In situ fluorescence imaging of glutamate‐evoked mitochondrial Na<sup>+</sup> responses in astrocytes

Bernardinelli, Yann; Azarias, Guillaume; Chatton, Jean–Yves

doi:10.1002/glia.20387

Cited by 52 publications

(73 citation statements)

References 34 publications

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“…We used real-time fluorescence imaging to follow intracellular ionic alterations during glutamate superfusion and studied the impact of glutamate on the metabolism of astrocyte mitochondria in situ. In previous studies, we have shown that the mitochondrial Na ϩ concentration increases as a result of glutamate transporter activity (Bernardinelli et al, 2006). In the present study, using a fluorescent pH biosensor targeted to the mitochondrial matrix, we show that glutamate, released from neurons or applied by superfusion, induces a dosedependent proton transfer into mitochondria that depends on glutamate transporter activity.…”

Section: Introductionsupporting

confidence: 63%

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

Azarias¹,

Perreten²,

Lengacher³

et al. 2011

J. Neurosci.

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During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca 2ϩ -mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

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Section: Introductionsupporting

confidence: 63%

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

Azarias¹,

Perreten²,

Lengacher³

et al. 2011

J. Neurosci.

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“…Immediately after uncaging, the intracellular Na + level was elevated by adding gramicidin, monensin, and ouabain. The combination of these three ionophores is known to equilibrate the intracellular Na + concentration with extracellular concentration within several minutes (22,30). The influx of Na + from extracellular medium caused the fluorescence inside cells to gradually increase over a time course of 30 min (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Among the limited number of Na + sensors, such as sodium-binding benzofuran isophthalate (22), Sodium Green (23), CoroNa Green/Red (24,25), and Asante NaTRIUM Green-1/2 (26), most of them are not selective for Na + over K + (22-25, 27, 28) or have a low binding affinity for Na + (with a K d higher than 100 mM) (25,(27)(28)(29)(30)(31). Furthermore, the presence of organic solvents is frequently required to achieve the desired sensitivity and selectivity for many of the Na + probes (32)(33)(34), making it difficult to study Na + under physiological conditions.…”

mentioning

confidence: 99%

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Torabi¹,

Wu²,

McGhee³

et al. 2015

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

300

286

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Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na + ), has remained underdeveloped, even though Na + is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na + -specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k obs ) ∼0.1 min −1 ], and the transformation of this DNAzyme into a fluorescent sensor for Na + by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na + catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na + over competing metal ions and has a detection limit of 135 μM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na + -specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na + in living cells.M etal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1-4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na + ) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5-8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na + affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na + in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), Xray fluorescence microscopy (13), and 23 Na NMR (14) have been used to detect the concentration of intracellular Na + . However, it is difficult to use these methods to o...

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“…The transport of glutamate plus H ϩ against aspartate is electrogenic (3, 59 -61), and the mitochondrial membrane potential may be dissipated by Ca 2ϩ ϩ Na ϩ cycling, with Ca 2ϩ entry along the uniporter, and exit through the NCX coupled to efflux of Na ϩ back to the cytosol through Na ϩ /H ϩ exchange (62,63). This paradoxical effect of Na ϩ would not affect the OGC, which is electroneutral and thus independent upon ⌬p (64,65).…”

Section: Activitymentioning

confidence: 99%

Calcium Signaling in Brain Mitochondria

Contreras

Satrústegui

2009

Journal of Biological Chemistry

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Ca2؉ signaling in mitochondria has been mainly attributed to Ca 2؉ entry to the matrix through the Ca 2؉ uniporter and activation of mitochondrial matrix dehydrogenases. However, mitochondria can also sense increases in cytosolic Ca 2؉ and activation of mitochondrial dehydrogenases (mitDH) (1). However, mitochondria can also sense increases in cytosolic Ca 2ϩ through a mechanism that involves the aspartate-glutamate carriers (AGCs) and not the CaU (2-5). Aralar (Slc25a12), also named aralar1, the AGC isoform with predominant expression in brain (6 -8), is a component of the NADH malateaspartate shuttle (MAS), which in brain is activated by extramitochondrial Ca 2ϩ (S 0.5 324 nM) (4). Immunocytochemistry and in situ hybridization data and mRNA levels in acutely isolated brain cells indicate that aralar is localized preferentially in neurons (8 -12). This is consistent with a higher MAS activity in neuronal than astrocyte cultures (8) and with aralar being one of the more enriched proteins during differentiation of P19 cells to a neuronal phenotype (13). It is also consistent with the higher levels of aralar in total than in synaptosome-free mitochondrial fractions (9).Studies in cultured neurons, which have aralar as only AGC isoform, showed that small Ca 2ϩ signals that have limited access to mitochondria are able to activate the aralar-MAS pathway (4). However, large Ca 2ϩ signals that induce robust mitochondrial Ca 2ϩ transients fail to activate the pathway (4). This suggested that in neuronal mitochondria the aralar-MAS pathway is inhibited under conditions in which the CaUmitDH pathway is activated. This surprising result indicates that Ca 2ϩ activation of the malate aspartate shuttle and tricarboxylic acid cycle activity are somehow mutually exclusive in neurons. We have now studied the interplay between the AGG-MAS and CaU-mitDH pathways in brain mitochondria under Ca 2ϩ -stimulation conditions. Our results show that the shared metabolite ␣KG controls the relationship between the second transporter of MAS, the oxoglutarate carrier (OGC, Slc25a11), and ␣KGDH in the Krebs cycle, by virtue of the effects of Ca 2ϩ on the kinetics of ␣KGDH. Interestingly, the inhibition of OGC and MAS is fully reversible, in parallel with Ca 2ϩ egress from mitochondria. Behaviorally evoked brain activation results in an increased cerebral blood flow and increased glucose utilization, but paradoxically, this is not accompanied with an equivalent increase in oxygen utilization (14, 15). As a consequence, the oxygen glucose index, which is close to 6 when glucose is fully oxidized in resting conditions, falls to about 5 (16). This is accompanied by an increase in brain lactate production (14, 16 -19). Our results suggest that MAS inhibition during Ca 2ϩ -induced Krebs cycle activation would drive pyruvate to lactate formation and may play a role in lactate formation during brain activation. EXPERIMENTAL PROCEDURESAnimals and Materials-3-Month-old C57BL/6xSv129 mice were housed with a 12-h light cycle and fed ad libitum on standa...

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In situ fluorescence imaging of glutamate‐evoked mitochondrial Na⁺ responses in astrocytes

Cited by 52 publications

References 34 publications

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Calcium Signaling in Brain Mitochondria

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In situ fluorescence imaging of glutamate‐evoked mitochondrial Na+ responses in astrocytes

Cited by 52 publications

References 34 publications

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

Glutamate Transport Decreases Mitochondrial pH and Modulates Oxidative Metabolism in Astrocytes

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Calcium Signaling in Brain Mitochondria

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In situ fluorescence imaging of glutamate‐evoked mitochondrial Na⁺ responses in astrocytes