Extended Data Figure 10. Involvement of integrin-mediate src/syk mechanisms in astrocytic mitochondrial entry into neurons in vitro.
a and b, Cultured rat cortical astrocytes were stimulated by cADPR (1 μM) for 24 hours. Intracellular mitochondria labeled by mitotracker dye was significantly increased in astrocytes stimulated with cADPR (1 μM) (n=7). **P<0.01 vs 0h. c, Some of mitochondria were found outside of cells. d, FACS analysis revealed that approximately 5×105 mitochondria were contained in 1mL of astrocyte-derived conditioned media (n=6). cADPR (1 μM) significantly increased the number of mitochondria in the media (n=6). e, Experimental schedule to quantify the mitochondrial entry into neurons following oxygen-glucose deprivation. Rat cortical neurons (1×105 cells/well) were prepared in 24-well culture plate. ACM or cADPR-ACM (each 1 mL) was co-incubated with neurons for 18 hours. Mitochondrial entry into neurons were calculated by mitochondrial intensity measured before and after washing cells with PBS. Phenol red free culture media were used to decrease back ground signal. Back ground signal was subtracted from fluorescent intensity obtained from each sample. f, Oxygen-glucose deprivation for 2 hours decreased approximately 50% of mitochondria in neurons after 18 h reoxygenation (n=4). g, All data are expressed as relative values, with total neuronal mitochondria after 2 h OGD/18 h reoxygenation being 100%. Mitochondrial entry into neurons was slightly higher in cADPR-ACM treatment (18%) compared to ACM treatment (11%), although there was no statistically significance (n=4). h, There was no difference in the percentage of mitochondrial entry between ACM treatment and cADPR-ACM treatment (n=4). i, cADPR-ACM treatment supported neuronal viability better than ACM treatment (n=4). j, Co-culture between rat cortical astrocytes in the upper chamber and rat cortical neurons in the lower chamber was performed for 18 hours following oxygen-glucose deprivation for 2 hours in neurons. Then, mitochondrial entry into neurons was measured. k, Immediately after oxygen-glucose deprivation, dynasore (5 μM), RGDS peptide (50 μg/ml), or MNS (1 μM) was initially added in neurons for 30 min, then astrocyte co-culture was performed for 18 hours. The data are expressed as relative values, with astrocytic extracellular mitochondria plus entered mitochondria into neurons being 100%. RGDS peptide and MNS significantly decreased mitochondrial entry into neurons, but dynasore did not inhibit the entry. l, MNS treatment significantly decreased astrocyte-mediated neuroprotection (n=4). m, Dynasore (5 μM), RGDS peptide (50 μg/ml), or MNS (1 μM) did not affect neuronal viability after 2 h oxygen-glucose deprivation (n=4). All values are mean +/− SEM. These data suggest that astrocyte into neuron mitochondrial particle entry may involve integrin-mediate src/syk mechanisms. However, we acknowledge that these pathways may be multifactorial and deeper analyses are warranted to dissect entry mechanisms under various physiologic and pathologic conditions.