Dendrimer-mediated siRNA delivery knocks down Beclin 1 and potentiates NMDA-mediated toxicity in rat cortical neurons

J. Neurochem. (2012) 120, 259–268. Autophagy is an important process which plays a key role in cellular homeostasis by degrading cytoplasmic components in the lysosomes, which facilitates recycling. Alterations to normal autophagy have been linked to excitotoxicity, but the mechanisms governing its...

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Published in:Journal of neurochemistry 2012-01, Vol.120 (2), p.259-268
Main Authors: Pérez-Carrión, María D., Pérez-Martínez, Francisco C., Merino, Sonia, Sánchez-Verdú, Prado, Martínez-Hernández, José, Luján, Rafael, Ceña, Valentín
Format: Article
Language:English
Subjects:
Adenine - analogs & derivatives
Adenine - pharmacology
Animals
Apoptosis Regulatory Proteins - genetics
Apoptosis Regulatory Proteins - metabolism
Autophagy
Autophagy - drug effects
Beclin 1
Biological and medical sciences
Calcium - metabolism
Cells, Cultured
Central nervous system
Central neurotransmission. Neuromudulation. Pathways and receptors
Cerebral Cortex - cytology
Cytoplasm
Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases
dendrimer
Dendrites - drug effects
Dendrites - metabolism
Dendrites - ultrastructure
Dose-Response Relationship, Drug
Excitatory Amino Acid Agonists - toxicity
excitotoxicity
Fundamental and applied biological sciences. Psychology
Homeostasis
Intracellular Fluid - drug effects
Intracellular Fluid - metabolism
L-Lactate Dehydrogenase - metabolism
Medical sciences
Microscopy, Electron, Transmission
Microtubule-Associated Proteins - metabolism
N-Methylaspartate - toxicity
Neurochemistry
Neurology
Neurons - cytology
Neurons - drug effects
Rats
Ribonucleic acid
RNA
RNA, Small Interfering - metabolism
Signal transduction
siRNA
Transfection - methods
Vertebrates: nervous system and sense organs
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Summary:J. Neurochem. (2012) 120, 259–268. Autophagy is an important process which plays a key role in cellular homeostasis by degrading cytoplasmic components in the lysosomes, which facilitates recycling. Alterations to normal autophagy have been linked to excitotoxicity, but the mechanisms governing its signal transduction remain unclear. The aim of this study was to explore the role of autophagy in neuronal excitotoxic death by delivering small interfering RNA (siRNA) to rat cortical neurons, using a dendrimer to silence the autophagy‐related gene 6 (beclin 1) and to determine the role of autophagy in excitotoxicity. We have found that the dendrimer is very efficient to deliver siRNA to rat cortical neurons, leading to almost complete removal of the target protein Beclin 1. In addition, NMDA increases autophagy markers, such as the protein levels of Beclin 1, the microtubule‐associated light chain 3 (LC3) B‐II/LC3B‐I ratio, and monodansylcadaverine (MDC) labeling in rat cortical neurons. Moreover, NMDA also increases the formation of autophagosomes observed under a transmission electron microscope. Silencing beclin 1 expression blocked NMDA‐induced autophagy. Moreover, Beclin 1 removal potentiated NMDA‐induced neuronal death indicating that autophagy plays a protective role during excitotoxicity and suggesting that targeting autophagy might be a helpful therapeutic strategy in neurodegenerative diseases. siRNA unveils the role of autophagy in excitotoxic deathThis study was performed to identify the role of autophagy in excitotoxic death by knocking down autophagic proteins using dendrimer‐delivered siRNA. The most relevant finding is that autophagy plays a neuroprotective role during excitotoxicity and that it is possible to knock down neuronal proteins using a dendrimer‐delivered siRNA. The conclusion is relevant because it indicates that the autophagic pathway might be a therapeutic target in neurodegenerative diseases and that it is possible to dissect neuronal signaling pathways using dendrimer‐delivered specific siRNA.
ISSN:0022-3042
1471-4159
DOI:10.1111/j.1471-4159.2011.07556.x