WldS prevents axon degeneration through increased mitochondrial flux and enhanced mitochondrial Ca2+ buffering

Abstract

Wld(S) (slow Wallerian degeneration) is a remarkable protein that can suppress Wallerian degeneration of axons and synapses, but how it exerts this effect remains unclear. Here, using Drosophila and mouse models, we identify mitochondria as a key site of action for Wld(S) neuroprotective function. Targeting the NAD(+) biosynthetic enzyme Nmnat to mitochondria was sufficient to fully phenocopy Wld(S), and Wld(S) was specifically localized to mitochondria in synaptic preparations from mouse brain. Axotomy of live wild-type axons induced a dramatic spike in axoplasmic Ca(2+) and termination of mitochondrial movement-Wld(S) potently suppressed both of these events. Surprisingly, Wld(S) also promoted increased basal mitochondrial motility in axons before injury, and genetically suppressing mitochondrial motility in vivo dramatically reduced the protective effect of Wld(S). Intriguingly, purified mitochondria from Wld(S) mice exhibited enhanced Ca(2+) buffering capacity. We propose that the enhanced Ca(2+) buffering capacity of Wld(S+) mitochondria leads to increased mitochondrial motility, suppression of axotomy-induced Ca(2+) elevation in axons, and thereby suppression of Wallerian degeneration.

Figure 1. Mitochondria as a focal point for Wld^S-mediated axon protection

(A) Mouse Nmnat3::Myc localizes to mitochondria in Drosophila axons. 22a-Gal4 was used to drive UAS-Nmnat3::Myc and UAS-mito::GFP. Insets show boxed region. (B) Mitochondrial Nmnat3 fully mimics Wld^S in axon protective function. 22a-Gal4 was used to drive UAS-Nmnat3 or UAS-Wld^S in a background where axons were labeled with membrane-tethered GFP (UAS-mCD8::GFP). n≥20 antennal lobes for each. *** indicates p<0.001. Error bars represent ±SEM. (C) Wld^S-suppresses axotomy-induced termination of mitochondrial motility. Mitochondrial movement was assessed in live open-filet preparations of third instar Drosophila larvae immediately after axotomy for 5 minutes. Axotomy was induced by severing axons with a Micropoint laser ablation system and confirmed by a breakage of mCD8::mCherry-labeled axons. n≥10 live samples for each genotype and time point. ***, p<0.001.

Figure 2. In vivo laser axotomy induces a dramatic rise in axonal Ca²⁺ that is suppressed by Wld^S

(A) tdc2-Gal4 labels 3 axons in each peripheral nerve, only one segment is illustrated. (B) Axons were labeled with mCD8::mCherry, axonal Ca²⁺ was monitored by co-expressing GCaMP3 in the tdc2-Gal4⁺ subset of neurons. Note the breakage of the axon after laser axotomy (red, mCherry). Axonal Ca²⁺ levels one minute after axotomy (green, GCaMP3). (C,D) Representative traces showing Ca²⁺ responses in axon fragments distal to the injury site over time. Genotypes as indicated. (E) Quantification of peak Ca²⁺ intensities and time to ½ recovery from average peak intensity for each genotype listed. n≥5 live samples for each genotype and time point versus control. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 3. Wld^s increases mitochondrial flux, which is essential for neuroprotective function

(A) Mitochondrial flux was assayed in tdc2-Gal4-expressing neurons in live preparations of third instar larvae. Representative kymographs of mitochondrial movement are shown for control and Wld^S-expressing axons. Anterograde is to the right, retrograde is to the left. (B) Mitochondrial flux was assayed in axons expressing each of the following molecules: Nmnat1, DN16-Wld^S, Wld^S-dead, Wld^S, N16-Nmnat1, and Nmant3. n≥10 live samples for each genotype. *, p<0.05; ***, p<0.001. (C) Quantification of the movement of individual mitochondrial by binning into mobile, docked, or pausing/releasing during a 5 minute window. n=5 movies for each genotype. (D) ORN axotomy assays in Wld^S backgrounds and miro mutants. A single antennal lobe where ORN axons were severed is shown. (E) Quantification of data from (D). Age-matched uninjured controls at the same time points are shown at right. n≥15 samples for each genotype and time point. **, p<0.01; ***, p<0.001.

Figure 4. Wld^S brain mitochondria display higher Ca²⁺ load capacity than age matched wild type (NJ) controls

(A,B) TMRE (membrane potential indicator) and CaG5N (extra-mitochondrial Ca²⁺ indicator) fluorescence were monitored over time simultaneously for each sample of non-synaptic mitochondria. As illustrated in TMRE traces for the first three minutes, the addition of pyruvate and malate (PM) an oxidative substrate, causes a marked downward deflection at 1 minute due to increased mitochondrial membrane potential (Δψm). Following ADP (A) addition, the loss of Δψm is indicated by upward deflection at 2 mins as Δψm is utilized to phosphorylate ADP to ATP via proton flow thru the ATP synthase. The ATP synthase inhibitor, oligomycin (O) addition at 3 min results in maximum Δψm as proton flow is inhibited. The Ca²⁺ infusion began at 5 min (infusion rate 160 nmol of Ca²⁺/mg protein/min) and was monitored by CaG5N fluorescence, and is illustrated by the initial upward deflection followed by constant signal due to mitochondrial Ca²⁺ uptake into the matrix. The subsequent rise in CaG5N fluorescence accompanied by a loss of membrane potential signifies mitochondrial permeability transition and subsequent release of mitochondrial Ca²⁺. (C) Quantification of mitochondrial Ca²⁺ buffering capacity (nmols/mg protein) indicates that Wld^S non-synaptic mitochondria sequestered significantly higher amounts of Ca²⁺ compared to control group (n=6/group, * p<0.05, unpaired t-test).