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Real-Time Neuroimaging Technology Offers Glimpse Inside the Cell

By BiotechDaily International staff writers
Posted on 08 May 2014
Image: The micrograph shows a peripheral nerve, with the neuromuscular endplates stained in red. The nerve-cell mitochondria were imaged with a fluorescent redox sensor (green in the cytoplasm, yellow at the endplates) (Photo courtesy of M. Kerschensteiner and T. Misgeld).
Image: The micrograph shows a peripheral nerve, with the neuromuscular endplates stained in red. The nerve-cell mitochondria were imaged with a fluorescent redox sensor (green in the cytoplasm, yellow at the endplates) (Photo courtesy of M. Kerschensteiner and T. Misgeld).
Cutting-edge imaging technology provides insights into the role of redox signaling and reactive oxygen species in living neurons, in real time. German scientists have developed a new optical microscopy technique that provides insights into the role of oxidative stress in damaged as well as healthy nervous systems.

The study, performed by researchers from Technische Universität München (TUM; Germany) and the Ludwig-Maximilians-Universität München (LMU; Germany), was described in the April 2014 issue of the journal Nature Medicine.

Reactive oxygen species (ROS) are important intracellular signaling molecules, but their course of action is complicated: In low concentrations they control key aspects of cellular function and behavior, while at high concentrations they can cause oxidative stress, which damages DNA, organelles, and membranes. To examine how redox signaling unfolds in single cells and organelles in real-time, an innovative optical microscopy technique has been developed cooperatively by the teams of LMU Prof. Martin Kerschensteiner and TUM Prof. Thomas Misgeld, both investigators of the Munich Cluster for Systems Neurology (SyNergy).

“Our new optical approach allows us to visualize the redox state of important cellular organelles, mitochondria, in real time in living tissue,” Prof. Kerschensteiner said. In earlier studies, the investigators had obtained validation that oxidative damage of mitochondria might contribute to the destruction of axons in inflammatory diseases such as multiple sclerosis.

The new technology allows the scientists to monitor the oxidation states of individual mitochondria with high spatial and temporal resolution. Prof. Kerschensteiner explained the incentive behind the development of the technique. “Redox signals have important physiological functions, but can also cause damage, for example when present in high concentrations around immune cells.”

The scientists used redox-sensitive variants of the green fluorescent protein (GFP) as visualization tools. “By combining these with other biosensors and vital dyes, we were able to establish an approach that permits us to simultaneously monitor redox signals together with mitochondrial calcium currents, as well as changes in the electrical potential and the proton (pH) gradient across the mitochondrial membrane,” stated Prof. Misgeld.

The researchers have applied the technique to two experimental models, and have arrived at some unexpected insights. On the one hand, they have been able for the first time to study redox signal induction in response to neural damage—in this instance, spinal cord injury—in the mammalian nervous system. The observations revealed that severance of an axon results in a wave of oxidation of the mitochondria, which begins at the site of damage and is propagated along the fiber. Furthermore, a flood of calcium at the site of axonal resection was shown to be needed for the subsequent functional damage to mitochondria.

Quite possibly the most unexpected outcome of the new study was that the study’s first author, graduate student Michael Breckwoldt, was able to image for the first time, spontaneous contractions of mitochondria that are accompanied by a rapid shift in the redox state of the organelle.

Prof. Misgeld concluded, “This appears to be a fail-safe system that is activated in response to stress and temporarily attenuates mitochondrial activity. Under pathological conditions, the contractions are more prolonged and may become irreversible, and this can ultimately result in irreparable damage to the nerve process.”

Related Links:

Technische Universität München
Ludwig-Maximilians-Universität München



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