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

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

<i>In vitro</i> gene knockdown with siRNA oligonucleotides.

<p>Knockdown of (A) neuronal nitric oxide synthase (nNOS) and (B) glutathione peroxidase-1 (GPx-1) with <i>Silencer</i> pre-designed siRNAs (Ambion) in H19-7 rat hippocampal progenitor cells. All three nNOS and GPx-1 siRNA oligos significantly reduced gene expression in cultured cells.</p

Neurobehavioral assessment after AAV siRNA treatment.

<p>Neurobehavioral assessment after AAV siRNA treatment.</p

Inhibition of Rock and Lim kinase in Rho-Rock-Lim kinase pathway by AAV-nNOS siRNA suggests a mechanism for AAV nNOS-mediated neuroprotection.

<p>(T) = Traumatic brain injury, (N) = nNos (neuronal nitric oxide synthase), (G) = GPx-1 (Glutathione peroxidase-1). Within the pathways, genes that are labeled green are down-regulated and genes that are in red are considered up-regulated compared to uninjured control expression.</p

Gene expression analysis of AAV treated rat brains.

<p>(A) Heat map from microarray analysis (rat Agilent whole genome arrays) of hippocampal gene expression two weeks after siRNA virus treatment. NNOS knockdown has strong effects on injury-associated gene expression. The effects of GPx-1 knockdown were more subtle. (B) AAV mediated gene silencing was very specific; only nNOS knockdown had effects on the canonical nitric oxide signaling pathway. (C) Confirmation of virus induced changes in genes affected by AAV siRNA treatments. All selected gene targets of nNOS knockdown were confirmed and four of six gene targets of GPx-1 knockdown showed expression trends that matched the microarray results.</p

Stereological assessment of neuronal injury.

<p>GPx-1 (2) and nNOS (3) were selected for <i>in vivo</i> assessment of neurodegeneration based on initial results of the qPCR and neurobehavioral tests. Only AAV nNOS (3) significantly reduced numbers of degenerating, Fluoro-Jade-positive (FJ+) cells in the TBI rat hippocampus.</p

Quantitative real-time PCR analysis of laser captured hippocampal neurons.

<p>(A) Laser capture microdissection of AAV siRNA treated hippocampal pyramidal neurons for subsequent quantitative real time PCR (qPCR) analysis. Nissl stained image showing the precise capture of pyramidal neurons for subsequent qPCR analysis of nNOS or GPx-1 expression. (B, C) QPCR analysis of nNOS and GPx-1 expression in laser captured neurons from brains of all control, TBI and virus-treated rats two weeks after siRNA virus treatment.</p

Comparison of AAV-mediated gene silencing on three canonical downstream cell signaling pathways essential for neuronal function.

<p>(A) Ephrin receptor signaling (B) CREB signaling (C) GABA receptor signaling.</p