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

Nikolai Otmakhov, Elena V. Gorbacheva, Shaurav Regmi, Ryohei Yasuda, Andy Hudmon, John Lisman

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere0120881
JournalPLoS One
Volume10
Issue number3
DOIs
StatePublished - Mar 20 2015

Fingerprint

Calcium-Calmodulin-Dependent Protein Kinase Type 2
Neurons
neurons
Chemical activation
stroke
calcium
N-Methylaspartate
protein phosphorylation
cells
cytotoxicity
complement
proteins
image analysis
fluorescence
oxidation
toxicity
Cluster Analysis
receptors
Stroke
Mitochondrial Swelling

ASJC Scopus subject areas

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons. / Otmakhov, Nikolai; Gorbacheva, Elena V.; Regmi, Shaurav; Yasuda, Ryohei; Hudmon, Andy; Lisman, John.

In: PLoS One, Vol. 10, No. 3, e0120881, 20.03.2015.

Research output: Contribution to journalArticle

Otmakhov, Nikolai ; Gorbacheva, Elena V. ; Regmi, Shaurav ; Yasuda, Ryohei ; Hudmon, Andy ; Lisman, John. / Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons. In: PLoS One. 2015 ; Vol. 10, No. 3.
@article{3c565f14f65743539512e326d0814122,
title = "Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons",
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.",
author = "Nikolai Otmakhov and Gorbacheva, {Elena V.} and Shaurav Regmi and Ryohei Yasuda and Andy Hudmon and John Lisman",
year = "2015",
month = "3",
day = "20",
doi = "10.1371/journal.pone.0120881",
language = "English (US)",
volume = "10",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "3",

}

TY - JOUR

T1 - Excitotoxic insult results in a long-lasting activation of CaMKIIα and mitochondrial damage in living hippocampal neurons

AU - Otmakhov, Nikolai

AU - Gorbacheva, Elena V.

AU - Regmi, Shaurav

AU - Yasuda, Ryohei

AU - Hudmon, Andy

AU - Lisman, John

PY - 2015/3/20

Y1 - 2015/3/20

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84961290041&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84961290041&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0120881

DO - 10.1371/journal.pone.0120881

M3 - Article

C2 - 25793533

AN - SCOPUS:84961290041

VL - 10

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 3

M1 - e0120881

ER -