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Knocking Down HMGB1 Using Dendrimer-Delivered siRNA Unveils Its Key Role in NMDA-Induced Autophagy in Rat Cortical Neurons

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ABSTRACT

Purpose

To explore the role of the High Mobility Group Box 1 (HMGB1) protein in NMDA-mediated excitotoxicity in rat cortical neurons.

Methods

We knocked down HMGB1 using small-interfering RNA (siRNA) delivered into neurons by means of a dendrimer. We determined autophagy activation by measuring the ratio of light chain 3 protein isoforms (LC3B-I)/LC3B-II and by determining autophagolysosome labeling using the specific marker monodansyl cadaverine. Neuronal toxicity was induced by exposing the neurons to N-methyl-D-aspartate (NMDA) and it was determined by measuring Lactate dehydrogenase and MTT reduction.

Results

We found that NMDA receptor stimulation induced both neuronal death and autophagy in rat cortical neurons. In addition, NMDA also caused HMGB1 translocation from the neuronal nucleus to the cytoplasm where it formed a complex with Beclin1. HMGB1 was efficiently knocked down using a specific siRNA causing a blockade of NMDA-induced autophagy and potentiating NMDA-induced neuronal death.

Conclusions

Our study demonstrates that HMGB1 plays a relevant role in neuronal autophagy regulation and suggest a protective role of autophagy during excitotoxicity. In addition, the dendrimer that we have used here is a good vector for siRNA delivery to neurons allowing lack-of-function studies.

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Abbreviations

DTT:

Dithiothreitol

EGTA:

Ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid

HEPES:

N-2-hydroxyethylpiperazine-N′-2- ethanesulphonic acid

HMGB1:

High Mobility Group Box 1

HRP:

Horseradish peroxidase

LC3:

Microtubule-associated light chain 3

LDH:

Lactate dehydrogenase

MDC:

Monodansylcadaverine

MTT:

2,5-diphenyl-3-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide

NMDA:

N-methyl-D-aspartate

PAGE:

Polyacrylamide gel electrophoresis

PBS:

Phosphate-buffered saline

siRNA:

Small-interfering RNA

TGD:

Transgeden dendrimer

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ACKNOWLEDGMENTS AND DISCLOSURES

We thank Ana B. García for her technical assistance. M.D.P-C. is a recipient of a Torres Quevedo contract from Ministerio de Ciencia e Innovación (Spain) and NanoDrugs, S.L. This work has been supported, in part, by grants PI081434 from Fondo de Investigaciones Sanitarias, BFU2011-30161-C02-01 from Ministerio de Ciencia e Innovación and PII1I09-0163-4002 and POII10-0274-3182 from Consejería de Educación, JCCM and EuronanoMed projects DENANORNA and DENPEPTHIV to V.C.

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Authors and Affiliations

  1. Unidad Asociada Neurodeath, CSIC-Universidad de Castilla-La Mancha Departamento de Ciencias Médicas, Albacete, Spain

    María D. Pérez-Carrión & Valentín Ceña

  2. CIBERNED, Instituto de Salud Carlos III, Madrid, Spain

    María D. Pérez-Carrión & Valentín Ceña

  3. Unidad Asociada Neurodeath, Facultad de Medicina, Avda. Almansa, 14, 02006, Albacete, Spain

    Valentín Ceña

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  1. María D. Pérez-Carrión
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  2. Valentín Ceña
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Correspondence to Valentín Ceña.

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Pérez-Carrión, M.D., Ceña, V. Knocking Down HMGB1 Using Dendrimer-Delivered siRNA Unveils Its Key Role in NMDA-Induced Autophagy in Rat Cortical Neurons. Pharm Res 30, 2584–2595 (2013). https://doi.org/10.1007/s11095-013-1049-9

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