Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motoneurons. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor and has been implicated in the mechanisms of pathogenesis of ALS and other neurological diseases. The potential neuroprotective effects of VEGF in a rat spinal cord organotypic culture were studied in a model of chronic glutamate excitotoxicity in which glutamate transporters are inhibited by threohydroxyaspartate (THA). Particularly, we focused on the effects of VEGF in the survival and vulnerability to excitotoxicity of spinal cord motoneurons. VEGF receptor‐2 was present on spinal cord neurons, including motoneurons. Chronic (3 weeks) treatment with THA induced a significant loss of motoneurons that was inhibited by co‐exposure to VEGF (50 ng/mL). VEGF activated the phosphatidylinositol 3‐kinase/Akt (PI3‐K/Akt) signal transduction pathway in the spinal cord cultures, and the effect on motoneuron survival was fully reversed by the specific PI3‐K inhibitor, LY294002. VEGF also prevented the down‐regulation of Bcl‐2 and survivin, two proteins implicated in anti‐apoptotic and/or anti‐excitotoxic effects, after THA exposure. Together, these findings indicate that VEGF has neuroprotective effects in rat spinal cord against chronic glutamate excitotoxicity by activating the PI3‐K/Akt signal transduction pathway and also reinforce the hypothesis of the potential therapeutic effects of VEGF in the prevention of motoneuron degeneration in human ALS.

Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.IMPORTANCE In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

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