Lysosomes Dysfunction Causes Mitophagy Impairment in PBMCs of Sporadic ALS Patients

Abstract

Mitochondria alterations are present in tissues derived from patients and animal models, but no data are available for peripheral blood mononuclear cells (PBMCs) of ALS patients. This work aims to investigate mitophagy in PBMCs of sporadic (sALS) patients and how this pathway can be tuned by using small molecules. We found the presence of morphologically atypical mitochondria by TEM and morphological abnormalities by MitoTracker™. We found a decreased number of healthy mitochondria in sALS PBMCs and an impairment of mitophagy with western blot and immunofluorescence. After rapamycin treatment, we found a higher increase in the LC3 marker in sALS PBMCs, while after NH4Cl treatment, we found a lower increase in the LC3 marker. Finally, mTOR-independent autophagy induction with trehalose resulted in a significant decrease in the lysosomes level sALS PBMCs. Our data suggest that the presence of morphologically altered mitochondria and an inefficient turnover of damaged mitochondria in PBMCs of sALS patients rely on the impairment of the mitophagy pathway. We also found that the induction of the mTOR-independent autophagy pathway leads to a decrease in lysosomes level, suggesting a more sensitivity of sALS PBMCs to trehalose. Such evidence suggests that trehalose could represent an effective treatment for ALS patients.

Keywords: ALS; PBMCs; autolysosomes; mTOR; mitochondria; trehalose.

Figure 1

PBMCs of sALS patients show alteration in mitochondrial structural morphology. (a) TEM analysis of mitochondrial structural morphology: yellow arrows indicate mitochondria with deformed cristae, while red arrows indicate degenerated mitochondria; scale bar: 250 nm. N = 3 (b) Measure of the mean area of mitochondria (ImageJ software), in sALS patients’ mitochondria are larger with respect to controls (** p < 0.01). N = 3 (c) Measure of the long mitochondrial axis (nm). N = 3 (d) Analysis of mitochondrial spatial organization by immunofluorescence using MitoTracker™ Red Blue: DAPI, Red: MitoTracker™. Scale bar: 5 µm. N = 3 (e) Analysis of clusterization. Data are reported as mean (* p < 0.05) of the percentage of number/size in sALS PBMCs respect to controls. N = 3 (f,g) Flow Cytometry analysis of the ratio between healthy and damaged mitochondria. Data are reported as the mean (* p < 0.05) of the percentage of healthy mitochondria in sALS PBMCs with respect to controls. MitoTracker™ marks all mitochondria, while TMRE marks the healthy ones. N = 12.

Figure 2

Mitophagy pathway increases in PBMCs of sALS patients. (a) In PBMCs of sALS patients, we observed a statistically significant increase in PINK1 expression levels (** p < 0.01). N = 32 (b) In PBMCs of sALS patients we observed a statistically significant increase in LC3-II/LC3-I ratio (** p < 0.01). N = 32 (c) In PBMCs of sALS patients we observed a statistically significant increase in LC3-II expression levels (** p < 0.01). We used GAPDH as a loading control. N = 32 (d,e) Analysis of the co-localization (merge) between LC3 and PINK1 in PBMCs of healthy controls and sALS patients by confocal microscopy analysis. We found an increased co-localization in sALS PBMCs (* p < 0.05). Red: LC3, Green: PINK1. Magnification: 63X, scale bar: 5 µm. Zoom: X4, scale bar 1 µm. N = 3.

Figure 3

Inhibition of fusion between autophagosomes and lysosomes. (a) Immunofluorescence analysis with LysoTracker™ marker revealed an accumulation of lysosomes in sALS patients’ PBMCs. We found a statistical increase in the number of puncta in sALS compared to control (* p < 0.05). Scale bar: 20 µm. N = 3 (b) Immunofluorescence analysis with LC3 and LysoTracker™ revealed that the fusion between lysosomes and autophagosomes is inhibited. Green: LysoTracker™; Red: LC3. Scale bar: 10 µm. N = 3 (c) WB analysis revealed no statistically significant differences between levels of Beclin-1 in sALS patients and control. GAPDH was used as a loading control. N = 32 (d) Immunofluorescence analysis with ER-Tracker™ revealed that mitochondria (Red) and ER (green) do not co-localize. Scale bar: 10 µm. N = 3.

Figure 4

Dissection of autolysosomes generation sALS PBMCs vs. Controls. (a) Immunofluorescence analysis with LC3 revealed an accumulation of autophagosomes in sALS patients’ PBMCs in NT conditions. After rapamycin treatment we found an increased accumulation of LC3 in sALS PBMCs compared to controls. On the other hand, we found a higher increase in LC3 after NH4Cl treatment in healthy controls respect to sALS patients. Scale bar 20X: 20 µm, Scale bar 40X: 10 µm. N = 3 (b) ImageJ analysis of LC3 after rapamycin treatment. We found statistically significant differences between Ctrl NT and sALS NT (* p < 0.05), Ctrl NT and Ctrl RAPA, sALS NT and sALS RAPA (*** p < 0.001), and Ctrl RAPA and sALS RAPA (**** p < 0.0001). (c) ImageJ analysis of LC3 after NH4Cl treatment. We found statistically significant differences between Ctrl NT and sALS NT, Ctrl NH4Cl and sALS NH4Cl (* p < 0.05), Ctrl NT and Ctrl NH4Cl (**** p < 0.0001), and sALS NT and sALS NH4Cl (*** p < 0.001). (d) ImageJ analysis of LC3 after trehalose treatment. We found statistically significant differences between Ctrl NT and sALS NT (* p < 0.05), Ctrl TREH and sALS TREH (** p < 0.01), Ctrl NT and Ctrl TREH, and sALS NT and sALS TREH (*** p < 0.001).

Figure 5

Trehalose proves to help to remove accumulated lysosomes. (a) Immunofluorescence analysis of LysoTracker™ in sALS patients and healthy controls treated and non-treated with NH4Cl or Tehalose; Scale bar: 20 µm. N = 3 (b) ImageJ quantification of LysoTracker™ puncta. We found statistically significant differences between Ctrl NT and sALS NT, CTRL NT and CTRL NH4Cl (* p < 0.05) and Ctrl NH4Cl and sALS NH4Cl (**** p < 0.0001). (c) ImageJ analysis of LysoTracker™ puncta. We found statistically significant differences between Ctrl NT and sALS NT (* p < 0.05), CTRL TREH and SALS TREH (** p < 0.01) SALS NT and sALS TREH (**** p < 0.0001).