Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.

Piper, JA; Jansen, MI; Thomas Broome, S; Rodgers, KJ; Musumeci, G; Castorina, A

Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.

Piper, JA Jansen, MI Thomas Broome, S Rodgers, KJ Musumeci, G Castorina, A

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Publisher:: Horizon Scientific Press
Publication Type:: Journal Article
Citation:: Current Issues in Molecular Biology, 2022, 44, (10), pp. 4500-4516
Issue Date:: 2022-09-28

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Field	Value	Language
dc.contributor.author	Piper, JA
dc.contributor.author	Jansen, MI
dc.contributor.author	Thomas Broome, S
dc.contributor.author	Rodgers, KJ
dc.contributor.author	Musumeci, G
dc.contributor.author	Castorina, A https://orcid.org/0000-0001-7037-759X
dc.date.accessioned	2022-11-07T04:35:00Z
dc.date.available	2022-09-27
dc.date.available	2022-11-07T04:35:00Z
dc.date.issued	2022-09-28
dc.identifier.citation	Current Issues in Molecular Biology, 2022, 44, (10), pp. 4500-4516
dc.identifier.issn	1466-531X
dc.identifier.issn	1467-3045
dc.identifier.uri	http://hdl.handle.net/10453/163282
dc.description.abstract	L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0-2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1β, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.
dc.format	Electronic
dc.language	eng
dc.publisher	Horizon Scientific Press
dc.relation.ispartof	Current Issues in Molecular Biology
dc.relation.isbasedon	10.3390/cimb44100308
dc.rights	info:eu-repo/semantics/openAccess
dc.subject	1108 Medical Microbiology
dc.subject.classification	Microbiology
dc.title	Pro-Inflammatory and Pro-Apoptotic Effects of the Non-Protein Amino Acid L-Azetidine-2-Carboxylic Acid in BV2 Microglial Cells.
dc.type	Journal Article
utslib.citation.volume	44
utslib.location.activity	Switzerland
utslib.for	1108 Medical Microbiology
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - CHT - Health Technologies
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
pubs.organisational-group	/University of Technology Sydney/Centre for Health Technologies (CHT)
utslib.copyright.status	open_access	*
pubs.consider-herdc	false
dc.date.updated	2022-11-07T04:34:38Z
pubs.issue	10
pubs.publication-status	Published
pubs.volume	44
utslib.citation.issue	10

Abstract:

L-Azetidine-2-carboxylic acid (AZE) is a toxic non-protein coding amino acid (npAA) that is highly abundant in sugar and table beets. Due to its structural similarity with the amino acid L-proline, AZE can evade the editing process during protein assembly in eukaryotic cells and be misincorporated into L-proline-rich proteins, potentially causing protein misfolding and other detrimental effects to cells. In this study, we sought to determine if AZE treatment triggered pro-inflammatory and pro-apoptotic responses in BV2 microglial cells. BV2 microglial cells exposed to AZE at increasing concentrations (0-2000 µM) at 0, 3, 6, 12 and 24 h were assayed for cell viability (MTT) and nitric oxide release (Griess assay). Annexin V-FITC/propidium iodide (PI) staining was used to assess apoptosis. Real-time qPCR, Western blot and immunocytochemistry were used to interrogate relevant pro- and anti-inflammatory and other molecular targets of cell survival response. AZE (at concentrations > 1000 µM) significantly reduced cell viability, increased BAX/Bcl2 ratio and caused cell death. Results were mirrored by a robust increase in nitric oxide release, percentage of activated/polarised cells and expression of pro-inflammatory markers (IL-1β, IL-6, NOS2, CD68 and MHC-2a). Additionally, we found that AZE induced the expression of the extracellular matrix degrading enzyme matrix metalloproteinase 9 (MMP-9) and brain derived neurotrophic factor (BDNF), two critical regulators of microglial motility and structural plasticity. Collectively, these data indicate that AZE-induced toxicity is associated with increased pro-inflammatory activity and reduced survival in BV2 microglia. This evidence may prompt for an increased monitoring of AZE consumption by humans.

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