Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin

“…of Parkin on mitochondria before and after 15–20 min of Antimycin A (or mock) treatment, plots of fluorescence intensity versus distance along the axon were generated for both DsRed (mitochondria) and YFP (Parkin) channels using the line scan function of ImageJ software (Schneider et al., 2012).”

“GFPLC3 is a cytosolic protein, but upon induction of autophagy, it gets lipidated and incorporated into autophagosomes forming distinct puncta (Mizushima and Komatsu, 2011).”

“Unlike many cell lines where the entire mitochondrial network can be cleared by mitophagy (Narendra et al., 2008, 2010; Geisler et al., 2010), neurons are strictly dependent on mitochondrial ATP production (Almeida et al., 2001, 2004).”

“…2006; Navarro et al., 2009) and PD­like symptoms caused by mitochondrial drugs, such as Rotenone and MPTP (1­methyl­4­phenyl­1,2,3,6­tetrahydro­ pyridine; Betarbet et al., 2000; Przedborski et al., 2004), were the first lines of evidence linking mitochondrial dysfunction to the pathology of PD.”

“The final step of mitophagy involves of autophagosome/ lysosome fusion and enzymatic degradation of the mitochon­ drion (Youle and Narendra, 2011).”

“Mitochondria, particularly when dysfunctional, produce excessive reactive oxygen species (ROS; Wallace, 2005) and must be removed by quality control pathways.”

“Therefore, to rescue the recruitment of Parkin to depolarized mitochon dria in PINK1/ axons, we expressed PINK1FLAG at low mitochondrial depolarization (Narendra et al., 2010).”

“Unlike many cell lines where the entire mitochondrial network can be cleared by mitophagy (Narendra et al., 2008, 2010; Geisler et al., 2010), neurons are strictly dependent on mitochondrial ATP production (Almeida et al., 2001, 2004).”

“Degradation of autophagosomal cargoes requires fusion of au­ tophagosomes with lysosomes to form autolysosomes (Xie and Klionsky, 2007), and it was suggested that autophagosomes would have to translocate to neuronal soma to fuse with ly­ sosomes (Cai et al., 2012).”

“Genetic studies have indicated that PINK1 acts upstream of Parkin in the regulation of mitochondrial integrity (Clark et al., 2006; Park et al., 2006), and stabilization of PINK1 on the outer mitochondrial membrane is essential for Parkin re­ cruitment in nonneuronal cells (Geisler et al., 2010;…”

“Physiological Triggers of Parkin Translocation beyond CCCP It is experimentally convenient in cultured cell lines, in primary neurons (Ashrafi et al., 2014) and in induced pluripotent stem cells (iPSCs) differentiated into neurons (Rakovic et al.”

“Because CCCP is highly toxic and induces non-physiological levels of mitochondrial damage especially in neurons, antimycin A is often used to induce mitophagy in neuronal cells (Cai et al , 2012; Ashrafi et al , 2014).”

“While some degradation may occur locally within the axon (Ashrafi et al., 2014), >80% of axonal autophagosomes formed by constitutive autophagy travel toward the cell soma (Maday et al., 2012), indicating a dependence on long-range axonal transport for clearance pathways.”

“Moreover, when mitophagy is triggered, either by application of antimycin A to axons or by the production of reactive oxygen species by photobleaching ofmito-KillerRed, autophagosomes are recruited to the axonal mitochondria in a manner dependent on both PINK1 and Parkin (Ashrafi et al., 2014).”

“…are selectively damaged in neurites with selective OXPHOS inhibition or photodamage while sparing the soma, however, Parkin can be recruited to the damaged mitochondria and initiate their capture in LC3-conjugated autophagosomes for fusion with LAMP1-positive lysosomes or late endososomes 138 .”

“Before triggering mitophagy, LC3 foci were not detected along axons, consistent with the model that autophagosomes are generated on demand during selective autophagy (Ashrafi et al., 2014).”

“This depolarization-triggered mitochondrial arrest is achieved by removal of Miro from the damaged mitochondrial surface (Ashrafi et al., 2014; Liu et al., 2012; Wang et al., 2011).”

“Parkin and PINK1 are genetic factors in Parkinson's disease and play a protective role in neurons through mitophagy (Ashrafi et al., 2014).”

“Moreover, mitochondrial Parkin translocation was shown to occur as a slow and subcellularly restricted process in mature cortical neurons [133], and Parkin accumulation was observed on only a fraction of depolarized mitochondria in neuronal axons [134].”

“We achieved local inhibition of a mitochondrial compartment optogenetically by targeting the phototoxic protein KillerRed (KR) to the mitochondrial matrix (KillerRed-Mito) (Ashrafi et al., 2014).”

“As mitophagy occurs locally in distal neuronal axons, we cannot exclude accumulation of dysfunctional mitochondria within axons (Ashrafi et al., 2014).”

“Motor neurons and retinal ganglion cells affected in ALS and glaucoma are characterized by extended axonal processes; whether damaged mitochondria occupying distal axonal regions are subject to the same quality control mechanisms as those mechanisms near the cell body remains unclear (32, 33).”

