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Pharmaceutical Research

, Volume 30, Issue 10, pp 2584–2595 | Cite as

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

  • María D. Pérez-Carrión
  • Valentín Ceña
Research Paper

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.

KEY WORDS

autophagy dendrimer excitotoxicity neurons siRNA 

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

Notes

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.

Supplementary material

11095_2013_1049_MOESM1_ESM.doc (100 kb)
Supplementary Figure 1 (DOC 99.5 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • María D. Pérez-Carrión
    • 1
    • 2
  • Valentín Ceña
    • 1
    • 2
    • 3
  1. 1.Unidad Asociada Neurodeath, CSIC-Universidad de Castilla-La Mancha Departamento de Ciencias MédicasAlbaceteSpain
  2. 2.CIBERNED, Instituto de Salud Carlos IIIMadridSpain
  3. 3.Unidad Asociada Neurodeath, Facultad de MedicinaAlbaceteSpain

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