HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5085fjev1 20/7/1021    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jacquard, C. Articles by Brouillet, E. Search for Related Content PubMed PubMed Citation Articles by Jacquard, C. Articles by Brouillet, E.

Brain mitochondrial defects amplify intracellular [Ca²⁺] rise and neurodegeneration but not Ca²⁺ entry during NMDA receptor activation

Carine Jacquard^*, Yael Trioulier^*, François Cosker, Carole Escartin^*, Nicolas Bizat^*, Philippe Hantraye^*, José Manuel Cancela, Gilles Bonvento^* and Emmanuel Brouillet^*,1

^* Unité de Recherche Associée CEA-CNRS 2210, Service Hospitalier Frédéric Joliot, Département de Recherches Médicales, Direction des Sciences du Vivant, Commissariat à l’Energie Atomique, Orsay, France; and

Laboratoire de Neurobiologie cellulaire et moléculaire, CNRS, UPR 9040, Gif-sur-Yvette, France

¹Correspondence: URA CEA-CNRS 2210, Service Hospitalier Frédéric Joliot, DRM, DSV, CEA, 4 place du Général Leclerc, 91401 Orsay cedex, France. Email: brouille{at}shfj.cea.fr

SPECIFIC AIMS

Mitochondrial dysfunction might potentiate NMDA-receptor (R)-mediated toxicity in neurodegenerative diseases. The aim of this work was to investigate in vivo whether the mechanisms of potentiation of excitotoxicity by inhibition of mitochondrial complex II were associated with 1) an "all-or-nothing" process, 2) an elevation of cytosolic Ca²⁺ concentrations, and 3) an increase in the entry of Ca²⁺ into neurons.

PRINCIPAL FINDINGS
1. Inhibition of mitochondrial complex II greatly potentiates NMDA-R-mediated toxicity
We determined how the neurotoxicity of the NMDA-R agonist quinolinate (QA) was amplified by chronic administration of 3-nitropropionic acid (3NP), a selective inhibitor of mitochondrial complex II/ succinate dehydrogenase (SDH). As QA and 3NP produce striatal lesions that are reminiscent of those seen in Huntington’s disease (HD), we investigated these mechanisms in the rat striatum in vivo and in cultured striatal neurons.

To study the effects of partial SDH inhibition on QA toxicity, we chose a dose of 35 mg/kg/day 3NP, which does not lead to overt striatal degeneration. SDH inhibition on the sixth day of 3NP administration at this dose was similar in the striatum and cerebral cortex (44±3 and 49±2%, respectively, n=7 per group, n.s.).

We then verified that stereotactic intrastriatal injections of vehicle (PBS) on the fifth day of 3NP treatment produced no striatal degeneration. In contrast, injection of QA on the fifth day of 3NP treatment produced dose-dependent striatal lesions. The curve showing the proportion of lesioned animals plotted as a function of increasing QA doses was significantly shifted to the left by 3NP treatment. The vol of QA-induced striatal lesions was greater in 3NP-treated animals than in rats not treated with 3NP. However, visual observation of the TTC-stained brains clearly indicated that the "intensity" of QA-induced cell death was higher in 3NP-treated rats than in rats not treated with 3NP. Supporting this view, QA increased free oligonucleosome levels and the number of TUNEL positive nuclei 10 times more strongly in 3NP-treated rats than in rats without 3NP. Neuronal loss determined by immunohistochemistry 2 wk after QA injection was total in the core of the lesion, in 3NP-treated rats, whereas it was only partial in rats not treated with 3NP.

2. QA-induced striatal degeneration is amplified by 3NP in an "all-or-nothing" process
We plotted a toxicity curve for 40 nmol QA, using oligonucleosome levels as a function of SDH inhibition measured in rats after treatment with various doses of 3NP. No potentiation was seen at levels of SDH/inhibition < 35%. At inhibition levels of 35–50%, the potentiation of QA-induced striatal degeneration increased sharply with inhibition.

3. Massive activation of cytosolic calpains in the potentiation of QA toxicity by 3NP
We tested whether 3NP-induced potentiation of QA toxicity was associated with Ca²⁺ deregulation in vivo. We studied the Ca²⁺-activated protease calpain that we used as an in vivo index of Ca²⁺ deregulation. The injection of 40 nmol of QA into rats without 3NP led to a mild increase in the proteolytic activity of calpain, as seen in fluorimetric assays. A significant accumulation of the products of fodrin digestion by calpain was also observed, as a 145–150 kDa doublet on Western blots. In rats treated with 3NP only, no change was observed in calpain activity or cleavage of calpain substrates. In 3NP-treated rats, injection of QA significantly increased the proteolytic activity of calpain. Western blot analysis showed that the calpain-mediated breakdown products of fodrin, huntingtin, PSD-95 and NMDA-R NR2B subunit accumulated in significantly larger amounts in 3NP-treated than in untreated rats.