“A more targeted stimulus such as light induced reactive oxygen species generation may be more amenable (Yang and Yang, 2013; Ashrafi et al., 2014).”

“Therefore, turnover of synaptic proteins and the elimination of damaged mitochondria and oxidized DA by lysosomes via synaptic autophagy are critical in maintaining dopaminergic synapses [82,85].”

“There is also evidence that autophagosome formation can be induced along the axon in order to clear damaged mitochondria (Ashrafi et al. 2014).”

“One group reported localized mitophagy along the axons of hippocampal neurons upon induction of cellular stress via mitochondrial depolarization or ROS generation (Ashrafi et al. 2014).”

“Although groups have shown strong evidence that Parkin-mediated mitophagy occurs in neurons (Ashrafi et al., 2014; Bingol et al., 2014), the extensive studies of this pathway in cultured cells have failed to reveal how Parkin contributes to PD pathogenesis and affects dopaminergic neurons in vivo.”

“, mtKillerRed [12]), and even misfolded proteins in the mitochondrial matrix—as discussed later—also induce PINK1/Parkin-dependent mitophagy, emphasizing that various stresses can activate this pathway.”

“This result is consistent with other reports of formation along the mid-axon (Ashrafi et al., 2014), although we find these events to be infrequent under basal conditions.”

“…integrity and function by promoting the removal of damaged mitochondria via mitophagy (Geisler et al., 2010; Batlevi and La Spada, 2011; Ashrafi et al., 2014; Lazarou et al., 2015; Pickrell and Youle, 2015)—a selective form of autophagy whereby defective mitochondria are specifically…”

“Despite limited in vivo mitophagy studies likely due to the technical challenges, a few studies of mitophagy have been reported in mouse heart and muscle [98,99]; kidney [100]; lung [101] and neurons [102].”

“Strikingly, we observed a progressive reduction in Parkin levels in the brains from a total of 16 AD patients (Braak I & II: 0.50 ± 0.09; P < 0.01; Braak III & IV: 0.40 ± 0.069; P < 0.01; Braak V & VI: 0.057 ± 0.014; P < 0.001) (Fig.”

“Axonal mitochondrial quality control may also occur in a Parkin-dependent or -independent manner thereby providing rapid neuroprotection against stress conditions or early neurodegenerative disease stages [5, 6].”

“PINK1/parkinmediated mitophagy can be triggered in a variety of neuronal and non-neuronal cells in vitro, typically by exposure to mitochondrial depolarizing agents (Narendra et al., 2008; Geisler et al., 2010; Cai et al., 2012; Ashrafi et al., 2014; Cornelissen et al., 2014; Oh et al., 2017).”

“Similarly, Ashrafi et al [156] utilized mito-KillerRed (see Box 1) to selectively damage a subset of mitochondria in hippocampal axons and observed autophagosome and axonal lysosome recruitment to the damaged mitochondria that was both Parkin and PINK1 dependent.”

“Upon stimulation of autophagy, DFCP1 translocates to mitochondria-associated membranes and is a marker for omegasomes (Axe et al., 2008; Itakura et al., 2012).”

“Given the fact that (1) a small population of mito-autophagosomes is labeled with late endosome/lysosomal marker LAMP1 (Ashrafi et al., 2014); and (2) retrograde transport of autophagosomes is essential for maturation and degradation within acidic lysosomes in the proximal region of the neuron…”

“, 2009), damaged mitochondria may also recruit Parkin or be engulfed by autophagosomes (Cai et al., 2012a,b; Ashrafi et al., 2014).”

“This leads to an increase in Parkin ligase activity.”

“However, the extent to which neuronal cells undergo Parkin and PINK1-dependent mitophagic flux is controversial, in part because of the finding that PINK1 / mice do not have obvious defects in mitophagic flux and also because most mitophagy studies in cultured neurons have not used flux reporters (Ashrafi et al., 2014…”

“Axonal autophagy is dependent on specialized enzymatic apparatuses, including the PINK1-PRKN complex for localized mitophagy [24], KIF1A/ UNC-104 for autophagosome formation at the synapse [22,25],”

“…mechanistic rationale to explain how damaged segments of the mitochondrial reticulum (Jin and Youle, JCB • Volume 213 • NumBer 2 • 2016168 2013) are selectively sensitized for DRP1-mediated fission during basal mitophagy, a fundamental process that occurs in cells and neurons (Bingol et al., 2014).”

“The selective mitophagy of damaged mitochondria may occur locally in distal neuronal axons, primed by PINK1/Parkin pathways in cultured rat neurons [64].”

“In spite of reports that PINK1 is absolutely critical for astrogliogenesis [38] and that it phenocopies PARKIN (encoded by PARK2) function [8], there are no direct reports associating PINK1 with αSyn pathogenesis.”

“Although mitophagy has been observed locally in axons [20], it is still assumed that mitochondria must travel to the cell body for complete degradation [21–23].”

“This mitophagy process is closely regulated by PINK1 and Parkin (Ashrafi et al., 2014; Auberger et al., 2014), two interacting proteins that relocalize to mitochondria to stimulate deficient proton gradients.”

