Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons

“Line scans In Fiji, line scans were generated using a pixel line width to trace OPTN-positive mitochondria and a three-pixel line width to trace axons and dendrites.”

“Mitophagy is primarily regulated by two proteins linked to familial PD, PTEN-Induced Putative Kinase 1 (PINK1) and Parkin (Kitada et al., 1998; Narendra et al., 2008; Narendra et al., 2010b; Valente et al., 2004; Wang and Klionsky, 2011; Youle and Narendra, 2011).”

“PINK1 recruits Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et al., 2015…”

“Mitophagy is primarily regulated by two proteins linked to familial PD, PTEN-Induced Putative Kinase 1 (PINK1) and Parkin (Kitada et al., 1998; Narendra et al., 2008; Narendra et al., 2010b; Valente et al., 2004; Wang and Klionsky, 2011; Youle and Narendra, 2011).”

“Since rounding and fragmentation are hallmarks of mitochondrial damage (Twig et al., 2008; Westermann, 2010; Youle and Narendra, 2011), we examined morphology following treatments by either staining with tetramethyl rhodamine ethyl ester (TMRE), a vital dye used to measure mitochondrial potential (Figure 1C–D), or by…”

“In non-neuronal cells, OPTN, NDP52, and TAX1BP1 are proposed to mediate the autophagic clearance of damaged mitochondria, while p62 clusters organelles without directly facilitating mitophagy (Heo et al., 2015; Lazarou et al., 2015; Moore and Holzbaur, 2016; Narendra et al., 2010a; Wong and Holzbaur, 2014a).”

“Since rounding and fragmentation are hallmarks of mitochondrial damage (Twig et al., 2008; Westermann, 2010; Youle and Narendra, 2011), we examined morphology following treatments by either staining with tetramethyl rhodamine ethyl ester (TMRE), a vital dye used to measure mitochondrial potential (Figure 1C–D), or by…”

“PINK1 recruits Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et al., 2015…”

“…contrast, KO rat models of PINK1 exhibited significant dopaminergic neuron loss, and null or mutant flies of PINK1 or Parkin display morphological defects in dopaminergic neurons (Cha et al., 2005; Clark et al., 2006; Dave et al., 2014; Park et al., 2006; Whitworth et al., 2005; Yang et al., 2006).”

“PINK1 recruits Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et al., 2015…”

“We also observed no significant differences in the expression levels of ATG5 and ATG16L1, proteins required for autophagy initiation (Figure 1—figure supplement 1B-C; Mizushima et al., 2001; Suzuki et al., 2001).”

“Expression of an ALS-associated mutation in OPTN is sufficient to disrupt mitochondrial health under basal conditions and this is exacerbated by oxidative stress.”

“…Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et…”

“OPTN is a heterozygous missense mutation identified in both sporadic and familial cases of ALS (Figure 8A; Maruyama and Kawakami, 2013; Maruyama et al., 2010).”

“Subsequently, microtubule-associated protein light chain 3 (LC3), is targeted to the ubiquitinated mitochondria via the LC3 interacting region (LIR) of OPTN (Stolz et al., 2014; Wild et al., 2011), leading to engulfment of the organelle inside an LC3-positive autophagosome.”

“In addition, several reports have described arrested axonal mitochondrial motility after damage due to the removal of Miro, a mitochondrial motor adaptor protein (Hsieh et al., 2016; Liu et al., 2012; Wang et al., 2011).”

“We also observed no significant differences in the expression levels of ATG5 and ATG16L1, proteins required for autophagy initiation (Figure 1—figure supplement 1B-C; Mizushima et al., 2001; Suzuki et al., 2001).”

“PINK1 recruits Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et al., 2015…”

“, 2002), is recruited to the mitochondrial surface where it interacts with ubiquitin through its ubiquitin binding in ABIN and NEMO (UBAN) domain (Gleason et al., 2011; Heo et al., 2015; Lazarou et al., 2015; Moore and Holzbaur, 2016; Rogov et al., 2014; Wild et al., 2011; Wong and Holzbaur, 2014a; Zhu et al., 2007).”

