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Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7- and calpain-mediated caspase-12 activation

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Abstract

Glucose is the main energy source in brain and it is critical for correct brain functioning. Type 1 diabetic patients might suffer from severe hypoglycemia if exceeding insulin administration, which can lead to acute brain injury if not opportunely corrected. The mechanisms leading to hypoglycemic brain damage are not completely understood and the role of endoplasmic reticulum (ER) stress has not been studied. ER stress resulting from the accumulation of unfolded or misfolded proteins in the ER is counteracted by the unfolded protein response (UPR). When the UPR is sustained, apoptotic death might take place. We have examined UPR activation during glucose deprivation (GD) in hippocampal cultured neurons and its role in the induction of apoptosis. Activation of the PERK pathway of the UPR was observed, as increased phosphorylation of eIF2α and elevated levels of the transcription factor ATF4, occurred 30 min after GD and the levels of the chaperone protein, GRP78 and the transcription factor CHOP, increased after 2 h of GD. In addition, we observed an early activation of caspase-7 and 12 during GD, while caspase-3 activity increased only transiently during glucose reintroduction. Inhibition of caspase-3/7 and the calcium-dependent protease, calpain, significantly decreased caspase-12 activity. The ER stress inhibitor, salubrinal prevented neuronal death and caspase-12 activity. Results suggest that the PERK pathway of the UPR is involved in GD-induced apoptotic neuronal death through the activation of caspase-12, rather than the mitochondrial-dependent caspase pathway. In addition, we show that calpain and caspase-7 are soon activated after GD and mediate caspase-12 activation and neuronal death.

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Abbreviations

ER:

Endoplasmic reticulum

UPR:

Unfolded protein response

GD:

Glucose deprivation

GR:

Glucose reintroduction,

GRP78:

78-kDa Glucose-regulated protein

eIF2α:

Eukaryotic translation initiation factor-2

ATF4:

Activating transcription factor 4

CHOP:

C/EBP homologous protein

QVDOPH:

Q-Val-Asp-OPH

DEVDCHO:

Acetyl-Asp-Glu-Val-Asp-aldehyde

MDL-28170:

N-Benzyloxycarbonylvalylphenylalaninal

Q-ATAD:

Q-Ala-Thr-Ala-Asp(OMe)-OPH

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

STS:

Staurosporine

Tg:

Thapsigargin

Tm:

Tunicamycin

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Acknowledgments

This work was supported by S112179 CONACyT and IN211710-3 PAPIIT (UNAM) Grants to L.M. and 221026 CONACyT fellowship to S.G.C The authors thank Teresa Montiel for her technical assistance. This study was performed in partial fulfillment of the requirements for the PhD degree in Ciencias Bioquímicas of S. García de la Cadena at the Universidad Nacional Autónoma de México.

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Authors and Affiliations

  1. División de Neurociencias, Departamento de Neuropatología Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, AP 70-253, Mexico, DF, 04510, Mexico

    Selene García de la Cadena & Lourdes Massieu

  2. Departamento de Neuroquímica, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Mexico, DF, Mexico

    Karla Hernández-Fonseca

  3. Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Mexico, DF, Mexico

    Ignacio Camacho-Arroyo

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  1. Selene García de la Cadena
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  2. Karla Hernández-Fonseca
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  3. Ignacio Camacho-Arroyo
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  4. Lourdes Massieu
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Correspondence to Lourdes Massieu.

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Fig. S1

Inhibition of caspase-7, calpain and reticular stress suppresses caspase-12 processing. Caspase-12 proteolysis into its active fragment (36 kDa) was determined by Western blot. Cells were exposed to 1 h GD in the presence or the absence of DEVDCHO (25 μM), MDL-28170 (MDL, 50 μM) or salubrinal (50 μM). A representative Western blot is shown. Results represent mean ± SEM of three independent experiments and are expressed as the optical density of the active fragment (36 kDa) band/β-Actin (42 kDa). Data were analyzed by one-way ANOVA followed by a Fisher’s least significant difference test. * P < 0.05 versus 1 h GD and & P < 0.05 versus control. Supplementary material 1 (TIFF 12,198 kb)

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de la Cadena, S.G., Hernández-Fonseca, K., Camacho-Arroyo, I. et al. Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7- and calpain-mediated caspase-12 activation. Apoptosis 19, 414–427 (2014). https://doi.org/10.1007/s10495-013-0930-7

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