4. Mitochondrial defects do not increase QA-induced neuronal activation in vivo
We then investigated whether the exacerbation of Ca²⁺ deregulation by a combination of QA plus 3NP was dependent on NMDA-R hypersensitivity. We tested this hypothesis by determining whether 3NP treatment exacerbated the QA-induced increase in glucose (Glc) uptake in the striatum: a direct index displaying 1:1 stoichiometry with glutamatergic activity in vivo. We injected the positron emitter [¹⁸F]fluorodeoxyglucose (FDG) 10 min after intrastriatal injection of QA to study the early pharmacological effects of QA. The administration of 40 nmol of QA significantly increased FDG uptake. This increase was of a similar (nonsignificant by Mann-Whitney test) extent in 3NP-treated rats (+81±8%) and in rats not treated with 3NP (+65±5%).

5. Mitochondrial defects do not increase the amount of Ca²⁺ entering the cell in response to NMDA-R stimulation
We used microscopy Fura-2 ratio imaging to dynamically study variation of cytosolic Ca²⁺ concentrations before and during QA treatment. Basal levels of cytosolic Ca²⁺ concentrations were similar in control and 3NP-treated cells. Perfusion of control striatal cultures with 5 mM QA produced a rapid rise in cytosolic Ca²⁺ that remained relatively stable thereafter. In cultures that were pretreated with 75 µM 3NP, the QA-induced rise in cytosolic Ca²⁺ was significantly higher than that seen in cells not treated with 3NP.

To investigate the origin of Ca²⁺ deregulation, we assessed the effect of 3NP on NMDA-R sensitivity to QA by measuring net influx of ⁴⁵Ca²⁺ in cultured striatal cells.

We first set up in vitro conditions reproducing our in vivo observations. We identified a 3NP concentration (75 µM) that substantially inhibited SDH (74%) without triggering overt degeneration. In striatal cells not treated with 3NP, 5 mM QA increased cell death levels slightly over those observed in the control. In contrast, QA-induced neurodegeneration levels in cultures treated with 3NP were 2.3 times higher than those in cells treated with QA alone and 3.2 times higher than those in control cells. We measured ⁴⁵Ca²⁺ accumulation in this cellular model (Fig. 1 ). 3NP did not significantly affect ⁴⁵Ca²⁺ accumulation in cells treated with PBS. Incubation with 5 mM QA increased ⁴⁵Ca²⁺ accumulation by a factor of seven over than in PBS-treated-cells. This entry of ⁴⁵Ca²⁺ was totally blocked by MK801 (Fig. 2A ). In 3NP-treated cells, QA increased ⁴⁵Ca²⁺ accumulation by a factor of only three with respect to basal conditions (Fig. 1B ). Apparent ⁴⁵Ca²⁺ efflux evaluated at the end of the period of accumulation after stimulation by QA was similar in 3NP-treated cells and untreated cells. Thus, net ⁴⁵Ca²⁺ entry produced by QA is not increased by 3NP treatment.

View larger version (15K): [in this window] [in a new window]   Figure 1. QA-induced 45Ca2+ entry in primary striatal cells cultured with or without 3NP. Intracellular accumulation of radioactivity was measured after 10 min of 45Ca2+ incubation with QA (5 mM), PBS (CTL) or QA + MK801 (10 µM). A) Effect of MK801 pretreatment on QA-induced entry of 45Ca2+. B) Effect of 3NP on basal and QA-induced 45Ca2+ entry. 3NP (75 µM) was applied 5 h before QA treatment. Data are mean ± SE determined for 4 culture wells per condition. Note that 3NP attenuates QA-induced increase in 45Ca2+ entry. *P < 0.001, and **P < 0.0001 vs. control (CTL). P < 0.0001 vs. QA+MK801. #P < 0.001 vs. QA, ANOVA, and Fisher’s post hoc PLSD test.

View larger version (45K): [in this window] [in a new window]   Figure 2. Scheme summarizing the hypothetical mechanisms involved in the potentiation of NMDA-R excitotoxicity by mitochondrial defects. Although potentiation is associated with intracellular [Ca2+] deregulation, the present results suggest that mitochondrial dysfunction does not lead to an increase in Ca2+ entry into striatal neurons. A), Control situation. B) Activation of NMDA-R (by QA) leading to an increased entry of Ca2+ into striatal neurons, calpain (CLP) activation, and cell death. C) Indirect excitotoxicity hypothesis where mitochondrial defects increase entry of Ca2+ through NMDA-R as a result of loss of membrane potential (V), leading to major Ca2+ deregulation. Gray lines crossing in C indicate that the present results are not consistent with this hypothesis. D) Hypothetical mechanisms we propose where Ca2+ deregulation associated with potentiation does not result from an increase in cation entry but perturbation of Ca2+ sequestration by mitochondria.

