Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo

Breckwoldt, Michael O.; Pfister, Franz; Bradley, Peter; Marinković, Petar; Williams, Paul R.; Brill, Monika S.; Plomer, Barbara; Schmalz, Anja; Clair, Daret K. St.; Naumann, Ronald; Griesbeck, Oliver; Schwarzländer, Markus; Godinho, Leanne; Bareyre, Florence M.; Dick, Tobias P.; Kerschensteiner, Martin; Misgeld, Thomas

doi:10.1038/nm.3520

Cited by 153 publications

(196 citation statements)

References 48 publications

Supporting

Mentioning

177

Contrasting

Order By: Relevance

“…1C, sectors I and III), implying that single neuronal mitochondria perform specialized functions. This finding confirms the functional heterogeneity of mitochondria, which has been proposed for various cell types (22)(23)(24)(25). The preferential presence of UCP4 or ATP synthase in mitochondria can be rationalized by different energy demands in various neuronal areas.…”

Section: Resultssupporting

confidence: 87%

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Klotzsch

Smorodchenko²,

Löfler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F 0 F 1 -ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F 0 F 1 -ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F 0 F 1 -ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. mitochondrial membrane proteins | proton diffusion | direct stochastic optical reconstruction microscopy | uncoupling | reactive oxygen species M itochondria are involved in a wide range of cell functions, including fatty acid oxidation, calcium homeostasis, apoptosis, reactive oxygen species (ROS) signaling, and above all, production of ATP (1, 2). In neurons, these organelles are transported along neuronal processes to provide energy for areas of high energy demand, such as synapses (3). To support their functions, mitochondria exhibit a complex morphology consisting of separate and functionally distinct outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). The latter is structurally organized into two domains: an inner boundary membrane (IBM) and a cristae membrane (CM) (4). The current hypotheses imply that the morphology/topology of the IMM is tightly related to biochemical function, the energy state, and the pathophysiological state of mitochondria (5). Whereas the OMM contains porins [e.g., voltage-dependent anion channel (VDAC)], which mediate its permeability to molecules up to 10 kDa, the IMM topology is highly complex. It is comprised of different transport proteins, the ATP synthase (complex V), and complexes I, III, and IV of the electron transport chain, which are responsible for generating the proton motive force (pmf); pmf represents the driving force for not only ATP synthesis, but also other protein-mediated transport activities (for example, phosphate, pyruvate, and glutamate transport). Uncoupling protein 1 (UCP1; thermogenin), a memb...

show abstract

Section: Resultssupporting

confidence: 87%

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Klotzsch

Smorodchenko²,

Löfler

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…To date, just a limited number of mouse models making use of genetically encoded redox biosensors for quantifying the redox potential have been developed. [9][10][11][12] Here, we demonstrate the successful establishment of cardiac transgenic expression of Grx1-roGFP2 for in vitro and in vivo monitoring of the glutathione redox potential of isolated cardiac myocytes and the whole heart.…”

Section: Discussionmentioning

confidence: 92%

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Swain

Kesemeyer

Meyer-Roxlau

et al. 2016

Circulation Research

View full text Add to dashboard Cite

Rationale: Changes in redox potentials of cardiac myocytes are linked to several cardiovascular diseases. Redox alterations are currently mostly described qualitatively using chemical sensors, which however do not allow quantifying redox potentials, lack specificity, and the possibility to analyze subcellular domains. Recent advances to quantitatively describe defined redox changes include the application of genetically encoded redox biosensors. Objective: Establishment of mouse models, which allow the quantification of the glutathione redox potential ( E GSH ) in the cytoplasm and the mitochondrial matrix of isolated cardiac myocytes and in Langendorff-perfused hearts based on the use of the redox-sensitive green fluorescent protein 2, coupled to the glutaredoxin 1 (Grx1-roGFP2). Methods and Results: We generated transgenic mice with cardiac myocyte–restricted expression of Grx1-roGFP2 targeted either to the mitochondrial matrix or to the cytoplasm. The response of the roGFP2 toward H 2 O 2 , diamide, and dithiothreitol was titrated and used to determine the E GSH in isolated cardiac myocytes and in Langendorff-perfused hearts. Distinct E GSH were observed in the cytoplasm and the mitochondrial matrix. Stimulation of the cardiac myocytes with isoprenaline, angiotensin II, or exposure to hypoxia/reoxygenation additionally underscored that these compartments responded independently. A compartment-specific response was also observed 3 to 14 days after myocardial infarction. Conclusions: We introduce redox biosensor mice as a new tool, which allows quantification of defined alterations of E GSH in the cytoplasm and the mitochondrial matrix in cardiac myocytes and can be exploited to answer questions in basic and translational cardiovascular research.

show abstract

“…It may be achieved by manually tuning laser wavelengths and acquiring sequential image pairs, as we and others have shown earlier (11,42). More advanced is the use of two separate lasers, which we have now introduced.…”

Section: Discussionmentioning

confidence: 99%

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

Wagener

Kolbrink

Dietrich

et al. 2016

Antioxidants & Redox Signaling

View full text Add to dashboard Cite

Aims: Reactive oxygen species (ROS) and downstream redox alterations not only mediate physiological signaling but also neuropathology. For long, ROS/redox imaging was hampered by a lack of reliable probes. Genetically encoded redox sensors overcame this gap and revolutionized (sub)cellular redox imaging. Yet, the successful delivery of sensor-coding DNA, which demands transfection/transduction of cultured preparations or stereotaxic microinjections of each subject, remains challenging. By generating transgenic mice, we aimed to overcome limiting cultured preparations, circumvent surgical interventions, and to extend effectively redox imaging to complex and adult preparations. Results: Our redox indicator mice widely express Thy1-driven roGFP1 (reduction-oxidation-sensitive green fluorescent protein 1) in neuronal cytosol or mitochondria. Negative phenotypic effects of roGFP1 were excluded and its proper targeting and functionality confirmed. Redox mapping by ratiometric wide-field imaging reveals most oxidizing conditions in CA3 neurons. Furthermore, mitochondria are more oxidized than cytosol. Cytosolic and mitochondrial roGFP1s reliably report cell endogenous redox dynamics upon metabolic challenge or stimulation. Fluorescence lifetime imaging yields stable, but marginal, response ranges. We therefore developed automated excitation ratiometric 2-photon imaging. It offers superior sensitivity, spatial resolution, and response dynamics. Innovation and Conclusion: Redox indicator mice enable quantitative analyses of subcellular redox dynamics in a multitude of preparations and at all postnatal stages. This will uncover cell-and compartment-specific cerebral redox signals and their defined alterations during development, maturation, and aging. Cross-breeding with other disease models will reveal molecular details on compartmental redox homeostasis in neuropathology. Combined with ratiometric 2-photon imaging, this will foster our mechanistic understanding of cellular redox signals in their full complexity. Antioxid. Redox Signal. 25, 41-58.

show abstract

Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo

Cited by 153 publications

References 48 publications

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

Contact Info

Product

Resources

About

Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo

Cited by 153 publications

References 48 publications

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

Redox Imaging Using Cardiac Myocyte-Specific Transgenic Biosensor Mice

Redox Indicator Mice Stably Expressing Genetically Encoded Neuronal roGFP: Versatile Tools to Decipher Subcellular Redox Dynamics in Neuropathophysiology

Contact Info

Product

Resources

About

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria

Superresolution microscopy reveals spatial separation of UCP4 and F ₀ F ₁ -ATP synthase in neuronal mitochondria