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• W2945482967 abstract "•A glutathione S-transferase (GST), Gfzf, decreases mitochondrial fusion in axons•Gfzf loss causes hyperfusion through enhanced oxidation of glutathione•GST loss or enhanced oxidation of glutathione increases the function of mitofusin•Hyperfusion was associated with functional changes but not neurodegeneration Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer’s. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link. Mitochondria are essential in long axons to provide metabolic support and sustain neuron integrity. A healthy mitochondrial pool is maintained by biogenesis, transport, mitophagy, fission, and fusion, but how these events are regulated in axons is not well defined. Here, we show that the Drosophila glutathione S-transferase (GST) Gfzf prevents mitochondrial hyperfusion in axons. Gfzf loss altered redox balance between glutathione (GSH) and oxidized glutathione (GSSG) and initiated mitochondrial fusion through the coordinated action of Mfn and Opa1. Gfzf functioned epistatically with the thioredoxin peroxidase Jafrac1 and the thioredoxin reductase 1 TrxR-1 to regulate mitochondrial dynamics. Altering GSH:GSSG ratios in mouse primary neurons in vitro also induced hyperfusion. Mitochondrial changes caused deficits in trafficking, the metabolome, and neuronal physiology. Changes in GSH and oxidative state are associated with neurodegenerative diseases like Alzheimer’s. Our demonstration that GSTs are key in vivo regulators of axonal mitochondrial length and number provides a potential mechanistic link. Most neurons are generated during embryogenesis and are subsequently maintained throughout the entire life of an organism. Mitochondria are integral to sustaining neuronal health, and the long polarized processes of neurons pose a unique challenge for adequate mitochondrial positioning and maintenance. Cellular homeostasis and adequate ATP production is thought to be achieved in axons through regulation of several essential mitochondrial processes, including mitochondrial fusion, fission, biogenesis, degradation, and transport (Harbauer, 2017Harbauer A.B. Mitochondrial health maintenance in axons.Biochem. Soc. Trans. 2017; 45: 1045-1052Crossref PubMed Scopus (12) Google Scholar). The major pathways responsible for regulating these have been increasingly well described over the past decade and are highly conserved from humans to Drosophila (Hewitt and Whitworth, 2017Hewitt V.L. Whitworth A.J. Mechanisms of Parkinson’s disease: lessons from Drosophila.Curr. Top. Dev. Biol. 2017; 121: 173-200Crossref PubMed Scopus (59) Google Scholar, Vanhauwaert and Verstreken, 2015Vanhauwaert R. Verstreken P. Flies with Parkinson’s disease.Exp. Neurol. 2015; 274: 42-51Crossref PubMed Scopus (26) Google Scholar), but whether they uniformly regulate mitochondrial biology similarly in all neuronal compartments remains an open question. Rapid and dynamic changes in mitochondrial length occur in response to the ever-changing environment of the cell (van der Bliek et al., 2013van der Bliek A.M. Shen Q. Kawajiri S. Mechanisms of mitochondrial fission and fusion.Cold Spring Harb. Perspect. Biol. 2013; 5: a011072Crossref PubMed Scopus (505) Google Scholar). Conditions that increase mitochondrial ATP consumption lead to enhanced fusion, allowing for the mixing of mtDNA and proteins, whereas metabolic signals that grossly uncouple the mitochondria may result in fusion inhibition and occur as a prerequisite to mitophagy and/or neurodegeneration (van der Bliek et al., 2013van der Bliek A.M. Shen Q. Kawajiri S. Mechanisms of mitochondrial fission and fusion.Cold Spring Harb. Perspect. Biol. 2013; 5: a011072Crossref PubMed Scopus (505) Google Scholar). Increased mitochondrial length can be classically achieved through increased function of the mitochondrial fusion proteins mitofusin (MFN) and optic atrophy 1 (OPA1) or decreased activity of the mitochondrial fission factors dynamin-related protein 1 (DRP1) and fission mitochondrial 1 (FIS1) (van der Bliek et al., 2013van der Bliek A.M. Shen Q. Kawajiri S. Mechanisms of mitochondrial fission and fusion.Cold Spring Harb. Perspect. Biol. 2013; 5: a011072Crossref PubMed Scopus (505) Google Scholar). The precise cellular processes that control the expression and function of these molecules for the dynamic regulation of mitochondria in long axon stretches are not well understood under physiological conditions or in complex diseases. Few key upstream modulating factors of the fission-fusion machinery have been identified (Anding et al., 2018Anding A.L. Wang C. Chang T.