Neurological diseases are often accompanied by neuronal loss. As neurons can only regenerate to a very limited extent, deciphering the mechanisms that cause neurodegeneration is an important step towards targeted therapies. Reactive oxygen species (ROS) have been implicated in neurodegeneration as possible molecular mediators of cellular damage. ROS are generated during the inflammatory burst of neutrophils and macrophages/microglia, as well as during mitochondrial oxidative phosphorylation. ROS can be detrimental to cells and tissues by oxidizing biomolecules like DNA, lipids or proteins, but can also act as cellular signaling molecules under physiological conditions. The study of ROS is notoriously difficult as ROS form intricate networks of short-lived and inter-convertible species. Therefore it was until recently not possible to directly visualize ROS and their effects in vivo.
In my thesis work, I aimed to establish such in vivo methods in order to measure redox changes that are caused by ROS in intact neurons. For this purpose, I generated with cooperating partners novel transgenic mouse lines (Thy1-mito-Grx1-roGFP2 and Thy1-mito-roGFP2-Orp1), which express redox sensors in their neuronal mitochondria. Additionally, I developed further strategies to co-visualize mitochondrial pH, calcium and potential dynamics. By using wide-field, confocal and 2 photon in vivo microscopy, I could quantify redox changes in single mitochondria under physiological conditions, as well as in murine disease models of acute neurodegeneration (axon transection, nerve crush, spinal cord injury), chronic neurodegeneration (amyotrophic lateral sclerosis) and in neuroinflammatory conditions (experimental autoimmune encephalitis, a model of multiple sclerosis). I obtained the following results:
(1) Under physiological conditions and in the absence of activity, the mitochondrial glutathione pool is almost completely reduced, indicating that mitochondrial redox levels are tightly regulated and independent of organelle location or movement. When axons are electrically stimulated, the mitochondrial redox potential is elevated in axons and synapses. (2) Mitochondria in PNS and CNS axons show spontaneous redox signals, which are accompanied by reversible shape changes and that we dubbed “mitochondrial contractions.” During these contractions, mitochondria transiently shorten by ~ 50% of their relaxed length and their glutathione pool reversibly oxidizes. A reversible drop of the mitochondrial inner membrane potential and a short alkaline spike of the matrix pH followed by a long lasting matrix acidification accompany the contraction. Matrix calcium levels do not change during the contraction. The frequency of mitochondrial contractions increases during extrinsic and intrinsic oxidative stress as well as during electrical activity. (3) Mechanistically, complex I activity is necessary for the initiation of contractions as well as for the oxidative shift of the redox potential. (4) Pharmacological manipulations inducing and blocking uncoupling proteins (UCPs) showed that these are activated during the contraction and that they sustain the long lasting drop in the mitochondrial inner membrane potential and the acidification. (5) Under pathological conditions e.g. during laser axotomy as well as during crush injury to peripheral nerve axons, a spreading wave of mitochondrial oxidation is initiated at the injury site. In damaged axons, the mitochondrial contraction frequency is increased ~4-fold, before mitochondria round up and fragment permanently and acute axonal degeneration sets in. A number of mitochondria also show an increase in contraction length of which some become irreversible. (6) In vivo imaging during laser axotomy in the spinal cord of living mice established a similar sequence of events and revealed further mechanisms: First, the presence of extracellular calcium and its injury-induced influx into axons is necessary and sufficient to cause mitochondrial oxidation and rounding. Knock-out of cyclophilin D, which is part of the mitochondrial transition pore, reduces the mitochondria pathology seen after axotomy. Preventing the oxidation of the mitochondrial glutathione pool by genetic and pharmacological means does not prevent mitochondrial shape changes. (7) In a model of ALS (SODG93A), mitochondria show severe oxidative changes that correlate with the disease course. In SODG93A mice, mitochondrial contractions are increased in frequency and last longer compared to wild-type controls.
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Neurological diseases are often accompanied by neuronal loss. As neurons can only regenerate to a very limited extent, deciphering the mechanisms that cause neurodegeneration is an important step towards targeted therapies. Reactive oxygen species (ROS) have been implicated in neurodegeneration as possible molecular mediators of cellular damage. ROS are generated during the inflammatory burst of neutrophils and macrophages/microglia, as well as during mitochondrial oxidative phosphorylation. ROS...
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