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Cleavage of the Vesicular GABA Transporter under Excitotoxic Conditions Is Followed by Accumulation of the Truncated Transporter in Nonsynaptic Sites.

Rodrigues Gomes, Joao Carlos; Lobo, Andrea C; Melo, Carlos V; Inacio, Ana LU ; Takano, Jiro; Iwata, Nobuhisa; Saido, Takaomi C; de Almeida, Luís P; Wieloch, Tadeusz LU and Duarte, Carlos B (2011) In The Journal of neuroscience : the official journal of the Society for Neuroscience 31(12). p.4622-4635
Abstract
GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in... (More)
GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
The Journal of neuroscience : the official journal of the Society for Neuroscience
volume
31
issue
12
pages
4622 - 4635
publisher
Society for Neuroscience
external identifiers
  • WOS:000288750700028
  • PMID:21430162
  • Scopus:79953008096
ISSN
1529-2401
DOI
10.1523/JNEUROSCI.3541-10.2011
language
English
LU publication?
yes
id
2736c924-8713-4af1-bbcd-2359866d18f3 (old id 1883551)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/21430162?dopt=Abstract
date added to LUP
2011-04-01 21:00:00
date last changed
2016-11-06 04:28:44
@misc{2736c924-8713-4af1-bbcd-2359866d18f3,
  abstract     = {GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.},
  author       = {Rodrigues Gomes, Joao Carlos and Lobo, Andrea C and Melo, Carlos V and Inacio, Ana and Takano, Jiro and Iwata, Nobuhisa and Saido, Takaomi C and de Almeida, Luís P and Wieloch, Tadeusz and Duarte, Carlos B},
  issn         = {1529-2401},
  language     = {eng},
  number       = {12},
  pages        = {4622--4635},
  publisher    = {ARRAY(0x86bb330)},
  series       = {The Journal of neuroscience : the official journal of the Society for Neuroscience},
  title        = {Cleavage of the Vesicular GABA Transporter under Excitotoxic Conditions Is Followed by Accumulation of the Truncated Transporter in Nonsynaptic Sites.},
  url          = {http://dx.doi.org/10.1523/JNEUROSCI.3541-10.2011},
  volume       = {31},
  year         = {2011},
}