“…to the mitochondrial surface where it interacts with ubiquitin through its ubiquitin binding in ABIN and NEMO (UBAN) domain (Gleason et al., 2011; Heo et al., 2015; Lazarou et al., 2015; Moore and Holzbaur, 2016; Rogov et al., 2014; Wild et al., 2011; Wong and Holzbaur, 2014a; Zhu et al., 2007).”

“A constitutive pathway for axonal autophagy has been reported in neurons both in vitro (Maday and Holzbaur, 2014; Wong and Holzbaur, 2014b) and in vivo (Neisch et al., 2017; Soukup et al., 2016; Stavoe et al., 2016).”

“…cells, OPTN, NDP52, and TAX1BP1 are proposed to mediate the autophagic clearance of damaged mitochondria, while p62 clusters organelles without directly facilitating mitophagy (Heo et al., 2015; Lazarou et al., 2015; Moore and Holzbaur, 2016; Narendra et al., 2010a; Wong and Holzbaur, 2014a).”

“…contrast, KO rat models of PINK1 exhibited significant dopaminergic neuron loss, and null or mutant flies of PINK1 or Parkin display morphological defects in dopaminergic neurons (Cha et al., 2005; Clark et al., 2006; Dave et al., 2014; Park et al., 2006; Whitworth et al., 2005; Yang et al., 2006).”

“…damage to a mitochondrion, phospho-ubiquitination of outer mitochondrial membrane proteins mediated by PINK1-recruited Parkin increases the abundance of ubiquitin chains, which recruit OPTN and TBK1 to the organelle (Figure 2A; Harper et al., 2018; Ordureau et al., 2018; Richter et al., 2016).”

“However, the use of another acid-sensitive mt-Keima probe illustrated insufficiencies in basal mitophagy in dopaminergic neurons of Parkin-deficient flies (Cornelissen et al., 2018; Katayama et al., 2011; Sun et al., 2015).”

“The current model suggests that following damage to a mitochondrion, phospho-ubiquitination of outer mitochondrial membrane proteins mediated by PINK1-recruited Parkin increases the abundance of ubiquitin chains, which recruit OPTN and TBK1 to the organelle (Figure 2A; Harper et al., 2018; Ordureau et al., 2018; Richt…”

“Autophagosomes mature during trafficking along the axon to the soma by fusion with lysosomes, which leads to efficient cargo degradation (Cheng et al., 2015; Hollenbeck, 1993; Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012; Neisch et al., 2017).”

“The use of the acid-sensitive mitoQC mitophagy probe also did not reveal deficits in tissues of high metabolic demand in mice (mitoQC Pink1) or in dopaminergic neurons of PINK1 and Parkin mutant flies (Lee et al., 2018; McWilliams et al., 2018).”

“Thus, we could visualize both LAMP1 localization and acidification, as the fluorescence signal of the SEP will quench in acidic environments while the RFP signal will persist (pH <6; Sankaranarayanan et al., 2000).”

“Under these conditions, as we demonstrate in vitro and was previously shown in vivo (Devireddy et al., 2015; McWilliams et al., 2016; Sung et al., 2016), the PINK1/Parkin mitophagy pathway is compartmentally restricted to the soma.”

“The fluorescence signal of EGFP, but not mCherry, quenches in acidic environments and thus the shift in fluorescence from yellow to red is commonly used to measure the efficiency of mitophagy (Allen et al., 2013; Lee et al., 2018; McWilliams et al., 2016; Rojansky et al., 2016).”

“PINK1 and Parkin knock-out (KO) mice display mild phenotypes with no dopaminergic neuron loss, a hallmark of PD, implying there are additional compensatory pathways contributing to neuronal mitophagy in vivo (Akundi et al., 2011; Goldberg et al., 2003; Kitada et al., 2007; Perez and Palmiter, 2005).”

“We hypothesize that either long-term exposure to low doses (>2 hr) or short periods of high concentration of AA (Ashrafi et al., 2014) may disrupt mitochondrial bioenergetics causing axonal mitochondria to undergo mitophagy locally to preserve the health of the neuron.”

