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Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons.

Otmakhov N, Gorbacheva EV, Regmi S, Yasuda R, Hudmon A, Lisman J - PLoS ONE (2015)

Bottom Line: Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma.Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation.The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brandeis University, Waltham, Massachusetts, 02454, United States of America.

ABSTRACT
Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1-3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.

No MeSH data available.


Related in: MedlinePlus

NMDA treatment resulted in a persistent mitochondrial swelling following Camui activation and clustering.(A) Summary graph showing persistent and transient increase in the fluorescence lifetime of Camui after NMDA treatment (grey bar). (B) Representative images of mitochondrial marker (mito-Red) and Camui intensity (SPC) at different times: before, during, and after NMDA application (in minutes). (C) Bar diagram indicating average times for Camui clustering (Camui clust.) and mitochondrial swelling (m. swelling). (D) Bar diagram showing the percentage of cells with CaMKII clustering after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation. (E) Bar diagram indicating the percentage of cells with mitochondrial swelling shortly after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation.
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pone.0120881.g006: NMDA treatment resulted in a persistent mitochondrial swelling following Camui activation and clustering.(A) Summary graph showing persistent and transient increase in the fluorescence lifetime of Camui after NMDA treatment (grey bar). (B) Representative images of mitochondrial marker (mito-Red) and Camui intensity (SPC) at different times: before, during, and after NMDA application (in minutes). (C) Bar diagram indicating average times for Camui clustering (Camui clust.) and mitochondrial swelling (m. swelling). (D) Bar diagram showing the percentage of cells with CaMKII clustering after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation. (E) Bar diagram indicating the percentage of cells with mitochondrial swelling shortly after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation.

Mentions: Ischemia and excitotoxicity are known to produce delayed neuronal cell injury and death [5]. Mitochondrial lesions occur during this period and are thought to be one of the main causes of the ischemic cell injury in both the brain and the heart [39,59]. Therefore, we monitored mitochondrial morphological integrity by expressing fluorescent mitochondrial marker mito-dsRed. The marker clearly labeled mitochondria in somas and dendrites of CA1 neurons without almost any background fluorescence (S7 Fig.). We coexpressed Camui and mito-dsRed and monitored Camui reactivity and mitochondrial integrity simultaneously in neuronal somas before and after the 7.5 min NMDA insult. Under basal conditions, mitochondrial labeling revealed a dense mesh of thin (< 1 μM in diameter) string-like structures filling the cytoplasmic region of the neuronal soma, excluding the nucleus (Fig. 6B, S7 Fig.). NMDA application produced strong Camui activation (Fig. 6A, n = 40) followed by Camui redistribution in a string-like pattern and subsequently in small clusters, as described earlier. Clusters were formed, on average, 8 min (8.3 ± 0.3 min, n = 37) after the start of the NMDA application. At this time, no clear change of mitochondrial morphology was observed (Fig. 6B, 8’). One to five minutes later, however (on average at 10.3 ± 0.4 min, n = 37, after the start of NMDA, Fig. 6C), clear signs of change in mitochondrial morphology were evident: string-like structures seemed to become shorter but larger in diameter (Fig. 6B, 13’, S7 Fig.) [60]. This change in mitochondrial morphology after ischemia has been characterized as swelling [39] although the actual volume could be smaller [61]. The somatic Camui clustering occurred in 100% (n = 27) of experiments in the group of the persistent Camui activation and remained until the end of the experiment in all of these cells (data not shown). The mitochondrial swelling also occurred in all cells of this group, and it persisted until the end of the experiment in 96% of them. In the group with transient Camui activation, clustering occurred in 92% (n = 13) and remained in 85% of them until the end of experiment, while mitochondrial swelling occurred in 77% and remained until the end only in 46% (n = 13) of cells (Fig. 6D, E). Therefore, our protocol for the NMDA-induced toxicity was effective in producing clear signs of prolonged (> 1 hour) mitochondrial damage. Importantly, the magnitude of these toxicity signs correlated with the persistence of Camui activation (Fig. 6E).


Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons.

Otmakhov N, Gorbacheva EV, Regmi S, Yasuda R, Hudmon A, Lisman J - PLoS ONE (2015)

NMDA treatment resulted in a persistent mitochondrial swelling following Camui activation and clustering.(A) Summary graph showing persistent and transient increase in the fluorescence lifetime of Camui after NMDA treatment (grey bar). (B) Representative images of mitochondrial marker (mito-Red) and Camui intensity (SPC) at different times: before, during, and after NMDA application (in minutes). (C) Bar diagram indicating average times for Camui clustering (Camui clust.) and mitochondrial swelling (m. swelling). (D) Bar diagram showing the percentage of cells with CaMKII clustering after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation. (E) Bar diagram indicating the percentage of cells with mitochondrial swelling shortly after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4368532&req=5

