Homozygous CbCln3Δex7/8 cells show abnormal membrane organelles
The abnormal transport and processing of cathepsin D suggested membrane trafficking disruptions in homozygous CbCln3Δex7/8 cells; therefore, we surveyed the subcellular distribution and morphology of membrane organelles. Components of the secretory pathway, including the ER, cis-Golgi, and trans-Golgi, did not appear altered from wild-type appearance when labeled with the respective markers, protein disulfide isomerase (PDI), GM130, and VVL (data not shown). By contrast, the lysosomal markers, Lysotracker and Lamp 2 had significantly altered signal in homozygous CbCln3Δex7/8 cells, versus wild-type cells. Wild-type cells exhibited brightly stained lysosomes that were large and clustered in the perinuclear region whereas homozygous CbCln3Δex7/8 lysosomes were lightly stained, smaller vesicles that were more diffusely scattered in the cytoplasm of the cell (Fig. 6). Lamp 1 distribution was also altered, as previously noted (Fig. 3). However, Lamp 1 and Lamp 2 total protein levels were similar in wild-type and homozygous CbCln3Δex7/8 cells by immunoblot analysis, indicating the altered signal likely reflects dispersed lysosomes or altered localization and/or epitope availability (data not shown). It is noteworthy that Lysotracker dye, which selectively accumulates in acidic compartments, exhibited the most marked reduction in lysosomal labeling. This observation may reflect altered lysosomal pH, an established finding in JNCL [10,15].
Figure 6  Lysotracker and Lamp 2 labeling of wild-type and homozygous CbCln3Δex7/8 lysosomes Lysosomal labeling of wild-type and homozygous CbCln3Δex7/8 precursor cells with lysotracker and Lamp 2 antibody is shown. Lysotracker dye (top panels) labeled large, perinuclear-clustered lysosomes and scattered lysosomes in the periphery of wild-type cells (CbCln3+/+). Lysotracker stain was dramatically reduced in homozygous mutant cells (CbCln3Δex7/8/Δex7/8), with smaller labeled vesicles and less apparent perinuclear clustering. Lamp 2 (bottom panels) immunostaining also showed reduced signal intensity with less perinuclear clustering in homozygous CbCln3Δex7/8 cells, although the effect was somewhat less dramatic than that observed with Lysotracker dye. Wild-type and homozygous CbCln3Δex7/8 confocal images were captured with identical exposure settings. 60 × magnification. Consistent with the altered early endosome marker (EEA1) signal observed by immunostaining (Fig. 3), fluid-phase endocytosis was also altered in homozygous CbCln3Δex7/8 cells, as measured by dextran-FITC uptake (Fig. 7). Following a 15-minute incubation in media containing dextran-FITC, wild-type and heterozygote cells displayed brightly stained, large endocytic vesicles that were clustered in the perinuclear region. However, homozygous CbCln3Δex7/8 cells were less brightly stained with most dextran-FITC signal localizing to smaller vesicles scattered throughout the cytoplasm of the cell.
Figure 7  Endocytosis in wild-type, heterozygous and homozygous CbCln3Δex7/8 cells Dextran-FITC uptake in wild-type, heterozygous and homozygous CbCln3Δex7/8 precursor cells is shown. In wild-type (CbCln3+/+, left panel) and heterozygous (CbCln3+/Δex7/8, middle panel) cells, dextran-FITC label was observed in a perinuclear-clustered vesicular pattern with scattered labeled vesicles also present in the periphery. In contrast, dextran-FITC label of homozygous mutant (CbCln3Δex7/8/Δex7/8, right panel) cells was reduced overall and exhibited smaller stained vesicles with less perinuclear clustering. Confocal images were captured with identical exposure settings. 40 × magnification. Finally, because subunit c is a mitochondrial protein and its turnover proceeds through autophagic engulfment of mitochondria [13], we analyzed homozygous CbCln3Δex7/8 cell mitochondrial morphology and function. Mitochondrial distribution in homozygous CbCln3Δex7/8 cells was indistinguishable from wild-type and heterozygous cells; however, homozygous CbCln3Δex7/8 mitochondria appeared more elongated by grp75 marker immunostaining and TEM analysis (Fig. 8a). 72% of homozygous mutant mitochondria were greater than 0.6 μm in length (range = 0.26 μm to 2.75 μm), while fewer wild-type mitochondria (51%) reached this length (range = 0.15 μm to 2.29 μm). Mitochondrial width was not altered in homozygous CbCln3Δex7/8 cells (data not shown). Moreover, compared to wild-type or heterozygous cells, homozygous CbCln3Δex7/8 cells had significantly reduced cellular ATP levels (1.3 fold less, Fig. 8b) and exhibited reduced survival following hydrogen peroxide treatment (~50% of wild-type survival, Fig. 8c), suggesting impaired energy metabolism and oxidative stress response. Taken together, these data support impaired mitochondrial function in homozygous CbCln3Δex7/8 cells.
Figure 8  Mitochondrial morphology and function in wild-type, heterozygous and homozygous CbCln3Δex7/8 cells a. Confocal and TEM micrographs of wild-type and homozygous CbCln3Δex7/8 mitochondrial morphology are shown. Immunostaining with the inner mitochondrial membrane marker, grp75 (top panels) highlighted elongated mitochondria in homozygous mutant cells (CbCln3Δex7/8/Δex7/8), relative to wild-type mitochondria (CbCln3+/+) (insets, zoom = 2.75x). Mitochondrial distribution was not altered from the wild-type pattern. Elongated homozygous CbCln3Δex7/8 mitochondria were also observed by TEM analysis. 60 × magnification. b. Cellular ATP levels in wild-type, heterozygous and homozygous CbCln3Δex7/8 precursor cells are shown. Wild-type (open bar) and heterozygous (gray bar) CbCln3Δex7/8 cells contained ~39 μM ATP, while homozygous CbCln3Δex7/8 cells (black bar) contained ~1.3 fold reduced levels of ATP (~30 μM), which was statistically significant in a t-test (p < 0.0001). Wild-type and heterozygous CbCln3Δex7/8 cellular ATP levels were not statistically different from each other (p > 0.4). A representative of triplicate experiments is shown (n = 6 in each experiment). c. Cell survival following 24-hour hydrogen peroxide treatment is shown. Homozygous CbCln3Δex7/8 cells were ~2-fold more sensitive to oxidative stress by hydrogen peroxide treatment. Wild-type (circle) and heterozygous (triangle) CbCln3Δex7/8 cells exhibited ~50% survival rates with 75–100 μM H2O2, whereas homozygous CbCln3Δex7/8 cells (squares) had a ~50% survival rate with 50 μM H2O2. A representative of triplicate experiments is shown (n = 4 in each experiment).