-K. Sliter D.A. Powers C.M. Hofmann K. Youle R.J. Baehrecke E.H. Vps13D encodes a ubiquitin-binding protein that is required for the regulation of mitochondrial size and clearance.Curr. Biol. 2018; 28: 287-295.e6Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, van der Bliek et al., 2013van der Bliek A.M. Shen Q. Kawajiri S. Mechanisms of mitochondrial fission and fusion.Cold Spring Harb. Perspect. Biol. 2013; 5: a011072Crossref PubMed Scopus (505) Google Scholar, Burman et al., 2017Burman J.L. Pickles S. Wang C. Sekine S. Vargas J.N.S. Zhang Z. Youle A.M. Nezich C.L. Wu X. Hammer J.A. Youle R.J. Mitochondrial fission facilitates the selective mitophagy of protein aggregates.J. Cell Biol. 2017; 216: 3231-3247Crossref PubMed Scopus (252) Google Scholar, Farmer et al., 2017Farmer T. Reinecke J.B. Xie S. Bahl K. Naslavsky N. Caplan S. Control of mitochondrial homeostasis by endocytic regulatory proteins.J. Cell Sci. 2017; 130: 2359-2370Crossref PubMed Scopus (28) Google Scholar, Otera and Mihara, 2011Otera H. Mihara K. Molecular mechanisms and physiologic functions of mitochondrial dynamics.J. Biochem. 2011; 149: 241-251Crossref PubMed Scopus (204) Google Scholar, Otera et al., 2013Otera H. Ishihara N. Mihara K. New insights into the function and regulation of mitochondrial fission.Biochim. Biophys. Acta. 2013; 1833: 1256-1268Crossref PubMed Scopus (329) Google Scholar). Oxidation and reduction (redox) reactions of the glutathione (GSH) pathway take place in all cells and are essential for vital metabolic processes, including the production of ATP. Glutathione S-transferases (GSTs), reductases (GR), and peroxidases (GPx) control redox homeostasis and tightly balance the ratio of the antioxidant GSH and the toxic species oxidized glutathione (GSSG) (Aquilano et al., 2014Aquilano K. Baldelli S. Ciriolo M.R. Glutathione: new roles in redox signaling for an old antioxidant.Front. Pharmacol. 2014; 5: 196Crossref PubMed Scopus (469) Google Scholar). Mitochondria can be affected by GSH redox changes that manifest in neurological diseases and stress response conditions by several mechanisms. First, mitochondria cannot produce GSH themselves, so they rely on its import from the cytoplasm for the adequate detoxification of reactive oxygen species (ROS) (Marí et al., 2009Marí M. Morales A. Colell A. García-Ruiz C. Fernández-Checa J.C. Mitochondrial glutathione, a key survival antioxidant.Antioxid. Redox Signal. 2009; 11: 2685-2700Crossref PubMed Scopus (667) Google Scholar, Ribas et al., 2014Ribas V. García-Ruiz C. Fernández-Checa J.C. Glutathione and mitochondria.Front. Pharmacol. 2014; 5: 151Crossref PubMed Scopus (323) Google Scholar). Second, GSSG accumulation can cause widespread oxidation of proteins and lipids, including essential mitochondrial proteins of the electron transport chain (Marí et al., 2009Marí M. Morales A. Colell A. García-Ruiz C. Fernández-Checa J.C. Mitochondrial glutathione, a key survival antioxidant.Antioxid. Redox Signal. 2009; 11: 2685-2700Crossref PubMed Scopus (667) Google Scholar, Ribas et al., 2014Ribas V. García-Ruiz C. Fernández-Checa J.C. Glutathione and mitochondria.Front. Pharmacol. 