Mitochondrial redox and pH signaling occurs in axonal and synaptic organelle clusters

Redox switches are important mediators in neoplastic, cardiovascular and neurological disorders. We recently identified spontaneous redox signals in neurons at the single mitochondrion level where transients of glutathione oxidation go along with shortening and re-elongation of the organelle. We now have developed advanced image and signal-processing methods to re-assess and extend previously obtained data. Here we analyze redox and pH signals of entire mitochondrial populations. In total, we quantified the effects of 628 redox and pH events in 1797 mitochondria from intercostal axons and neuromuscular synapses using optical sensors (mito-Grx1-roGFP2; mito-SypHer). We show that neuronal mitochondria can undergo multiple redox cycles exhibiting markedly different signal characteristics compared to single redox events. Redox and pH events occur more often in mitochondrial clusters (medium cluster size: 34.1 ± 4.8 μm2). Local clusters possess higher mitochondrial densities than the rest of the axon, suggesting morphological and functional inter-mitochondrial coupling. We find that cluster formation is redox sensitive and can be blocked by the antioxidant MitoQ. In a nerve crush paradigm, mitochondrial clusters form sequentially adjacent to the lesion site and oxidation spreads between mitochondria. Our methodology combines optical bioenergetics and advanced signal processing and allows quantitative assessment of entire mitochondrial populations.

Suppl. Fig.9 sparse non-clustered activity sparse clustered activity    Fig. 1: Signal characterization Example trace of Grx1-roGFP2 oxidation during a single contraction illustrates the determination of up and down slope, signal amplitude and time intervals. For each mitochondrial signal s(t) manual trace characterization determined the start and end points of the corresponding signal rise and decay time-intervals (∆tup, ∆tdown). The amplitude (∆A) is the maximum signal rise above baseline. The corresponding rise (decay) slopes were determined with a linear (exponential) fit of s(t) according to s(t) = s0 + c•t (s(t) = s0 + b•exp(-a•t)).

Suppl. Fig. 2: Various signal forms of oxidizing mitochondria
Example traces of Grx1-roGFP2 mitochondria show a variety of traces that include no signal, single, double or multiple oxidations of single organelles.
Suppl. Fig. 3: Cervical spinal cord, peripheral nerve and NMJ of a wildtype mouse injected with AAV-mito-SypHer Representative image of a horizontal spinal cord section of a wildtype mouse, which was bilaterally injected with AAV-mito-SypHer two weeks prior (a). Magnified images show motorneurons in the ventral horn with long projections that give rise to intercostal axons. Three axons in the intercostal nerve with virally labelled mitochondria (b). Note that there is also fluorescent protein in the cytoplasm of the axons. Image of a virally labelled NMJ (c). Scale bar is 200 µm in (a), 10 µm in close ups and 2 µm in (b) and (c).

Suppl. Fig. 5: Baseline oxidation and pH of contracting and non-contracting mitochondria.
Baseline EGSH oxidation and mitochondrial matrix pH of contracting and non-contraction mitochondria. *p<0.05, **p<0.01. Fig. 6: Signal characteristics of redox and pH signals Non-, single-and multi-event intensity traces for individual mitochondria as well as their associated absolute squared wavelet transforms (lower panels) are shown for Grx1-roGFP2 (a) and SypHer (b), respectively. While non-events do not exhibit any relevant frequency features, single-event signals are associated with a wavelet transform that is smeared around the inverse signal length (~ 5-10 mHz for Grx1-roGFP2 and ~ 50-100 mHz for SypHer). For multi-event traces, the wavelet transform illustrates the additional frequency of subsequent events (e.g. ~ 8 mHz for Grx1-roGFP2 doublet and ~ 10-20 mHz for Grx1-roGFP2 multi-events, and ~ 10-30 mHz for the SypHer multi-event trace; see also dashed line in wavelet transforms). Fig. 7: Illustration of mitochondrial scaling Single trace of a representative pH spike exhibiting frequency scaling behavior (a). The absolute squared wavelet transform of the main signal (~ 30 mHz) contains high-frequency content in the buildup to the signal peak at ~180 s (b). Rise time distribution of SypHer signals (c).

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Suppl. Fig. 8: Spatial clustering of mitochondrial signals Representative axons and NMJ are shown with respective spatial clusters of mitochondrial signals (lower panel). Mitochondria that exhibit an event during the recording are depicted in blue when they are part of a spatial cluster, in black when they are not part of a spatial cluster, and in grey when they do not exhibit any event. Red outline around mitochondria indicates the respective cluster. Dashed line shows the axon / NMJ border. Scale bars are 2 µm.
Suppl. Fig. 9: Isochrone analysis Some axons show only sparse clustered activity or no apparent event clustering. This might be due to mitochondrial signals in the axon being normally distributed with local hotspots and less active regions.