Local ROS and inefficient autophagosome formation limit Optineurin-mediated mitophagy in neurons

Key results

1. ShownAt 6 h, most OPTN-positive mitochondrial fragments had not yet recruited LC3B, so autophagosome initiation and engulfment, rather than lysosomal fusion, is the stalled step; bulk autophagosomes formed and acidified normally, but those at OPTN+ mitochondria acidified less.

   ~0.6 of OPTN+ events were LC3B-negative and ~0.15 fully engulfed in both control and AOF (n = 47-114 events, 3 biological replicates); in the DMSO/BafA1 experiment most events stayed LC3B-negative in both conditions and LC3B+/CatD+ events were rare (0.06); AOF did not change total LC3B puncta (n = 71 neurons, 7 replicates); the acidified mCherry+ LC3B fraction was significantly lower at OPTN+ events than elsewhere. · Autophagosomes fail to form in OPTN-mediated…; Autophagosome initiation and formation are…; Figure legends

2. ShownDelivery of OPTN+ mitochondria to lysosomes takes days in neurons, whereas in HeLa cells most OPTN+ events colocalize with lysosomes within 2 h even under mild stress; a SNAP-tag pulse-chase separating old from new mitochondria showed lysosomal colocalization rising between 24 and 72 h after AOF.

   At 6 h the vast majority of neuronal OPTN+ events were LysoTracker-negative, versus ~0.6 LysoTracker-positive in HeLa at 0.5 or 4 µM AA/OA (n = 30-32 cells). Pulse-chase validation: 96.5% dual-labeled with co-added ligands, 87.5% first-pulse-only when sequential; LysoT+ fraction of old OPTN+ events rose from 24 to 72 h under AOF (p < 0.05; n = 24-60 events). · Autophagosome initiation and formation limit the…; “Aged” mitochondria remain in hippocampal…; Figure legends

3. ShownOPTN+ mitochondria are depolarized and carry locally elevated mitochondrial ROS relative to the rest of the network in both control and AOF neurons, while ATP at these sites did not differ from the soma.

   Mito-Grx1-roGFP2 405/488 ratio significantly higher at OPTN+ events than in the network (n = 19-36 events, 3 biological replicates; p < 0.01 to p < 0.0001); TMRE significantly lower at events (p < 0.0001; n = 14-17 events); QUEEN-37C 400/494 ratio not different between soma and events (n = 4 events in 48 neurons). · AOF treatment induces low levels of ROS and…; Local ROS is elevated at OPTN+ mitophagy events; Figure legends

4. SuggestedScavenging ROS for 24 h after AOF increased the fraction of OPTN+ mitochondria inside acidic lysosomes without changing how many neurons had OPTN+ events, implicating the local redox state in the delay.

   NAC (500 µM) lowered MitoSOX (p < 0.01) and raised LysoT+ OPTN+ events (p < 0.05; n = 44-55 events, 70 neurons, 4 biological replicates); EUK-134 (1 µM) lowered MitoSOX (p < 0.0001) with a similar rise in LysoT+ events (p = 0.0507; n = 47-64 events). · Scavenging mitochondrial ROS accelerates…; Mitochondrial damage and treatments; Figure legends

5. ShownBoosting autophagy by 24 h partial nutrient deprivation (equal parts medium and Hanks' Balanced Salt Solution, HBSS) increased lysosomal delivery of OPTN+ mitochondria without altering mitophagy induction or the local ROS elevation.

   HBSS lowered pS6 and raised LC3B-II:LC3B-I; total LC3B puncta (p < 0.0001) and acidified fraction (p < 0.01) rose (n = 49-50 neurons); mitochondrial volume and OPTN+ event counts unchanged; CatD+ fraction of OPTN+ events increased significantly (n = 17-27 events, 5 biological replicates). A 4 h deprivation lowered pS6 without changing LC3B-II:LC3B-I. · Partial nutrient deprivation induces autophagy…; Figure legends