2014; 5: 151Crossref PubMed Scopus (323) Google Scholar). While large shifts in intracellular redox are detrimental to cells, small shifts in the GSH:GSSG ratio have been reported to induce mitochondrial-associated changes that are not associated with toxicity in vitro (Shutt et al., 2012Shutt T. Geoffrion M. Milne R. McBride H.M. The intracellular redox state is a core determinant of mitochondrial fusion.EMBO Rep. 2012; 13: 909-915Crossref PubMed Scopus (181) Google Scholar). In this study, we developed a high-throughput method to identify new molecules required for axonal mitochondrial maintenance in vivo. We discovered that a novel GST, Gfzf, homologous to GSTT1 in humans, regulates mitochondrial length in axons and functions by altering redox balance. Ablation of gfzf was found to cause the oligomerization of the outer mitochondrial membrane fusion factor Marf, a fly homolog of MFN, to initiate the fusion process and induce a complete fusion of the inner membrane through a secondary OPA1-mediated response. We show that mitochondrial fusion events also require the coordinated action of the specific GPx Jafrac1 and can be ameliorated by the expression of the GR TrxR-1. Loss of this GST caused significant functional changes within the neuron, including mitochondrial trafficking and electrophysiology. GSTs can therefore be classified as new essential upstream regulators of redox-mediated mitochondrial fusion. Mitochondrial maintenance processes present in non-polarized cells may occur in the axonal compartment of neurons (Amiri and Hollenbeck, 2008Amiri M. Hollenbeck P.J. Mitochondrial biogenesis in the axons of vertebrate peripheral neurons.Dev. Neurobiol. 2008; 68: 1348-1361Crossref PubMed Scopus (117) Google Scholar, Ashrafi and Schwarz, 2015Ashrafi G. Schwarz T.L. PINK1- and PARK2-mediated local mitophagy in distal neuronal axons.Autophagy. 2015; 11: 187-189PubMed Google Scholar, Ashrafi et al., 2014Ashrafi G. Schlehe J.S. LaVoie M.J. Schwarz T.L. Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin.J. Cell Biol. 2014; 206: 655-670Crossref PubMed Scopus (360) Google Scholar, Cartoni et al., 2016Cartoni R. Norsworthy M.W. Bei F. Wang C. Li S. Zhang Y. Gabel C.V. Schwarz T.L. He Z. The mammalian-specific protein Armcx1 regulates mitochondrial transport during axon regeneration.Neuron. 2016; 92: 1294-1307Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, Harbauer, 2017Harbauer A.B. Mitochondrial health maintenance in axons.Biochem. Soc. Trans. 2017; 45: 1045-1052Crossref PubMed Scopus (12) Google Scholar, Lee et al., 2018Lee J.J. Sanchez-Martinez A. Zarate A.M. Benincá C. Mayor U. Clague M.J. Whitworth A.J. Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin.J. Cell Biol. 2018; 217: 1613-1622Crossref PubMed Scopus (179) Google Scholar, Misgeld and Schwarz, 2017Misgeld T. Schwarz T.L. Mitostasis in neurons: maintaining mitochondria in an extended cellular architecture.Neuron. 2017; 96: 651-666Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, Schwarz, 2013Schwarz T.L. Mitochondrial trafficking in neurons.Cold Spring Harb. Perspect. Biol. 2013; 5: a011304Crossref PubMed Scopus (224) Google Scholar), but the similarities or differences remain poorly defined. We devised an in vivo forward genetic screen to identify new factors controlling mitochondrial dynamics in axons with single axon and mitochondrial resolution using Drosophila (Figure 1A). We used the MARCM system to visualize a subset of glutamatergic neurons in the adult Drosophila wing (Neukomm et al., 2014Neukomm L.J. Burdett T.C. Gonzalez M.A. Züchner S. Freeman M.R. Rapid in vivo forward genetic approach for identifying axon death genes in Drosophila.Proc. Natl. Acad. Sci. USA. 2014; 111: 9965-9970Crossref PubMed Scopus (42) Google Scholar) and simultaneously labeled mitochondria (Figure 1B). Flies were fed the chemical mutagen ethyl methane sulfonate (EMS) and crossed to generate progeny containing MARCM clones in the F1 generation (Neukomm et al., 2017Neukomm L.J. Burdett T.C. Seeds A.M. Hampel S. Coutinho-Budd J.C. Farley J.E. Wong J. Karadeniz Y.B. Osterloh J.M. Sheehan A.E. Freeman M.R. Axon death pathways converge on axundead to promote functional and structural axon disassembly.Neuron. 