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Neuron-specific oxidative stress using a novel RNAi strategy in vivo causes cognitive impairment and carbonylation of key dendrite proteins

AutorFernandez-Fernandez, Seila; Requejo-Aguilar, Raquel; Ros, Joaquim; Almeida, Angeles; Bolaños, Juan P.
Fecha de publicación2013
EditorSociedad Española de Bioquímica y Biología Molecular
CitaciónXXXVI Congreso SEBBM (2013)
ResumenExcess reactive oxygen species is behind the causes of neurological disorders, but the lack of tissue-specific experimental models of oxidative stress has complicated in vivo demonstration of proof-of-concept and mechanisms. Here, we designed and constructed a DNA vector with two unique restriction sites ready to insert any small hairpin RNA (shRNA) sequence in the opposite strand orientation, flanked by LoxP and Lox2272 sites, and governed by the polymerase III (H1) promoter. This DNA construct, after insertion of any shRNA, is suitable to trigger protein knockdown-mediated loss-of-function after tissue specific Cre-mediated recombination in vitro or in vivo. Using this strategy, we inserted a previously validated shRNA sequence targeted against glutamate-cysteine ligase catalytic subunit (GCL), the rate-limiting step in glutathione biosynthesis, into the above-mentioned DNA vector. With this shGCLfloxed DNA construct, we generated several transgenic mice founders, one of which harbored a unique insertion site and showed no apparent biochemical or phenotypic alterations. This shGCLfloxed mouse was crossed with mice harboring Cre recombinase governed by the neuron-specific CaMKIIα promoter (CaMKIIα-Cre), which is active after two postnatal weeks. The resulting CaMKIIα-shGCL mouse showed decreased GCL protein levels and increased signs of oxidative stress in hippocampal neurons during adulthood in vivo, as well as cognitive impairment. Oxy-proteome analysis of this region revealed a 3-fold increase in carbonylation of several proteins, of which at least one is involved in dendrite formation. To the best of our knowledge, this is the first evidence showing that neuron-specific oxidative stress in vivo oxidizes a key protein involved in memory formation leading to signs of dementia.
DescripciónResumen del póster presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular celebrado en Madrid del 3 al 6 de septiembre de 2013.
Aparece en las colecciones: (IBFG) Comunicaciones congresos
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