Dendrimer-mediated siRNA delivery knocks down Beclin 1 and potentiates NMDA-mediated toxicity in rat cortical neurons.
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.
NanoDrugs, S.L. Parque Científico y Tecnológico, Albacete, Spain.
María D
MD
Pérez-Carrión
Francisco C
FC
Pérez-Martínez
Sonia
S
Merino
Prado
P
Sánchez-Verdú
José
J
Martínez-Hernández
Rafael
R
Luján
Valentín
V
Ceña
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects; D000071186:Beclin-1; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D003712:Dendrites / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D004305:Dose-Response Relationship, Drug; D018690:Excitatory Amino Acid Agonists / Q000633:toxicity*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D016202:N-Methylaspartate / Q000633:toxicity*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism*; D051381:Rats; D014162:Transfection / Q000379:methods
D000225:Adenine; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D002118:Calcium; D018690:Excitatory Amino Acid Agonists; D007770:L-Lactate Dehydrogenase; D008869:Microtubule-Associated Proteins; D016202:N-Methylaspartate; D034741:RNA, Small Interfering; C512829:Becn1 protein, rat; C526109:Anxa3 protein, rat; C025946:3-methyladenine