CONCLUSION AND SIGNIFICANCE
This study demonstrates that moderate NMDA-R activation combined with nontoxic mitochondrial SDH defects can synergistically trigger massive striatal degeneration. The results presented here are novel in that they show that QA toxicity is exacerbated for a particular "window" of SDH inhibition. Indeed, no potentiation was detected for levels of inhibition < 35%. Once this threshold had been exceeded, QA toxicity rapidly increased with the concentration of SDH inhibition through an almost "all-or-nothing" process.

We show here that QA activates cytosolic calpain more strongly in 3NP-treated rats than in untreated rats, suggesting major increase in cytosolic Ca²⁺ concentrations in vivo. In line with this, we found that the rise in cytosolic Ca²⁺ concentration after QA stimulation was greater in cultured cells treated with 3NP compared with untreated cells. We investigated two major mechanisms that could explain Ca²⁺ deregulation: 1) increases in Ca²⁺ entry and 2) changes in the sequestration of Ca²⁺ to intracellular stores.

The potentiation of QA toxicity by 3NP may depend on an increase in Ca²⁺ entry into striatal neurons. Indeed, according to the hypothesis of "indirect excitotoxicity" in energy-deficient situations, ATP availability is reduced and the plasma membrane Na⁺/K⁺-ATPase cannot maintain the resting membrane potential. Partial depolarization renders NMDA-R hypersensitive to agonists by relieving the voltage-dependent Mg²⁺ blockade of the cation channel. In the presence of even low (physiological) concentrations of glutamate, this would lead to an increase in Ca²⁺ influx and cell death (Fig. 2 C).

Although this popular hypothesis is often suggested to explain how energy compromise triggers or potentiates excitotoxic process, our results show that NMDA-R did not seem to become hypersensitive to QA in 3NP-treated rats. Indeed, we directly showed that the QA-induced influx of ⁴⁵Ca²⁺ into cultured striatal neurons was not increased by 3NP, suggesting the absence of a hypersensitivity of NMDA-R. As similar analysis of Ca²⁺ entry could not be performed in vivo, we used an indirect index of NMDA-R activation. We found that 3NP did not enhance the QA-induced increase in Glc consumption, indicating that the primary effect of NMDA-R activation by QA (ionic imbalance across the plasma membrane) remains essentially unchanged by 3NP-induced SDH inhibition in vivo. Thus, "mild" mitochondrial dysfunction potentiates NMDA-R-mediated excitotoxicity through a process that does not involve the "indirect" excitotoxicity mechanism. Possibly, more severe mitochondrial defects are necessary to trigger "indirect" excitotoxicity.

Finally, SDH inhibition may modify the sequestration of Ca²⁺ entering through NMDA-R in intracellular pools. It has been demonstrated that Ca²⁺ entering through NMDA-R is rapidly sequestered by mitochondria. It is possible that subtoxic 3NP treatment modifies the ability of mitochondria to buffer cytosolic Ca²⁺ entering through NMDA-R (Fig. 2D ). The present findings, showing that synaptic proteins are massively cleaved by calpain in 3NP-treated rats receiving QA support this hypothesis. Indeed, these proteins are preferentially localized in dendrites of striatal neurons in vivo. As dendrites are remote from the reticulum and nucleus (2 important pools of Ca²⁺) but contain many mitochondria, calpain-dependent cleavage of synaptic proteins suggests preferential Ca²⁺ deregulation in the vicinity of mitochondria.

In conclusion, the present results provide new insights into the mechanisms associated with potentiation of NMDA-R-mediated excitotoxicity by mitochondrial defects and support the view that a similar potentiation might play an important role in neurodegenerative disorders, such as HD in particular.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5085fje

This article has been cited by other articles:

				A. Benchoua, Y. Trioulier, E. Diguet, C. Malgorn, M.-C. Gaillard, N. Dufour, J.-M. Elalouf, S. Krajewski, P. Hantraye, N. Deglon, et al. Dopamine determines the vulnerability of striatal neurons to the N-terminal fragment of mutant huntingtin through the regulation of mitochondrial complex II Hum. Mol. Genet., May 15, 2008; 17(10): 1446 - 1456. [Abstract] [Full Text] [PDF]

				S. N. Blythe, J. F. Atherton, and M. D. Bevan Synaptic Activation of Dendritic AMPA and NMDA Receptors Generates Transient High-Frequency Firing in Substantia Nigra Dopamine Neurons In Vitro J Neurophysiol, April 1, 2007; 97(4): 2837 - 2850. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5085fjev1 20/7/1021    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jacquard, C. Articles by Brouillet, E. Search for Related Content PubMed PubMed Citation Articles by Jacquard, C. Articles by Brouillet, E.