“Further, stable isotope labeling of flies showed a slowing in the turnover of mitochondrial proteins isolated from the heads of Parkin null flies (Vincow et al., 2013).”

“Autophagosomes mature during trafficking along the axon to the soma by fusion with lysosomes, which leads to efficient cargo degradation (Cheng et al., 2015; Hollenbeck, 1993; Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012; Neisch et al., 2017).”

“…for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b; Ordureau et al., 2015; Ordureau et al., 2014; Poole et al., 2010; Sarraf et al., 2013; VivesBauza et al., 2010).”

“These data are consistent with previous reports that autophagosomes effectively fuse with lysosomes to degrade cargo (Boland et al., 2008; Kimura et al., 2008; Lee et al., 2011; Maday et al., 2012).”

“, 2014) and studies indicating that axonal mitochondria motility is arrested following mitochondrial damage (Hsieh et al., 2016; Liu et al., 2012; Wang et al., 2011), necessitating a local clearance mechanism.”

“First, OPTNE478G fails to translocate to damaged organelles, which may perturb the mitophagy pathway in a dominant-negative manner (Moore and Holzbaur, 2016).”

“The fluorescence signal of EGFP, but not mCherry, quenches in acidic environments and thus the shift in fluorescence from yellow to red is commonly used to measure the efficiency of mitophagy (Allen et al., 2013; Lee et al., 2018; McWilliams et al., 2016; Rojansky et al., 2016).”

“DOI: https://doi.org/10.7554/eLife.50260 2 of 30 inhibitor) displayed enhanced retrograde mitochondrial motility, suggesting damaged organelles are trafficked back to the soma for degradation (Cai et al., 2012; Miller and Sheetz, 2004).”

“The fluorescence signal of EGFP, but not mCherry, quenches in acidic environments and thus the shift in fluorescence from yellow to red is commonly used to measure the efficiency of mitophagy (Allen et al., 2013; Lee et al., 2018; McWilliams et al., 2016; Rojansky et al., 2016).”

“These puncta may represent either the self-association of OPTN or the recruitment of OPTN to other autophagy substrates, such as protein aggregates (Korac et al., 2013).”

“PINK1 recruits Parkin to phospho-ubiquitinate outer mitochondrial membrane proteins, which act as a specific cue for organelle degradation (Geisler et al., 2010; Kane et al., 2014; Kazlauskaite et al., 2014; Koyano et al., 2014; Matsuda et al., 2010; Narendra et al., 2008; Narendra et al., 2010b;…”

“A constitutive pathway for axonal autophagy has been reported in neurons both in vitro (Maday and Holzbaur, 2014; Wong and Holzbaur, 2014b) and in vivo (Neisch et al., 2017; Soukup et al., 2016; Stavoe et al., 2016).”

“Autophagosomes mature during trafficking along the axon to the soma by fusion with lysosomes, which leads to efficient cargo degradation (Cheng et al., 2015; Hollenbeck, 1993; Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012; Neisch et al., 2017).”

“However, the use of another acid-sensitive mt-Keima probe illustrated insufficiencies in basal mitophagy in dopaminergic neurons of Parkin-deficient flies (Cornelissen et al., 2018; Katayama et al., 2011; Sun et al., 2015).”

“DOI: https://doi.org/10.7554/eLife.50260 20 of 30 was generated from site-directed mutagenesis, untagged-Parkin (mCherry-Parkin was removed), YFP-NavII-III (Addgene 26056), GFP-Ub (Addgene 11928), TBK1 (Addgene 23851) was recloned into a pSNAPf vector (New England Biolabs), LAMP1-EGFP was subcloned from Addgene 1817 w…”

“Autophagosomes mature during trafficking along the axon to the soma by fusion with lysosomes, which leads to efficient cargo degradation (Cheng et al., 2015; Hollenbeck, 1993; Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012; Neisch et al., 2017).”

“Only partial rescue was observed with the Parkinson’s disease-linked mutant mCherry-Parkin (Sriram et al., 2005).”

“, 2019) and NIX/BNIP3L-dependent mitophagy (Bellot et al., 2009; Koentjoro et al., 2017; Real et al., 2005; Sowter et al., 2001) support mitochondrial clearance.”

