Fig 2: Within area penumbra cells, lysosomal-associated membrane protein 1 (LAMP1) molecules are reduced and dissipated from vacuoles. Graph (A) reports a reduced amount of LAMP1 molecules counted in the cytosol of neurons from the area penumbra (Ipsi/Ischemia) compared with the amount measured within neurons from homologous regions of the contralateral side (Contra/Ischemia) and from sham-operated mice (Sham). Graph (B) reports a massive decrease in the amount LAMP1 molecules counted within vacuoles of neurons from the area penumbra (Ipsi/Ischemia) compared with the amount measured within vacuoles from neurons from homologous regions of the contralateral side (Contra/Ischemia) and sham-operated mice (Sham). Graph (C) reports a decrease in the ratio between vacuolar and cytosolic amount of LAMP1 molecules within neurons from the area penumbra (Ipsi/Ischemia) compared with the ratio measured within neurons from homologous regions of the contralateral side (Contra/Ischemia) and from sham-operated mice (Sham). Graph (D) reports the decrease in LAMP1-positive vacuoles, which are counted within neurons from the area penumbra (Ipsi/Ischemia) compared with the amount measured within neurons from homologous regions of the contralateral side (Contra/Ischemia) and sham-operated mice (Sham). Values are reported as the mean ± S.E.M. per cell counted in 120 cells for each group. * p ≤ 0.05 compared with other groups.

Fig 3: Compartmentalization of specific autophagy molecules in physiological conditions (A) and within the area penumbra neurons (B). In detail, in control conditions (A), LC3 (1), which stains the autophagosomes, co-localizes with HSP70 (2) within the same vacuoles. In ischemia (B), there is a loss of both LC3 (1) and HSP70 (2) in the vacuoles as shown by the downward arrows, while both molecules greatly increase within cytosol (as shown by triple upward arrows in B). In the lysosome compartment both LAMP1 (3) and cathepsin-D (CAT D) (4) can be found in control conditions (A). During ischemia (B), there is a loss of LAMP1 (3) both in vacuoles (double downward arrows) and cytosol (double downward arrows). In ischemia (B), cathepsin-D (4) is reduced within vacuoles (double downward arrow) while it markedly increases in the cytosol (double upward arrows). In these conditions, the co-localization of the autophagosome marker LC3 (1) with the lysosome marker LAMP1 (3) within vacuoles is suppressed. This occurs more markedly than the suppression of each molecule from autophagosome and lysosome, respectively. This suggests that the massive defect in autophagolysosome formation is also due to a decrease in merging between vacuoles (5).

Fig 4: Within the area penumbra, lysosomal-associated membrane protein 1 (LAMP1) decreases, while Heat Shock Protein 70 (HSP70) increases and their co-localization is suppressed. (A) shows representative pictures show LAMP1 immunofluorescence (red), HSP70 immunofluorescence (green) and merge of both antigens. Arrows point to the merge of LAMP1 and HSP70. Graph (B) reports a decrease in LAMP1-positive cells within the ischemic area penumbra (Ischemia) compared with the control (Sham). Graph (C) reports the increase in HSP70-positive cells within the ischemic area penumbra (Ischemia) compared with the control (Sham). Graph (D) reports a massive decrease in LAMP1- + HSP70-positive cells within the ischemic area penumbra (Ischemia) compared with the control (Sham). Values are reported as percentage of control and values are given as the mean ± S.E.M. of 12 values for each group (n = 3 slices × N = 4 mice). * p ≤ 0.05 compared with other groups. Scale bar = 50 µm; 10 µm (inserts) (A).

Fig 5: Within the area penumbra, microtubule-associated protein I/II-Light Chain 3 (LC3) + lysosomal-associated membrane protein 1 (LAMP1) co-localization is suppressed. (A) shows representative pictures of LC3 immunofluorescence (red), LAMP1 immunofluorescence (green) and merge of both antigens. Arrows point to the merge of LC3 and LAMP1. Graph (B) reports the increase in LC3-positive cells within the area penumbra (Ischemia) compared with the control (Sham). Graph (C) reports the decrease in LAMP1-positive cells within the area penumbra (Ischemia) compared with the control (Sham). Graph (D) reports the suppression of LC3- + LAMP1-positive cells within the ischemic area penumbra (Ischemia) compared with the control (Sham). Values are reported as percentage of control and are given as the mean ± S.E.M. of 12 values for each group (n = 3 slices × N = 4 mice). * p ≤ 0.05 compared with other groups. Scale bars = 50 µm; 10 µm (inserts) (A).