Research ReportNeighbor effects of neurons bearing protective transgenes
Introduction
Neurological insults such as seizures or hypoxia–ischemia, can cause devastating damage. This is principally due to excitotoxicity, the result of excessive levels of the major excitatory neurotransmitter in the brain, glutamate, which leads to pathological overactivation of glutamate receptors. Gene therapy in the CNS can prevent neuron death from excitotoxic insults (Sapolsky, 2003) initiated by hypoxia–ischemia or occurring during seizures. Different transgenes have targeted various cellular processes, including metabolism, oxidation, calcium and glutamate trafficking, neural repair, inflammation and apoptosis. As neurons dying necrotically can damage neighbors (e.g., by releasing neurotoxic quantities of glutamate, calcium and reactive oxygen species), saving a neuron from death with gene therapy might aid transgene-negative neighbors. We tested the hypothesis that such transgene-bearing cells would, in effect, constitute “good neighbors.”
We used HSV amplicon vectors to overexpress genes that lessen excitotoxicity by blunting two facets of neuron death (Lawrence et al., 1996, Lee et al., 2003). The first coded for Bcl-2, the anti-apoptotic mitochondrial membrane protein (Hockenbery et al., 1990). The second was a calcium-activated potassium channel, SK2, a transmembrane protein that mediates the Imedium after hyperpolarization of the action potential (Hammond et al., 2006). Overexpression prolongs the refractory period, thus damping excitation. SK2 overexpression has also been shown to limit glutamatergic EPSP responses in hippocampal neurons (Hammond et al., 2006).
We determined whether, following an excitotoxic insult, being in close proximity to an infected neuron increases survival of uninfected neurons, and if neurons overexpressing a protective transgene release fewer neurotoxic soluble factors that would otherwise damage neighbors. Our data suggest that transgene-bearing hippocampal neurons protect their neighbors, and that this “good neighbor” effect does not require cell–cell contact.
Section snippets
Increased neuron survival after excitotoxicity within a defined distance from a transgene-positive neuron
Following excitotoxic insults, we quantified the number of NeuN+ (a neuronal marker) cells within a radius of 5 neuronal nuclei or a generous maximum of 2 cell diameters (∼ 67 μm) of a neuron expressing a protective transgene (identified by GFP expression) (Fig. 1a; henceforth, we will refer to being within that radius as being within the “neighborhood” of the infected neuron). This distance is an estimation of the maximum distance that reactive oxygen species (ROS) may travel after release from
Discussion
To our knowledge, this study is the first to show that proximity to a therapeutically manipulated neuron increases survival of its neighbors in the face of excitotoxic insult. Previous gene therapy studies have focused on cell population benefits, and behavioral effects. This report presents evidence that preventing excitotoxic neuron death by inhibiting apoptosis (which has been shown for both Bcl-2 (Zhao et al., 2003, Zhao et al., 2004) and SK2 (Lee et al., 2003)) or decreasing neuronal
Virus preparation
Bcl-2, SK2, and glucose transporter (GT) vectors were purified as previously described (Lawrence et al., 1996). (Note: GT was previously named GLUT-1.) These were bi-promoter, constitutively active vectors expressing both the therapeutic protein along with a reporter, either eGFP or βgal. Negative control plasmid was pα22eGFP, or for a subset of the laminar culture experiments, pα22βgal (β-Galactosidase); either one expresses the reporter gene only. After packaging these amplicons into modified
Acknowledgments
The authors would like to thank Sheila Brooke, Daniela Kaufer, and Ki Goosens for valuable advice and discussion, Ilona Zemlyak and Hui Wang for cell culture assistance, and John P. Bowman for statistical assistance.
Funding: This work was supported by National Research Service Award 5F32NS10879-02 (A.L.L.), Exploratory/Developmental Research Grant Award MH70815 (awarded to K.G., supported A.L.L.), and Research Project Grant Award AG020633 (R.M.S.) from the National Institutes of Health.
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