pone.0120881.g006: NMDA treatment resulted in a persistent mitochondrial swelling following Camui activation and clustering.(A) Summary graph showing persistent and transient increase in the fluorescence lifetime of Camui after NMDA treatment (grey bar). (B) Representative images of mitochondrial marker (mito-Red) and Camui intensity (SPC) at different times: before, during, and after NMDA application (in minutes). (C) Bar diagram indicating average times for Camui clustering (Camui clust.) and mitochondrial swelling (m. swelling). (D) Bar diagram showing the percentage of cells with CaMKII clustering after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation. (E) Bar diagram indicating the percentage of cells with mitochondrial swelling shortly after the NMDA treatment and at the end of experiments for cells with persistent and transient Camui activation.
Mentions: Ischemia and excitotoxicity are known to produce delayed neuronal cell injury and death [5]. Mitochondrial lesions occur during this period and are thought to be one of the main causes of the ischemic cell injury in both the brain and the heart [39,59]. Therefore, we monitored mitochondrial morphological integrity by expressing fluorescent mitochondrial marker mito-dsRed. The marker clearly labeled mitochondria in somas and dendrites of CA1 neurons without almost any background fluorescence (S7 Fig.). We coexpressed Camui and mito-dsRed and monitored Camui reactivity and mitochondrial integrity simultaneously in neuronal somas before and after the 7.5 min NMDA insult. Under basal conditions, mitochondrial labeling revealed a dense mesh of thin (< 1 μM in diameter) string-like structures filling the cytoplasmic region of the neuronal soma, excluding the nucleus (Fig. 6B, S7 Fig.). NMDA application produced strong Camui activation (Fig. 6A, n = 40) followed by Camui redistribution in a string-like pattern and subsequently in small clusters, as described earlier. Clusters were formed, on average, 8 min (8.3 ± 0.3 min, n = 37) after the start of the NMDA application. At this time, no clear change of mitochondrial morphology was observed (Fig. 6B, 8’). One to five minutes later, however (on average at 10.3 ± 0.4 min, n = 37, after the start of NMDA, Fig. 6C), clear signs of change in mitochondrial morphology were evident: string-like structures seemed to become shorter but larger in diameter (Fig. 6B, 13’, S7 Fig.) [60]. This change in mitochondrial morphology after ischemia has been characterized as swelling [39] although the actual volume could be smaller [61]. The somatic Camui clustering occurred in 100% (n = 27) of experiments in the group of the persistent Camui activation and remained until the end of the experiment in all of these cells (data not shown). The mitochondrial swelling also occurred in all cells of this group, and it persisted until the end of the experiment in 96% of them. In the group with transient Camui activation, clustering occurred in 92% (n = 13) and remained in 85% of them until the end of experiment, while mitochondrial swelling occurred in 77% and remained until the end only in 46% (n = 13) of cells (Fig. 6D, E). Therefore, our protocol for the NMDA-induced toxicity was effective in producing clear signs of prolonged (> 1 hour) mitochondrial damage. Importantly, the magnitude of these toxicity signs correlated with the persistence of Camui activation (Fig. 6E).

Bottom Line: Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma.Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation.The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury.

View Article: PubMed Central - PubMed

Affiliation: Biology Department, Brandeis University, Waltham, Massachusetts, 02454, United States of America.

ABSTRACT
Over-activation of excitatory NMDA receptors and the resulting Ca2+ overload is the main cause of neuronal toxicity during stroke. CaMKII becomes misregulated during such events. Biochemical studies show either a dramatic loss of CaMKII activity or its persistent autonomous activation after stroke, with both of these processes being implicated in cell toxicity. To complement the biochemical data, we monitored CaMKII activation in living hippocampal neurons in slice cultures using high spatial/temporal resolution two-photon imaging of the CaMKIIα FRET sensor, Camui. CaMKII activation state was estimated by measuring Camui fluorescence lifetime. Short NMDA insult resulted in Camui activation followed by a redistribution of its protein localization: an increase in spines, a decrease in dendritic shafts, and concentration into numerous clusters in the cell soma. Camui activation was either persistent (> 1-3 hours) or transient (~20 min) and, in general, correlated with its protein redistribution. After longer NMDA insult, however, Camui redistribution persisted longer than its activation, suggesting distinct regulation/phases of these processes. Mutational and pharmacological analysis suggested that persistent Camui activation was due to prolonged Ca2+ elevation, with little impact of autonomous states produced by T286 autophosphorylation and/or by C280/M281 oxidation. Cell injury was monitored using expressible mitochondrial marker mito-dsRed. Shortly after Camui activation and clustering, NMDA treatment resulted in mitochondrial swelling, with persistence of the swelling temporarily linked to the persistence of Camui activation. The results suggest that in living neurons excitotoxic insult produces long-lasting Ca2+-dependent active state of CaMKII temporarily linked to cell injury. CaMKII function, however, is to be restricted due to strong clustering. The study provides the first characterization of CaMKII activation dynamics in living neurons during excitotoxic insults.

No MeSH data available.


Related in: MedlinePlus