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

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.

Diphenyl diselenide protects neuronal cells against oxidative stress and mitochondrial dysfunction: Involvement of the glutathione-dependent antioxidant system

“…Therefore, our results indicate that the positive modulation of Gpx1 expression would be a key component for the protection afforded by (PhSe) 2 in HT22 cells. It should be mentioned the importance of GPx activity, since its decrease promote susceptibility to oxidative stress by allowing the accumulation of harmful oxidants [58] , [59] , and because other selenoproteins cannot replace its function in protecting from generalized oxidative stress [5] . Gpx4 gene expression was not upregulated after (PhSe) 2 incubation and the results of decrease observed in the mtGpx4 expression would not represent a biological significance event according to functional thresholds used in quantitative PCR analyses [60] .…”

Mechanistic insights gained from cell and molecular analysis of the neuroprotective potential of bioactive natural compounds in an immortalized hippocampal cell line

“…To test if CM could be neuroprotective in a rodent model of TBI, the number of dying/injured, Fluoro-Jade-positive neurons in the rat hippocampus was determined as previously described in Boone et al, [ 27 ]. Briefly, animals were survived for 24 hours, sacrificed and brains dissected out and frozen immediately on dry ice.…”

“…10μm coronal rat brain tissue sections were collected on Superfrost Plus slides, every 15 th section for 10 sections through the hippocampus. Sections were stained with 0.0001% FJC (a marker for neuronal injury) and neurons were counted in the CA1/2 & CA3 regions using stereological methods [ 27 ].…”

Strategies for Oligodendrocyte and Myelin Repair in Traumatic CNS Injury

“…Functional testing was not performed in this experiment, but a similar study silencing IRF5 in macrophages in a mouse model of SCI found significant motor recovery as well as decreases in demyelination and inflammation (Li et al, 2016 ). Another experiment silencing nitric oxide synthase in a TBI model showed decreased neuronal degeneration in addition to improved working memory (Boone et al, 2017 ). As of now, most experiments using RNAi in TBI models focus on minimizing post-injury edema (Fukuda and Badaut, 2013 ; Xu et al, 2014 ; Guan et al, 2020 ).…”