Journal Article

1069. The HSV-2 Vector DeltaRR Prevents Excitotoxicity-Induced Neuronal Cell Loss through Activation of Survival Pathways and Glial Cell Modulation

Gober, Michael D, Laing, Jennifer M, Aurelian, Laure

2006

Overview

Gene therapy of neurodegenerative diseases is of significant current interest, but the identification of appropriate gene targets and effective delivery platforms has proven to be a major stumbling block. We constructed a novel, growth compromised HSV-2 based vector, ΔRR, which utilizes the viral anti-apoptotic gene ICP10PK to protect neurons from excitotoxic injury in animal models of temporal lobe epilepsy. In this model, intranasally delivered ΔRR evidenced no toxicity and the anti-apoptotic gene ICP10PK was expressed in tissues along the olfactory bulb route as determined by immunoblotting and/or immunohistochemistry. In the hippocampus, expression began on day 2 after ΔRR administration and was independed of virus replication, as evidenced by plaque assays and the failure to detect the viral protein, VP5, the expression of which requires viral DNA replication. ICP10PK expression lasted for at least 42 days, and it prevented kainic acid (KA) induced neuronal cell loss in the hippocmapus. An HSV-2 vector deleted in ICP10PK(ΔPK) failed to protect from KA induced seizures and neuronal loss. Studies of organotypic hippocampal cultures (OHC) indicated that protection depended on ΔRR induced activation of the MEK/ ERK and PI3K/Akt survival pathways, which override KA-induced apoptosis. Brains from ΔRR treated animals were also protected from KA induced inflammation, including microglial expression of TNFa, and astrocyte upregulation of glial fibrillary protein (GFAP). This may reflect the anti-inflammatory activity of ΔRR, because the levels of TNFα and RANTES in supernatants from ΔRR treated primary microglia cultures (114.4 pg/ml and 34.2 pg/ml, respectively) were lower than those in supernatants from ΔPK treated microglia (586.8 pg/ml and 321.7 pg/ml, respectively). In addition, ΔRR treated microglia induced neuroprotective factors. Thus, neurons exposed to the excitotoxin NMDA (5uM; 3hrs) were protected from apoptosis when co-cultured with ΔRR treated microglia as evidenced by double immunofluorescence with FITC-dUTP (TUNEL) and Texas red labeled NeuN antibody. Protection was mediated by a soluble factor, because similar results were obtained in cultures in which the ΔRR treated microglia were physically separated from the neurons using the Co-Star transwell insert co-culturing system. Protection was not seen with ΔPK or PBS treated microglia (10.4±3.3%, 53.2±6.5%, and 60±4.3%, TUNEL+cells for ΔRR, ΔPK, and PBS respectively, p<0.001). ΔRR (but not ΔPK) also induced expression of the neuroprotective transforming growth factorβ (TGFβ) and nerve growth factor (NGF) in primary astrocyte cultures. Collectively, the data indicate that ΔRR protects neurons from excitotoxic cell death by overriding apoptotic pathways, as well as modulating support cells in the CNS to create a more favorable microenviornment for neuron survival.

• Publisher: Elsevier Limited
• Subject: Apoptosis / Artificial chromosomes / Ataxia / Gene therapy / Growth factors / Herpes viruses / Neurons / Proteins / Vectors (Biology)