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Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction
Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimen...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9313431/ https://www.ncbi.nlm.nih.gov/pubmed/35883560 http://dx.doi.org/10.3390/biom12071004 |
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author | Polster, Brian M. Mark, Karla A. Arze, Rafael Hudson, Derek |
author_facet | Polster, Brian M. Mark, Karla A. Arze, Rafael Hudson, Derek |
author_sort | Polster, Brian M. |
collection | PubMed |
description | Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases protease activity in neuronal cell bodies prior to DCD. Treatment of rat cortical neurons with excitotoxic (100 µM) glutamate increased the low baseline rate of intracellular cA-TAT proteolysis by approximately three-fold prior to DCD and by approximately seven-fold upon calcium deregulation. The glutamate-induced rate enhancement prior to DCD was suppressed by glutamate receptor antagonists, but not by calpain or proteasome inhibitors, whereas DCD-stimulated proteolysis was partly attenuated by the proteasome inhibitor MG132. Further suggesting that cA-TAT cleavage is calpain-independent, cA-TAT fluorescence was observed in immortalized Capn4 knockout fibroblasts lacking the regulatory calpain subunit. About half of the neurons lost calcium homeostasis within two hours of a transient, 20 min glutamate receptor stimulation. These neurons had a significantly (49%) higher mean baseline cA-TAT proteolysis rate than those maintaining calcium homeostasis, suggesting that the unknown protease(s) cleaving cA-TAT may influence DCD susceptibility. Overall, the results indicate that excitotoxic glutamate triggers the activation of calpain-independent neuronal protease activity prior to the simultaneous loss of calcium homeostasis and mitochondrial bioenergetic function. |
format | Online Article Text |
id | pubmed-9313431 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-93134312022-07-26 Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction Polster, Brian M. Mark, Karla A. Arze, Rafael Hudson, Derek Biomolecules Article Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases protease activity in neuronal cell bodies prior to DCD. Treatment of rat cortical neurons with excitotoxic (100 µM) glutamate increased the low baseline rate of intracellular cA-TAT proteolysis by approximately three-fold prior to DCD and by approximately seven-fold upon calcium deregulation. The glutamate-induced rate enhancement prior to DCD was suppressed by glutamate receptor antagonists, but not by calpain or proteasome inhibitors, whereas DCD-stimulated proteolysis was partly attenuated by the proteasome inhibitor MG132. Further suggesting that cA-TAT cleavage is calpain-independent, cA-TAT fluorescence was observed in immortalized Capn4 knockout fibroblasts lacking the regulatory calpain subunit. About half of the neurons lost calcium homeostasis within two hours of a transient, 20 min glutamate receptor stimulation. These neurons had a significantly (49%) higher mean baseline cA-TAT proteolysis rate than those maintaining calcium homeostasis, suggesting that the unknown protease(s) cleaving cA-TAT may influence DCD susceptibility. Overall, the results indicate that excitotoxic glutamate triggers the activation of calpain-independent neuronal protease activity prior to the simultaneous loss of calcium homeostasis and mitochondrial bioenergetic function. MDPI 2022-07-20 /pmc/articles/PMC9313431/ /pubmed/35883560 http://dx.doi.org/10.3390/biom12071004 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Polster, Brian M. Mark, Karla A. Arze, Rafael Hudson, Derek Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title | Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title_full | Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title_fullStr | Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title_full_unstemmed | Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title_short | Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction |
title_sort | calpain-independent intracellular protease activity is elevated in excitotoxic cortical neurons prior to delayed calcium deregulation and mitochondrial dysfunction |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9313431/ https://www.ncbi.nlm.nih.gov/pubmed/35883560 http://dx.doi.org/10.3390/biom12071004 |
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