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Redox Biol
2018 Jun 01;16:344-351. doi: 10.1016/j.redox.2018.03.012.
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Mitochondrial ROS cause motor deficits induced by synaptic inactivity: Implications for synapse pruning.
Sidlauskaite E
,
Gibson JW
,
Megson IL
,
Whitfield PD
,
Tovmasyan A
,
Batinic-Haberle I
,
Murphy MP
,
Moult PR
,
Cobley JN
.
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Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS-using mitochondria-targeted Paraquat (MitoPQ)-recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features.
Fig. 1. α-BTX induced motor deficits are redox regulated. (A) α-BTX mechanism of action scheme. Left: skeletal muscle activity stems from acetylcholine (ACh) binding to post-synaptic nicotinic acetylcholine receptor (nAChR) to permit Na+ entry. Right: α-BTX ligates the nAChR to render it impermeable to Na+ leading to forced post-synaptic inactivity induced motor deficits. (B) Evoked swimming frequency (expressed as %δ control) by condition (α-BTX, α-BTX plus: MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- or MitoTempol; n = 10 in each condition). (C) Mito-SOX oxidation (expressed as %δ control) by condition (n = 10 per condition). (D) MitoPY1 oxidation (expressed as %δ control) by condition (n = 10 per condition). Error bars are SEM. Concentrations and incubations: α-BTX (8 µM for 30 min), MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- conditions = 1 µM for 30 min. MitoTempol = 20 µM for 30 min. # denotes significant difference vs control. * denotes significant difference vs α-BTX.
Fig. 2. α-LTX induced motor deficits are redox regulated. (A) α-LTX mechanism of action scheme. α-LTX binds to nerve terminals to cause mass neurotransmitter vesicle depletion, leading to pre-synaptic inactivity. (B) Evoked swimming frequency (expressed as %δ control) by condition (α-LTX, α-LTX plus: MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- or MitoTempol; n = 10 in each condition). (C) Mito-SOX oxidation (expressed as %δ control) by condition (n = 10 per condition). (D) MitoPY1 oxidation (expressed as %δ control) by condition (n = 10 per condition). Error bars are SEM. Concentrations and incubations: α-LTX (15 nM for 30 min), MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- conditions = 1 µM for 30 min. MitoTempol = 20 µM for 30 min. # denotes significant difference vs control. * denotes significant difference vs α-LTX.
Fig. 3. SPARC induced motor deficits are redox regulated. (A) SPARC mechanism of action scheme. SPARC binds to weak synaptic inputs to selectively induce pruning—via a yet to be fully elucidated mechanism. Withdrawing inputs (retraction bulbs) are inactive; hence, global SPARC treatment can silence multiple NMJs to induce motor deficits. (B) Evoked swimming frequency (expressed as %δ control) by condition (SPARC, SPARC plus: MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- or MitoTempol; n = 10 in each condition). (C) Mito-SOX oxidation (expressed as %δ control) by condition (n = 10 per condition). (D) MitoPY1 oxidation (expressed as %δ control) by condition (n = 10 per condition). Error bars are SEM. Concentrations and incubations: SPARC (50 nM for 30 min), MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- conditions = 1 µM for 30 min. MitoTempol = 20 µM for 30 min. # denotes significant difference vs control. * denotes significant difference vs SPARC.
Fig. 4. MitoPQ induced motor deficits are redox regulated. (A) MitoPQ mechanism of action scheme. Within the mitochondrial matrix, MitoPQ accepts an electron from complex I to yield a MitoPQ radical. The MitoPQ radical reacts with molecular oxygen (O2) to generate O2.-, leading to an increase in matrix [O2.- and H2O2] (the latter as a consequence of MnSOD mediated O2.- dismutation). (B) Mito-SOX oxidation (expressed as %δ control) by condition (MitoPQ, MitoPQ plus: MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- or MitoTempol n = 10 per condition). (C) MitoPY1 oxidation (expressed as %δ control) by condition (n = 10 per condition). (D) Evoked swimming frequency (expressed as %δ control) by condition (n = 10 in each condition). Error bars are SEM. Concentrations and incubations: MitoPQ (5 µM for 60 min), MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- conditions = 1 µM for 30 min. MitoTempol = 20 µM for 30 min. #denotes significant difference vs control. *denotes significant difference vs MitoPQ.
Supplementary Fig. 1. No statistical difference in cell viability between MitoPQ vs control treated primary muscle-nerve co-cultures.
Supplementary Fig. 2. Representative florescent images of Mito-SOX oxidation in vivo by synaptic inactivity model (α-BTX, α-LTX, SPARC and MitoPQ) and treatment condition (control, MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- and Mito-Tempol) taken from the intermyotomal region of stage 37–38 X. laevis tadpoles using whole-mount redox imaging. Scale bar is 0.1 mm.
Supplementary Fig. 3. Representative florescent images of MitoPY1 oxidation in vivo by synaptic inactivity model (α-BTX, α-LTX, SPARC and MitoPQ) and treatment condition (control, MnTE-2-PyP5+, MnTnBuOE-2-PyP5+, MnTBAP3- and Mito-Tempol) taken from the intermyotomal region of stage 37–38 X. laevis tadpoles using whole-mount redox imaging. Scale bar is 0.1 mm.
Supplementary Fig. 4. A stage 37–38 X. laevis tadpole with the location of a representative intermyotomal region sampled (red box) with florescent images of Mito-SOX and MitoPY1 oxidation shown below.
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