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;…”

“Most neuronal mitochondria reside in distal dendritic and axonal processes, far from the cell body where ly sosomes are abundant (Parton et al., 1992; Craig and Banker, 1994).”

“The involvement of PINK1 and Par­ kin in regulation of mitochondrial dynamics further supports the classification of PD as a mitochondrial disease (Chen and Chan, 2009; Wang et al., 2011).”

“We have now established that (a) mitophagy of damaged mitochondria occurs locally in distal neuronal axons and (b) that axonal mitophagy depends on both PINK1 and Parkin.”

“…at 400 g for 2–3 min, resuspended at a final concentration of 4 × 10 4 /µl in Neurobasal medium supplemented with l -glutamine and penicillin/ streptomycin, plated into one of the somal compartments at a density of 5 × 10 6 /cm 2 , and cultured as previously described (Taylor et al., 2005).”

“Untreated mitochondria in PINK1  /  axons were smaller than in PINK1 +/+ axons as previously reported (Dagda et al., 2009), and expression of PINK1­FLAG restored the wild­type mitochondrial size in the PINK1  /  background.”

“129S4-Park2 tm1Shn (exon 3 deletion), and their control mice have been previously described (Goldberg et al., 2003).”

“Accumulation of mitochondrial damage in the brains of PD patients (Keeney et al., 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…”

“Antimycin A induces mi­ tochondrial ROS production in nonneuronal cells (Soubannier et al., 2012a) and depolarizes neuronal mitochondria (Cai et al., 2012).”

“PINK1 functions as a sensor for mitochondrial damage; its precursor is constitutively imported to the inner membrane of healthy mitochondria, where it is proteolytically cleaved (Greene et al., 2012) and eventually degraded by the protea­ some (Yamano and Youle, 2013).”

“PINK1 functions as a sensor for mitochondrial damage; its precursor is constitutively imported to the inner membrane of healthy mitochondria, where it is proteolytically cleaved (Greene et al., 2012) and eventually degraded by the protea­ some (Yamano and Youle, 2013).”

“S2, B and C) as previously observed (Maday et al., 2012).”

“S2, B and C) as previously observed (Maday et al., 2012).”

“…min, permeabilized with PBS containing 0.5% saponin for 1 h at room temperature, blocked with the permeabilization buffer + 1% BSA for 1 h, and incubated with mouse monoclonal anti-LAMP1 primary antibody (LY1C6; EMD Millipore) at 1:500 overnight at 4°C (Stowers et al., 2002; Glater et al., 2006).”

“…min, permeabilized with PBS containing 0.5% saponin for 1 h at room temperature, blocked with the permeabilization buffer + 1% BSA for 1 h, and incubated with mouse monoclonal anti-LAMP1 primary antibody (LY1C6; EMD Millipore) at 1:500 overnight at 4°C (Stowers et al., 2002; Glater et al., 2006).”

“Lightinduced activation of mtKR causes local ROSmediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012).”

“Quality control of axonal mitochondria, however, is of particular interest because axonal degeneration precedes the loss of the soma in neuro­ degenerative diseases, such as PD (Burke and O’Malley, 2013).”

“A prevalent model has favored transport of mitochondria toward the cell body for subsequent degradation (Miller and Sheetz, 2004).”

“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).”

“Similarly, PINK1 is re­ quired for basal turnover of mitochondrial respiratory chain subunits in Drosophila melanogaster (Vincow et al., 2013).”

“In our study, mitoautophagosomes were largely station ary; although in agreement with previous studies (Lee et al., 2011; Maday et al., 2012), nonmitophagic autophagosomes were highly motile.”

“Light­induced activation of mt­KR causes local ROS­mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012).”

“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).”

“1 vector with a CMV promoter (Weihofen et al., 2009), mt-DsRed with a CMV promoter (Takara Bio Inc.), mt-GFP in the pShooter vector with a CMV promoter (gift of M. Haigis, Harvard Medical School, Boston, MA), pEGFP-N1 with a CMV promoter (Takara Bio Inc.), pKillerRed-dMito with a CMV promoter (Evrogen), and LAMP1-YFP…”

“Veratridine, an activator of voltage­gated sodium channels, was applied to axons to increase firing and intracellular calcium (Chang et al., 2006).”

“…(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; Narendra et al., 2010) and cell bodies of induced pluripotent stem cell– derived neurons (Seibler et al., 2011).”