“…2002), is recruited to the mitochondrial surface where it interacts with ubiquitin through its ubiquitin binding in ABIN and NEMO (UBAN) domain (Gleason et al., 2011; Heo et al., 2015; Lazarou et al., 2015; Moore and Holzbaur, 2016; Rogov et al., 2014; Wild et al., 2011; Wong and Holzbaur,…”

“Previous studies have proposed that mitochondrial ubiquitin ligase 1 (MUL1) plays a role in mitochondrial quality control in neurons, where it functions as an additional E3 ubiquitin ligase in a parallel pathway to Parkin (Ambivero et al., 2014; Puri et al., 2019; Yun et al., 2014).”

“…LAMP1 is routinely used as a marker of lysosomes, recent work has demonstrated that a portion of LAMP1-positive organelles did not colocalize with lysosomal hydrolases, implying not all LAMP1 is on degradation-competent vesicles but also on late endosomes (Cheng et al., 2018; Yap et al., 2018).”

“In addition, we monitored expression level of Transcription Factor EB (TFEB), a master transcriptional regulator of genes that are involved in lysosomal biogenesis and function (Bajaj et al., 2019; Sardiello, 2016).”

“Alternatively, ion transporters or other proteins associated with this vesicular compartment could be differentially regulated (Majumdar et al., 2011), but further work will be required to determine the underlying cause.”

“Autophagosomes mature during trafficking along the axon to the soma by fusion with lysosomes, which leads to efficient cargo degradation (Cheng et al., 2015; Hollenbeck, 1993; Lee et al., 2011; Maday and Holzbaur, 2014; Maday et al., 2012; Neisch et al., 2017).”

“The spatial specificity of mitophagy was previously postulated based on observations in vivo in Drosophila (Devireddy et al., 2015; Sung et al., 2016), qualitative findings of acidified mitochondria only within the soma in mito-QC mice (McWilliams et al.”

“Previous studies have proposed that mitochondrial ubiquitin ligase 1 (MUL1) plays a role in mitochondrial quality control in neurons, where it functions as an additional E3 ubiquitin ligase in a parallel pathway to Parkin (Ambivero et al., 2014; Puri et al., 2019; Yun et al., 2014).”

“…of mitophagy in PINK1 or Parkin mutant flies found no enhanced accumulation of axonal mitochondria in motor neurons, but did report dramatic changes in the morphology of somal mitochondria, indicating neuronal mitophagy may be compartmentally restricted (Devireddy et al., 2015; Sung et al., 2016).”

“…studies have suggested that alternative pathways such as NIPSNAP/Parkin-mediated mitophagy (Nautiyal et al., 2010; Princely Abudu et al., 2019) and NIX/BNIP3L-dependent mitophagy (Bellot et al., 2009; Koentjoro et al., 2017; Real et al., 2005; Sowter et al., 2001) support mitochondrial clearance.”

“Expression of an ALS-associated OPTN mutant increases the neurons susceptibility to stress and damage Genetic studies have identified mutations in OPTN as causative for ALS and frontotemporal dementia (FTD; Ito et al., 2011; Maruyama and Kawakami, 2013; Pottier et al., 2015).”

“OPTN is a heterozygous missense mutation identified in both sporadic and familial cases of ALS (Figure 8A; Maruyama and Kawakami, 2013; Maruyama et al., 2010).”

“Other studies have suggested that alternative pathways such as NIPSNAP/Parkin-mediated mitophagy (Nautiyal et al., 2010; Princely Abudu et al., 2019) and NIX/BNIP3L-dependent mitophagy (Bellot et al., 2009; Koentjoro et al., 2017; Real et al., 2005; Sowter et al., 2001) support mitochondrial…”

“In cultured cells, acidification and degradation of mitochondria in the soma may be rate-limiting steps, suggesting that neurons may have comparatively low rates of mitophagic flux 137 .”

“A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript mitochondrial content determines the markers of mitochondrial quality (Evans and Holzbaur, 2020; Calkins et al., 2011; Wang et al., 2009).”