Figure 7. Impaired degradation of protein aggregates and damaged mitochondria in ATP13A2-null MEFs and dATP13A2 RNAi fly brains. (a) Impaired degradation of protein aggregates in ATP13A2-null cells. Control (WT, upper panel) and ATP13A2-null MEFs (KO, lower panel) were treated with MG132 for 12 h to induce protein aggregation followed by MG132 removal for 0 or 18 h. Protein aggregates were detected with an anti-ubiquitin antibody (Ubi, green). Nuclei were DAPI-stained (blue). Bar, 7.5 µm. (b) HDAC6 expression rescues impaired aggregate degradation. ATP13A2-null MEFs (KO) were transfected with HDAC6 (WT) or the HDAC6 H611A mutant. Cells were treated with MG132 for 12 h to induce protein aggregation followed by MG132 removal for 18 h. Protein aggregates (Ubi, green) and exogenous HDAC6 (FLAG, red; lower panel) were detected. Nuclei were DAPI-stained (blue). Dashed lines indicate transfectant of HDAC6 variants. Bar, 7.5 µm. (c and d) TEM analysis of fly brain. Brains were dissected from 3-d-old (c, upper panel) and 30-d-old (c, lower panel) flies followed by TEM analysis. Control flies expressing elav-gal4 only (Elav/+), flies expressing dATP13A2 RNAi (dATP13A2 RNAi), flies expressing both dATP13A2 RNAi and WT dHDAC6 (dATP13A2 RNAi, dHDAC6 WT), or flies expressing both dATP13A2 RNAi and dHDAC6 H664A mutant (dATP13A2 RNAi, dHDAC6 H664A) were analyzed. Bar, 1 µm. Quantitation of inclusion body diameter is shown (d). *, P < 0.05; **, P < 0.005; ***, P < 0.001. For 3 d, Elav/+: n = 51, dATP13A2 RNAi: n = 103, dATP13A2 RNAi/dHDAC6 WT: n = 48, dATP13A2 RNAi/dHDAC6 H664A: n = 63. For 30 d: Elav/+: n = 33, dATP13A2 RNAi: n = 70, dATP13A2 RNAi/dHDAC6 WT: n = 31, dATP13A2 RNAi/dHDAC6 H664A: n = 51. (e and f) Accumulation of insoluble proteins in dATP13A2-RNAi fly brains. Triton X-100–soluble (left panel) and –insoluble (right panel) proteins were extracted from dATP13A2 RNAi fly brains. Ubiquitinated proteins (Ubi) and actin were detected (e). Control flies expressing elav-gal4 only (Elav/+), flies expressing dATP13A2 RNAi (dATP13A2 RNAi), flies expressing both dATP13A2 RNAi and WT dHDAC6 (dATP13A2 RNAi, dHDAC6 WT), or flies expressing both dATP13A2 RNAi and dHDAC6 H664A mutant (dATP13A2 RNAi, dHDAC6 H664A) were analyzed. Quantitative analysis is shown (f). *, P < 0.05; ***, P < 0.001, n = 3. (g) Impaired clearance of damaged mitochondria in ATP13A2-null HEK293 cells. Control (ATP13A2, WT) and ATP13A2-null (ATP13A2, KO) HEK293 cells transfected with either pcDNA3.1 (HDAC6, –) or HDAC6 (HDAC6, +) were treated with either DMSO (filled bar) or CCCP (empty bar) for 12 h. mtDNA/nDNA ratios were calculated. **, P < 0.01; ***, P < 0.001, n = 3. (h) TEM analysis of CCCP-induced mitophagy in ATP13A2-null HEK293 cells. Control and ATP13A2-null (KO) HEK293 cells were treated with either DMSO or CCCP followed by TEM analysis. Representative images of damaged mitochondria in APS (white arrows) are shown. Bar, 0.5 µm.
Figure 7.

Impaired degradation of protein aggregates and damaged mitochondria in ATP13A2-null MEFs and dATP13A2 RNAi fly brains. (a) Impaired degradation of protein aggregates in ATP13A2-null cells. Control (WT, upper panel) and ATP13A2-null MEFs (KO, lower panel) were treated with MG132 for 12 h to induce protein aggregation followed by MG132 removal for 0 or 18 h. Protein aggregates were detected with an anti-ubiquitin antibody (Ubi, green). Nuclei were DAPI-stained (blue). Bar, 7.5 µm. (b) HDAC6 expression rescues impaired aggregate degradation. ATP13A2-null MEFs (KO) were transfected with HDAC6 (WT) or the HDAC6 H611A mutant. Cells were treated with MG132 for 12 h to induce protein aggregation followed by MG132 removal for 18 h. Protein aggregates (Ubi, green) and exogenous HDAC6 (FLAG, red; lower panel) were detected. Nuclei were DAPI-stained (blue). Dashed lines indicate transfectant of HDAC6 variants. Bar, 7.5 µm. (c and d) TEM analysis of fly brain. Brains were dissected from 3-d-old (c, upper panel) and 30-d-old (c, lower panel) flies followed by TEM analysis. Control flies expressing elav-gal4 only (Elav/+), flies expressing dATP13A2 RNAi (dATP13A2 RNAi), flies expressing both dATP13A2 RNAi and WT dHDAC6 (dATP13A2 RNAi, dHDAC6 WT), or flies expressing both dATP13A2 RNAi and dHDAC6 H664A mutant (dATP13A2 RNAi, dHDAC6 H664A) were analyzed. Bar, 1 µm. Quantitation of inclusion body diameter is shown (d). *, P < 0.05; **, P < 0.005; ***, P < 0.001. For 3 d, Elav/+: n = 51, dATP13A2 RNAi: n = 103, dATP13A2 RNAi/dHDAC6 WT: n = 48, dATP13A2 RNAi/dHDAC6 H664A: n = 63. For 30 d: Elav/+: n = 33, dATP13A2 RNAi: n = 70, dATP13A2 RNAi/dHDAC6 WT: n = 31, dATP13A2 RNAi/dHDAC6 H664A: n = 51. (e and f) Accumulation of insoluble proteins in dATP13A2-RNAi fly brains. Triton X-100–soluble (left panel) and –insoluble (right panel) proteins were extracted from dATP13A2 RNAi fly brains. Ubiquitinated proteins (Ubi) and actin were detected (e). Control flies expressing elav-gal4 only (Elav/+), flies expressing dATP13A2 RNAi (dATP13A2 RNAi), flies expressing both dATP13A2 RNAi and WT dHDAC6 (dATP13A2 RNAi, dHDAC6 WT), or flies expressing both dATP13A2 RNAi and dHDAC6 H664A mutant (dATP13A2 RNAi, dHDAC6 H664A) were analyzed. Quantitative analysis is shown (f). *, P < 0.05; ***, P < 0.001, n = 3. (g) Impaired clearance of damaged mitochondria in ATP13A2-null HEK293 cells. Control (ATP13A2, WT) and ATP13A2-null (ATP13A2, KO) HEK293 cells transfected with either pcDNA3.1 (HDAC6, –) or HDAC6 (HDAC6, +) were treated with either DMSO (filled bar) or CCCP (empty bar) for 12 h. mtDNA/nDNA ratios were calculated. **, P < 0.01; ***, P < 0.001, n = 3. (h) TEM analysis of CCCP-induced mitophagy in ATP13A2-null HEK293 cells. Control and ATP13A2-null (KO) HEK293 cells were treated with either DMSO or CCCP followed by TEM analysis. Representative images of damaged mitochondria in APS (white arrows) are shown. Bar, 0.5 µm.

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