2017; 95: 78-91.e5Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These animals were screened for changes in mitochondrial number, size, and position. This approach allowed us to discover both lethal and nonlethal mutants that regulate mitochondrial in adult post-mitotic neurons in vivo. To begin characterizing this system, we assayed the effects of loss-of-function manipulations of known molecules that regulate mitochondrial biology at 7 days post-eclosion (p.e.) (Figure 1C). Mitochondria in control neurons were distributed throughout the cell body and the axon and were stereotyped in size and density per unit axon area (Figure 1Ci). Induction of neuronal clones with a null mutation in the mitochondrial transport gene Milton resulted in the failure of mitochondria to enter the axonal compartment (Figure 1Cii), in agreement with Vagnoni et al., 2016Vagnoni A. Hoffmann P.C. Bullock S.L. Reducing Lissencephaly-1 levels augments mitochondrial transport and has a protective effect in adult Drosophila neurons.J. Cell Sci. 2016; 129: 178-190Crossref PubMed Scopus (29) Google Scholar. Mutations in mitochondrial fission gene Drp1 caused fusion of mitochondria in the axon and a hyperfusion phenotype in the cell body (Figure 1Ciii; Figure S1). Mutants lacking the fusion factor Marf exhibited a significant shortening of mitochondria within the axon and a diffuse appearance in the soma (Figure 1Civ). Given the high efficiency and resolution of this system for examining mitochondrial phenotypes in axons in an in vivo setting and the potential to identify new regulators of mitochondrial physiology, we screened through ∼8,000 mutagenized chromosomes on the left arm of the 3rd chromosome (representing ∼20% of the Drosophila genome). We discovered one lethal mutant, line #541, where mitochondria were significantly longer in axons compared to non-mutagenized controls, which we functionally characterize below. A combination of whole-genome sequencing and deficiency mapping was used to identify a premature stop mutation in GST-containing FLYWCH zinc-finger protein (gfzf), a GST gene, that caused the long mitochondrial phenotype. Outcrossed flies that retained the phenotype were always associated with lethality. We found three overlapping deficiencies that failed to complement #541 in the genomic region of gfzf mutation revealed by the sequencing. Based on this observation and rescue experiments (below) we refer to mutant #541 as gfzf−/−. Increased mitochondrial length in gfzf−/− mutants was found to be age dependent (Figure 2). While only a minimal phenotypic difference was observed at 2 days p.e., excessively long mitochondria were observed at both 7 and 28 days (Figure 2A). Mitochondrial morphology was also altered in the neuronal cell bodies of gfzf−/− mutant clones at later time points (Figure 2A). Quantification of mitochondrial length in axons revealed that at 7 and 28 days, mitochondria were on average 3–4 times longer than age-matched controls (Figure 2B), and the total number of mitochondria significantly decreased (Figure 2C). For instance, by 28 days p.e., one single long mitochondrion was observed in a 60 μm length of mutant axon, whereas control neurons contained ∼5 shorter mitochondria (Figure 2C). Analysis of the frequency distribution of mitochondrial length in gfzf−/− mutant axons revealed a shift in length from a typical control range of 0.1–2.61 μm to a much broader range of 0.41 to >5 μm (Figure 2D). In the experiments described above, mitochondrial lengths were analyzed in a defined region of the proximal wing (Figure 1A). However, given the substantial length of these glutamatergic neurons, we wished to determine whether the alterations in mitochondrial length observed in gfzf#541 mutants were present throughout the axon, and this was indeed the case (Figures S2A and S2B). A frequency distribution shift of mitochondrial length also manifested as animals aged (Figures S2C–S2E). Therefore, gfzf−/− mutants affected mitochondrial length in axons independent of their location relative to the soma, and the effect increases with age. The total percentage axonal area occupied by mitochondria in the distal wing was further quantified within the axon and cell body compartments and not significantly changed (Figures S2F and S2G). This indicated that the mitochondrial phenotype is likely a fission-fusion deficit that does not affect biogenesis or degradation pathways. To confirm that the stop mutation in gfzf−/− was indeed responsible for the alterations in mitochondrial size, we expressed a full-length wild-type gfzf cDNA in gfzf#541 mutant clones. Upstream activating sequence (UAS)-mediated expression of gfzf was sufficient to rescue long mitochondrial phenotypes observed in the axons and cell bodies at 7 days p.e. (Figure 3A). Expression of 5xUAS-gfzf significantly reduced mitochondrial length compared to baseline, equivalent to those observed in controls (Figures 3B and 3C). A genomic bacterial artificial chromosome (BAC) clone also fully rescued the mitochondrial phenotype in gfzf#541 mutants (Venken et al., 2009Venken K.J.T. Carlson J.W. Schulze K.L. Pan H. He Y. Spokony R. Wan K.H. Koriabine M. de Jong P.J. White K.P. et al.Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.Nat. Methods. 2009; 6: 431-434Crossref PubMed Scopus (280) Google Scholar), (Figures 3B and 3C). Autonomous expression of gfzfΔN, which still harbors the GST domain (but lacks the zinc finger [ZNF] domains), was also sufficient to rescue the mitochondrial phenotype in gfzf mutants. These findings argue that the GST domain is the key regulatory domain in Gfzf necessary to modulate mitochondrial length in axons. A range of other mutants of gfzf have been generated (Provost et al., 2006Provost E. Hersperger G. Timmons L. Ho W.Q. Hersperger E. Alcazar R. Shearn A. Loss-of-function mutations in a glutathione S-transferase suppress the prune-Killer of prune lethal interaction.Genetics. 2006; 172: 207-219Crossref PubMed Scopus (10) Google Scholar), outlined in (Figure S3A), but we found that the nature of these mutations was less severe than gfzf#541. gfzf#541 was the only allele that failed to complement the lethality of a deficiency lacking gfzf, or a transposon inserted in gfzf Mi(MIC)gfzfMI08697 (Figure S3B). We therefore propose that gfzf#541 is a null allele and will also refer to gfzf#541 as gfzf−/−. gfzfCZ811 also failed to complement most other alleles, including gfzf−/− and the Mi(MIC)gfzfMI08697 line, and showed increased mitochondrial length and decreased mitochondrial number (Figures S3C–S3E). No clones were observed gfzfCL1027, suggesting this chromosome arm may harbor an additional cell lethal mutation. This independently derived allele further confirms that loss of Gfzf causes increased mitochondrial length. Rescue experiments indicate that embryonic lethality caused by Gfzf loss was likely not the result of neuronal-specific deficits, decreased GST activity, or mitochondrial fusion perturbations (Figure S3F). Therefore, Gfzf seems to have a dual role: an essential role in fly development in other tissues that does not depend on GST activity and a role to maintain mitochondrial length in adult neurons in which GST is both necessary and sufficient. The GST domain of Gfzf has a 30% identity and 48% homology with the GST domain of human GSTT1 (Figure S3G). Cas9 and guide RNA (gRNA)-mediated targeting of the mammalian GST homolog GSTT1 was also sufficient to increase mitochondrial length in cultured neurons in vitro (Figure S4), arguing for strong conservation of GST-mediated regulation of mitochondrial size. Mitochondrial phenotypes were first rescued by expression of Gfzf in gfzf−/− clones confirming the causative gene (Figure 3A) before investigating the interaction with other GSTs. There are multiple GSTs in the Drosophila genome, all of which can likely regulate GSH:GSSG ratios. To determine whether the ability of the GST domain of Gfzf to regulate mitochondrial length was specific, we tested whether other cytoplasmic localized GSTs could compensate for Gfzf or directly regulate mitochondrial length in axons (Figures 3B and 3C). We found that the expression of dGstO1 could reduce mitochondrial length in gfzf−/− neurons (Figure 3B). This suggests that GST-domain-containing proteins in Drosophila have the general ability to regulate mitochondria and may function synergistically to determine axonal mitochondrial length. GST-domain-containing proteins are highly conserved between the fruit fly and humans. We further found that mitochondrial length in gfzf−/− clones was rescued by expression of hGSTT1, (Figure 3B), but not hGSTT2, hGSTO1, hGSTO2, and hGSTM1 (Table S1), arguing that human GSTT1 may be the most conserved GST-containing protein for maintaining mitochondria dynamics in neurons. Rescue