In vivo real-time dynamics of ATP and ROS production in axonal mitochondria show decoupling in mouse models of peripheral neuropathies

Abstract

Mitochondria are critical for the function and maintenance of myelinated axons notably through Adenosine triphosphate (ATP) production. A direct by-product of this ATP production is reactive oxygen species (ROS), which are highly deleterious for neurons. While ATP shortage and ROS levels increase are involved in several neurodegenerative diseases, it is still unclear whether the real-time dynamics of both ATP and ROS production in axonal mitochondria are altered by axonal or demyelinating neuropathies. To answer this question, we imaged and quantified mitochondrial ATP and hydrogen peroxide (H 2 O 2 ) in resting or stimulated peripheral nerve myelinated axons in vivo , using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. We found that ATP and H 2 O 2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H 2 O 2 productions but with different dynamics: ROS production peaked shortly and transiently after the stimulation while ATP production increased gradually for a longer period of time. In neuropathic MFN2 R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, elevated H 2 O 2 production was largely sustained. Finally, inducing demyelination with lysophosphatidylcholine resulted in a reduced level of ATP while H 2 O 2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity. Electronic supplementary material The online version of this article (10.1186/s40478-019-0740-4) contains supplementary material, which is available to authorized users.

The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

“…In wildtype neurons, a first burst generated a H2O2 but no ATP peak and a second burst applied 30 min later generated a second H2O2 peak followed by an ATP rise 20 min later. In neurons from R94Q mice, this ATP peak was greatly attenuated [15] which is in line with our findings that prior H2O2 exposure affects ATP production.…”

“…Maybe the hydrogen peroxide challenge affected succinate oxidation. Others also reported that R94Q mitochondria failed to upregulate ATP production following burst neuronal activation [15]. In wildtype neurons, a first burst generated a H2O2 but no ATP peak and a second burst applied 30 min later generated a second H2O2 peak followed by an ATP rise 20 min later.…”

“…Using the same model and elaborate techniques to quantify mitochondrial ATP and hydrogen peroxide in resting or stimulated peripheral nerve myelinated axons in vivo, it was recently demonstrated that R94Q mitochondria fail to upregulate ATP production following axonal activity whereas H2O2 production was largely unaffected. The authors concluded that neuropathic conditions uncouple the production of reactive oxygen species (ROS) and ATP thereby potentially compromising axonal function and integrity [15]. Finally, R94Q MFN2 appears to lead to reduction in MERCS in CMT2A patient-derived fibroblasts, in primary neurons and in vivo in motoneurons of the above-mentioned mouse model of CMT2A [16].…”

Long term AAV2/9-mediated silencing of PMP22 prevents CMT1A disease in rats and validates skin biomarkers as treatment outcome measure

“…AAV2/9 was significantly more efficient than AAV2/rh10 regarding the transduction rate at the injection site (83% vs 32% respectively on average). This high transduction rate is clearly correlated to the injection protocol as an intrathecal injection of the same vectors of newborn or adult mice resulted in the transduction of a large amount of neurons and glial cells [4,22,23,53] but no mSC in sciatic nerves. While we cannot rule out a transduction of mSC in nerve roots close to the spinal cord after intrathecal injection, intra nerve injection appears as the most efficient way to transduce these cells in vivo.…”

Quantitative Imaging of Changes in Astrocytic and Neuronal Adenosine Triphosphate Using Two Different Variants of ATeam

“…In the case of ATeam, binding of ATP to the sensor does not produce a hydrolysis of the molecule (Imamura et al, 2009), and there is no evidence that ATeam exerts a buffering effect, a phenomenon well described for chemical ion indicators of Ca 2+ (McMahon and Jackson, 2018). ATeam and its mitochondrial variant mito-ATeam were recently employed in vivo for imaging of ATP dynamics in cortical neurons and in peripheral axons (Baeza-Lehnert et al, 2019;Van Hameren et al, 2019). In summary, these results, together with the possibility to perform calibrations as demonstrated in the present study, show that ATeam and its variants are suitable tools for the detection of intracellular ATP in sub-cellular compartments, cell cultures, and tissue slices, as well as in vivo.…”