The number of Wipi2-positive puncta was not reduced but increased in Optn-deficient MEFs compared with wild type MEFs (Fig. Ser-177 was required for autophagosome formation but not for Optn recruitment to the phagophore. These results suggest that Optn potentiates LC3-II production and maturation of the phagophore into the autophagosome, by facilitating the recruitment of the Atg12-5-16L1 complex to Wipi2-positive phagophores. and mutant proteins (Huntington, TDP43, SOD1) that form aggregates associated with neurodegenerative diseases (33, 34). Along with other autophagy receptors, OPTN mediates autophagy of damaged mitochondria (35,C39). Although OPTN and NDP52 play a vital role in recruiting LC3 to damaged mitochondria, LC3-II production upon induction of mitophagy does not depend on these autophagy receptors (39). It has been shown that OPTN, along with other proteins, NDP52 and T6BP, plays a role in autophagosome maturation by linking myosin VI to autophagosomes (40). Mutations in OPTN are associated with adult-onset primary open-angle glaucoma and amyotrophic lateral sclerosis (ALS) (41, 42). Although not mutated, OPTN is also associated with pathological structures seen in many neurodegenerative diseases (43). Glaucoma-associated mutants of OPTN are generally single copy missense mutations, whereas ALS-causing mutations include missense, deletion, and truncation IL1B mutations (31). A glaucoma-associated mutant of OPTN, E50K, BETd-260 inhibits autophagy that contributes to apoptotic death of retinal cells (44, 45). Transgenic mice BETd-260 expressing BETd-260 E50K-OPTN show mitochondrial fission, mitophagy, and degeneration of retinal ganglion cells (46). Another variant, M98K-OPTN, causes enhanced autophagy that also results in death of retinal cells (47). The ALS-associated mutant, E478G-OPTN, is usually impaired in mediating autophagic clearance of aggregated proteins, bacteria, and damaged mitochondria (33, 34, 48). Thus, impaired autophagy appears to play an important role in the pathogenesis caused by OPTN mutants. Therefore, it is essential to understand in detail the role of Optn in autophagy. In this study, we have explored the role of Optn in autophagosome formation during basal and starvation-induced autophagy. Our results show that Optn deficiency leads to reduced LC3-II production and reduced formation of autophagosomes although the formation of phagophores (Wipi2-positive puncta) is not reduced. We have also investigated the role of Optn in the recruitment of Atg12-5-16L1 complex to Wipi2-positive phagophores. Our BETd-260 results suggest that Optn potentiates autophagosome formation by facilitating the recruitment of Atg12-5-16L1 complex to Wipi2-positive phagophore upstream of LC3-II production. We also show that an ALS-associated mutant, E478G-OPTN, is defective in autophagosome formation, and unlike optineurin, does not colocalize with Atg12-positive or Atg16L1-positive phagophores. Results Effect of Optn deficiency on basal and starvation-induced autophagy Optn gene was inactivated by replacing ATG made up of exon 2 with a cassette made up of -galactosidase reporter and neomycin resistance gene through homologous recombination (Fig. 1gene was confirmed by PCR and Southern blot using mouse tail DNA (Fig. BETd-260 1, and = 3 experiments; ***, 0.001; **, 0.01. = 8 for untreated and = 5 for EBSS-treated samples; **, 0.01. = 3 experiments with minimum 50 cells in each experiment; **, 0.01 wild type. = 4 for WT MEF KO MEF graph (UT and EBSS treated). For KO MEF HA-OPTN graph, = 5 for KO MEF and = 3 for HA-OPTN MEF (UT and EBSS treated). Each represents an experiment done with minimum 20 cells; ***, 0.001; **, 0.01; *, 0.05; scatter plots represent average S.D. To examine the contribution of Optn to basal and starvation-induced autophagy we.
The number of Wipi2-positive puncta was not reduced but increased in Optn-deficient MEFs compared with wild type MEFs (Fig