“Antimycin A induces mi­ tochondrial ROS production in nonneuronal cells (Soubannier et al., 2012a) and depolarizes neuronal mitochondria (Cai et al., 2012).”

“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).”

“1 vector with a CMV promoter (Weihofen et al., 2009), mt-DsRed with a CMV promoter (Takara Bio Inc.), mt-GFP in the pShooter vector with a CMV promoter (gift of M. Haigis, Harvard Medical School, Boston, MA), pEGFP-N1 with a CMV promoter (Takara Bio Inc.), pKillerRed-dMito with a CMV promoter…”

“…et al., 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.”

“…generated from the time-lapse videos and analyzed with a custom-made ImageJ macro Kymolyzer (Supplemental material; National Institutes of Health; Pekkurnaz et al., 2014). mt-KR activation Before imaging, hippocampal neurons expressing mt-KR were protected from light to prevent inadvertent ROS…”

“We there­ fore used Antimycin A, an inhibitor of respiratory complex III (Slater, 1973), to impair mitochondria.”

“Kymo-graphs were generated from the time-lapse videos and analyzed with a custom-made ImageJ macro Kymolyzer (Supplemental material; National Institutes of Health; Pekkurnaz et al., 2014).”

“Accumulation of mitochondrial damage in the brains of PD patients (Keeney et al., 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…”

“Light­induced activation of mt­KR causes local ROS­mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012).”

“The PINK1–Parkin pathway is primarily characterized in nonneuronal cells, and controversy surrounds whether it plays any role in mitochondrial quality control in neuronal processes (Van Laar et al., 2011; Wang et al., 2011; Cai et al., 2012; Grenier et al., 2013).”

“Indeed, most studies have focused on mitochondrial quality con trol in the soma (Seibler et al., 2011; Van Laar et al., 2011; Cai et al., 2012; Rakovic et al., 2013), and little is known about mitochondrial clearance from distal axons.”

“In several neurological diseases, such as PD, excessive neuronal firing is closely associated with neurodegeneration, and excitotoxicity­related increases in intracellular calcium can lead to ROS production and mitochondrial damage (Ambrosi et al., 2014).”

“…fluidic devices that allowed local application of Antimycin A. Compared with the nonspecific ionophore CCCP, Antimycin A is a milder depolarizer and has been previously used to modu late mitochondrial motility in neurons (Miller and Sheetz, 2004; Verburg and Hollenbeck, 2008; Wang et al., 2011).”

“Hippocampal neurons from embryonic day 18 mouse or rat embryos were dissected and dissociated as previously described (Nie and Sahin, 2012).”

“Despite the fact that dopami­ nergic neurons are the most pathologically affected cells in PD (Surmeier et al., 2010), the neuronal role of PINK1 and Par­ kin remained unclear.”

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

“…before rapid mitophagy, we reexamined the ques­ tion with locally restricted and near­physiological levels of our observations of mitochondrial motility arrest (Wang et al., 2011) and the accumulation of aged mitochondrial proteins in distal processes (Ferree et al., 2013) argue against this model.”

“However, flux measurements in zebrafish axons indicate a bias for mitochondrial movement in the anterograde direction, suggesting that local removal of distal mitochondria must occur to maintain mitochondrial density (Plucińska et al., 2012).”

“…antiapoptotic role by limiting the translo­ cation of proapoptotic Bax to the mitochondrial surface (Johnson et al., 2012a) and increasing the threshold for cytochrome c re­ lease from mitochondria (Berger et al., 2009), both under nor­ mal and stress conditions (Johnson et al., 2012b).”

“Light­induced activation of mt­KR causes local ROS­mediated damage to mitochondria (Bulina et al., 2006; Ertürk et al., 2014) and has been previously used to induce mitophagy in HeLa cells (Yang and Yang, 2011; Wang et al., 2012).”

“RFP­LC3 is more stable than GFP­LC3 and labels both autophagosomes and autolysosomes (Bains and Heidenreich, 2009), accounting for frequent double labeling of lysosomes with both LAMP1­ YFP and RFP­LC3.”

“Hippocampal neurons from embryonic day 18 mouse or rat embryos were dissected and dissociated as previously described (Nie and Sahin, 2012).”

“…in mitophagy, Parkin plays a PINK1independent, antiapoptotic role by limiting the translo cation of proapoptotic Bax to the mitochondrial surface (Johnson et al., 2012a) and increasing the threshold for cytochrome c re lease from mitochondria (Berger et al., 2009), both under nor mal and stress…”

“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).”