of mitochondrial length was always accompanied with a rescue in number per unit of axon length (Figure 3C), supporting a causal link between these phenotypes. Genetic manipulations that lead to dramatic changes in mitochondrial morphology are also tightly associated with perturbation of mitochondrial physiology and age-dependent neurodegeneration. We used in vivo live-cell imaging to assess mitochondrial Ca2+ levels, mitochondrial age and redox potential specific to GSSG and H2O2, in gfzf mutant clones, using 5xUAS-mito-GCaMP5, 5xUAS-mitoTimer, 5xUAS-mito-Grx1-roGFP2 and 5xUAS-mito-roGFP2-Orp1 respectively (Figures 3D–3G). We detected no significant differences in the fluorescence intensity of mitochondrial localized GCaMP5 (Figure 3D) and mitoTimer (Figure 3E). These data support the notion that mitochondria in gfzf−/− mutant axons remain broadly functional. There was a significant increase in the fluorescence intensity depicting GSSG within the mitochondria from 7 days, which was not detectable in controls (Figure 3F). The fluorescence signal denoting H2O2 levels remained unchanged (Figure 3G). Electroretinogram (ERG) recordings in aged flies further indicated that these dynamic mitochondrial changes were associated with neuronal response phenotypes (Figures 3H–3M). Amplitude of the off-transient was reduced, the time to reach half-maximal corneal depolarization was delayed, and the time to fully repolarize was shortened in gfzf−/− backgrounds relative to wild type (WT). Changes were rescued by a BAC clone or pan-neuronal knockdown of Marf, suggesting that physiological responses were directly related to the GSH pathway and mitochondrial hyperfusion (Figures S5A–S5C). gfzf−/− mutant axons remain intact in vivo, and baseline Ca2+ levels were not significantly different from control, yet a significant decrease in mitochondrial transport was observed (Figures S5D–S5G). This indicates that large mitochondria had significantly higher GSSG content, which may impact bioenergetics, transport, and neuronal function. We generated a transgenic stock containing 5xUAS-gfzf::GFP and drove its expression using the pan-neuronal synapsin1-Gal4 driver. Gfzf::GFP was observed throughout the neuron (Figure S6A). The localization of this molecule in cultured Drosophila S2 cells is also cytoplasmic (Dai et al., 2004Dai M.-S. Sun X.-X. Qin J. Smolik S.M. Lu H. Identification and characterization of a novel Drosophila melanogaster glutathione S-transferase-containing FLYWCH zinc finger protein.Gene. 2004; 342: 49-56Crossref PubMed Scopus (13) Google Scholar). Regulation of mitochondrial size by Gfzf was also observed in other neuronal subtypes (Figures S6B–S6E). Interestingly, morphological changes in mitochondria were not observed at synaptic terminals or in glial clones (Figures S7A and S7B), suggesting that synaptic and glial mitochondrial size may be regulated by different mechanisms or other GSTs. Finally, we examined other organelles (lysosomes, endosomes, and peroxisomes) and observed no differences in distribution, number, and size of these vesicles between genotypes (Figures S7C–S7E). Together these data indicate that the Gfzf selectively regulates mitochondria in a pan-neuronal fashion and contributes to shaping the mitochondrial network in the axonal and cell body compartments. To explore the mechanism by which long mitochondria are generated in gfzf mutant clones, we examined the epistatic relationship between Gfzf and known fission and fusion factors. We found that increased expression of Drp1, Marf, or Pink1 and RNAi-mediated knockdown of Marf or Opa1 in adult neurons in vivo were sufficient to rescue the long mitochondrial phenotype of gfzf−/− mutants (Figures 4A and 4B ). Knockdown of the mitochondrial fusion factors Opa1 and Marf caused a complete rescue of the long mitochondrial phenotype, which was also observed in double mutants of gfzf−/−, Opa1+/− (Figure 4B). In each case, factors that increased mitochondrial length resulted in a decrease in mitochondrial number (Figure 4C). Interestingly, we found that promoting fusion by expression of Marf caused a biphasic shift in mitochondrial length and number (Figures 4D and 4E) and ultimately age-dependent neurodegeneration (Figure 4F). We simultaneously knocked down Marf in neurons overexpressing Gfzf, and no additive effect was observed, providing further epistatic evidence that Gfzf and Marf act in the same genetic pathway (Table S2). These observations indicate the increased mitochondrial length observed in gfzf−/− mutant clones can be reversed by altering factors in the classical fission-fusion pathway and suggest that increased mitochondrial length in gfzf null clones is caused by hyperfusion of mitochondria. The GST activity of Gfzf is therefore a novel potential upstream modulator of inner and outer mitochondrial membrane fusion. GSTs bind GSH to oxidized molecules and lipids for the purpose of cellular detoxification (Marí et al., 2009Marí M. Morales A. Colell A. García-Ruiz C. Fernández-Checa J.C. Mitochondrial glutathione, a key survival antioxidant.Antioxid. Redox Signal. 2009; 11: 2685-2700Crossref PubMed Scopus (667) Google Scholar, Ribas et al., 2014Ribas V. García-Ruiz C. Fernández-Checa J.C. Glutathione and mitochondria.Front. Pharmacol. 2014; 5: 151Crossref PubMed Scopus (323) Google Scholar), producing GSSG as a byproduct. We speculated that GSTs might modulate the capacity of Marf to drive mitochondrial fusion through a shift in the GSH:GSSG ratio. To test this possibility, we used two small-molecule treatments to change the redox state in cultured mouse primary cortical neurons: L-buthionine-sulfoximine (BSO) to block GSH synthesis (Dai et al., 2004Dai M.-S. Sun X.-X. Qin J. Smolik S.M. Lu H. Identification and characterization of a novel Drosophila melanogaster glutathione S-transferase-containing FLYWCH zinc finger protein.Gene. 2004; 342: 49-56Crossref PubMed Scopus (13) Google Scholar) and diamide (Dia) to induce the rapid conversion of GSH to GSSG. We found that 48 h of BSO treatment or 1 h of Dia treatment resulted in clear morphological changes of mitochondria in cortical neuron projections (Figures 5A and 5B ). Under these conditions, there was no evidence of cell death (Table S3). Mitochondria in neurites were significantly longer following BSO or Dia treatments compared to vehicle (Figure 5C), and the number of mitochondria was reduced compared to vehicle treatment (Figure 5D), phenocopying genetic in vivo data. These results show that reducing GSH:GSSG ratios initiates increases in mitochondrial length in mammalian neuronal projections. To explore how increased GSSG might modulate known components of the mitochondrial fusion machinery, we examined mitofusin 2 (MFN2) localization in primary neuron cultures. Treatment with BSO or Dia resulted in MFN2-positive puncta that were larger in size and fewer in number compared to vehicle conditions (Figure 5E). This suggests that heightened GSSG levels might act by increasing oligomerization of MFN2 to promote fusion, as described previously (Shutt et al., 2012Shutt T. Geoffrion M. Milne R. McBride H.M. The intracellular redox state is a core determinant of mitochondrial fusion.EMBO Rep. 2012; 13: 909-915Crossref PubMed Scopus (181) Google Scholar). Biochemical analysis further showed that the redox state had no obvious effect on fission-fusion factors other than MFN2 (Figures 5F and 5G). These experiments support the notion that GST-dependent modulation of glutathione redox balance is an evolutionarily conserved mechanism by which MFN2 oligomerization can be altered to modulate mitochondrial dynamics in axons. The dramatic changes in mitochondrial size we observed by manipulating GSTs in vivo or the GSH:GSSG balance in vitro might be predicted to have significant effects on mitochondrial metabolism or physiology. Similar to our in vivo findings, pharmacological treatments with BSO and Dia did n" @default.
• W2945482967 created "2019-05-29" @default.
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• W2945482967 date "2019-07-01" @default.
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• W2945482967 title "Glutathione S-Transferase Regulates Mitochondrial Populations in Axons through Increased Glutathione Oxidation" @default.
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