“This protein has been targeted to mitochondria and used to locally ablate mitochondria in axons (Spillane et al., 2013; Ashrafi et al., 2014), providing a method for subcellular analysis of mitochondrial function.”

“Then, the autophagy receptors are recruited, such as p62/SQSTM1, and link to the LC3B II autophagophore to form autophagosomes [24,25].”

“Mitophagy in the axonal compartment of neurons has also been described, indicating that the PINK1–Parkin pathway is essential for distal mitophagy in hippocampal axons 104 .”

“This is consistent with the idea that lysosomal dysfunction contributes to axonopathy in prevalent neurological diseases (Ashrafi et al., 2014; Gowrishankar et al., 2015; Xie et al., 2015) and suggests that insights from HSP will inform our knowledge of these common disorders.”

“to mitochondria and the induction of mitophagy [44,58,59].”

“To test this hypothesis, we applied the Complex III inhibitor Antimycin A to trigger mitophagy [2, 19, 42] in iPSC-derived neurons.”

“Local autophagic degradation, for example of damaged mitochondria (Ashrafi et al., 2014), might be another important contributor to homeostasis in remote cellular regions such as distal axons, where most of the axonal autophagosomes form (Maday and Holzbaur, 2014).”

“Thus, it is unclear how mitochondria in distal axons are cleared in ischemic neurons (Ashrafi et al., 2014).”

“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.”

“, 2015), elimination of damaged mitochondria and local recycling of synaptic proteins through lysosomes were also reported in axonal terminals (Ashrafi et al., 2014; Jin et al., 2018b).”

“PINK1-PRKN-dependent mitoQC was reported to occur locally in distal axons as well as in the neuronal cell bodies upon retrograde transport in different neuronal cultures [43,44].”

“To test the effects of 298/9EE on mitochondrial dynamics when endogenous PINK1 was activated, we transfected rat hippocampal neurons with either with Myc-Miro or Myc-Miro298/9EE and applied 10 μM antimycin A to activate the PINK1/Parkin pathway (51).”

“In neurons, autophagasomes are continuously formed in the distal tips of neurites and are transported retrogradely to the cell body (Ashrafi et al., 2014; Maday & Holzbaur, 2012; Maday, Wallace, & Holzbaur, 2012).”

“…mitochondrial compartment, the autophagosome and the lysosome have, however, provided evidence in neurons for events of engulfment of mitochondria into autophagosomes, or of fusion of autophagosomes containing mitochondria with lysosomes (Cai et al. 2012, Ashrafi et al. 2014, Hsieh et al. 2016).”

“However, it has been reported that mitophagy can occur in distal axons [145,146], as well as mtDNA replication and translation of mitochondrial-encoded proteins implicated in mitochondrial biogenesis [45,147] and reviewed in [148].”

“predicted to provide rapid neuroprotection against oxidative stress by damaged mitochondria, without requirement for retrograde transport (Ashrafi et al. 2014; Ashrafi and Schwarz 2015).”

“are locally removed by autophagy in a PINK1 and Parkin-dependent manner [134].”

“Several other pathological pathways, such as impairment in axonal transport and synaptic function, oxidative stress, dysfunction of lysosomes and mitochondria [25,26], activation of microglial cells and protein aggregation, also contribute to neuronal damage [27].”

“Live imaging indicates LC3/Atg8-labeled structures, likely autophagosomes, form at synaptic terminals and they are transported along axons to the cell body (Hernandez et al, 2012; Maday et al, 2012; Ashrafi et al, 2014; Binotti et al, 2015; Wang et al, 2015; Soukup et al, 2016).”

“So far, three prominent examples of mitophagy have been identified: (1) the removal of mitochondria during reticulocyte development, (2) the selective elimination of paternal mitochondria from fertilized oocytes, and (3) the degradation of damaged mitochondria (Ashrafi and Schwarz 2013).”

“Panel B: (i) Under hypoxic conditions, BNIP3, NIX, or FUNDC1 can bin regulates ER-mitochondria contacts ubiquitinating MFN-2, resulting in its pr proteins for proteasomal degradation or resulting in AMBRA1-mediated recruit PHB2 exposure results in LC3 II-coated vesicle recruitment to the mitochondr response (mtUPR) us…”

“The localized mitophagy in the axon observed by Ashrafi et al. (2014) was dependent on PINK1 and Parkin.”

“In vitro studies have shown that Parkin-dependent mitophagy occurs locally in distal neuronal axons (83).”

“However, unlike many other cell types, specific Parkin-associated mitophagy itself has more rarely been clearly demonstrated in neurons (Ashrafi et al., 2014; Cai et al., 2012), although evidence of non-Parkin-associated neuronal mitophagy has also been observed (Chu et al.”

“, 2016); however, clearance of damaged mitochondria has also been shown to occur locally in distal axons in response to acute stress (Ashrafi et al., 2014).”

“In a study by Ashrafi et al. 2014, it was shown that mitophagy of damaged mitochondria takes place away from the center of neurons, and also depends on PINK1 and parkin [54].”

“There is also evidence that autophagosome formation can be induced along the axon in order to clear damaged mitochondria (Ashrafi et al., 2014).”