlated to the percentage of CD45 Annexin V (%CD45 MVs) by Spearman rank analysis. LMVs level was not associated to the densitometric level of misfolded SOD1 in ALS patients (Spearman test, r = 0.288, p = 0.123) (D). We instead found a strong correlation between misfolded SOD1 protein level and LMVs in slow progressing patients (r = 0.545, p = 0.033) (n = 12) (E). No statistically significant difference was found in fast progressing patients (r = –0.371, p = 0.234) (n = 6) (F).