Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Deborah R Boone,Tatsuo Uchida,Maria-Adelaide Micci,Jeanna M Leek,Douglas S Dewitt,Margaret A Parsley,Karen E O Torres,Michael T Falduto,Stacy L Sell,Jeremy C Cowart,Helen L Hellmich,Donald S Prough,Cesar V Borlongan

doi:10.1371/journal.pone.0185943

Abstract

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Highlights

Acute and chronic neurodegeneration and long term deficits in cognitive function are hallmarks of traumatic brain injury (TBI) survivors, there are currently no treatments to address these debilitating repercussions [1,2,3]
Rats were prepared for fluid percussion (FPI) TBI or sham injury, as previously described [37,41], were assigned into nine groups: 1) sham injury (SHAM); 2) TBI; 3) TBI + adenoassociated virus (AAV) scrambled virus (TBI+scrambled AAV (SV); control); 4–6) TBI + 3 neuronal nitric oxide synthase (nNOS) siRNA constructs; 7–9) TBI + 3 glutathione peroxidase 1 (GPx-1) siRNA constructs
RNA interference (RNAi) has been reported in studies of ischemic brain injury [43], in mice subjected to FPI [48], and in vitro stretch models of brain injury [49,50], this study is the first to report the consequences of in vivo RNAi in a rat TBI model

Summary

Introduction

Acute and chronic neurodegeneration and long term deficits in cognitive function are hallmarks of traumatic brain injury (TBI) survivors, there are currently no treatments to address these debilitating repercussions [1,2,3]. New therapeutic modalities to restore normal brain function are worth exploring, such as genomic engineering strategies [4]. We and others have shown that TBI-induced changes in many genes may be causally linked to neurodegeneration [5,6,7,8] and that these injury-induced changes are linked to cognitive. AAV-mediated nNOS and GPx-1 knockdown after TBI articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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