Sheri L. Fink

Defective herpes simplex virus vectors expressing the rat brain stress-inducible heat shock protein 72 protect cultured neurons from severe heat shock

Abstract: Recently, preinduction of the heat shock response has been shown to protect CNS neurons undergoing various stressful insults, e.g., heat, ischemia, or exposure to excitotoxins. However, it is not known which of the proteins induced by the heat shock response mediate the protective effects. Previous correlative evidence points to a role for the highly stress‐induced 72‐kDa heat shock protein (hsp72). However, it is not known whether hsp72 expression alone can protect against a range of acute neuronal insults. We constructed a herpes simplex virus‐1 vector carrying the rat brain stress‐inducible hsp72 gene and the Escherichia coli lacZ (marker) gene. Infection with the vector caused hippocampal neurons to coexpress hsp72 and β‐galactosidase. Infection with a control vector led to marker gene expression only. Overexpression of hsp72 protected cultured hippocampal neurons against a heat shock but not against the metabolic toxin 3‐nitropropionic acid or the excitotoxin glutamate. This is the first published report of protection following heat shock protein transfection in CNS neurons.

Defective herpes simplex virus vectors expressing the rat brain glucose transporter protect cultured neurons from necrotic insults

Abstract: Because neurons are postmitotic, they are irreplaceable once they succumb to necrotic insults such as hypoglycemia, ischemia, and seizure. A paucity of energy can exacerbate the toxicities of these insults; thus, a plausible route to protect neurons from necrotic injury would be to enhance their glucose uptake capability. We have demonstrated previously that defective herpes simplex virus (HSV) vectors overexpressing the rat brain glucose transporter (GT) gene (gt) can enhance glucose uptake in adult rat hippocampus and in hippocampal cultures. Furthermore, we have observed that such vectors can maintain neuronal metabolism during hypoglycemia and reduce kainic acid‐induced seizure damage. In this study, we have developed bicistronic vectors that coexpressed gt and Escherichia coli lacZ as a reporter gene, which allows us to identify directly neurons that are infected with the vectors. Overexpression of GT from these vectors protected cultured hippocampal, spinal cord, and septal neurons against various necrotic insults, including hypoglycemia, glutamate, and 3‐nitropropionic acid. Our observations demonstrate the feasibility of using HSV vectors to protect neurons from necrotic insults. Although this study has concentrated on the delivery of gt, other genes with therapeutic or protective capability might also be used.

Herpes simplex virus vector system: analysis of its in vivo and in vitro cytopathic effects

With its natural propensity to infect and establish life-long latency in neurons, herpes simplex virus type 1 (HSV-1) has been successfully employed by various laboratories as vectors for gene transfer into neurons. However, analysis of its cytopathic effects in vivo and in vitro has been limited. In this study, we examined the cytopathic effects of 2 HSV-1 alpha 4 mutants (ts756 and d120) on adult rat hippocampus and striatum and of d120 on hippocampal neurons in culture. We assessed damage by stringent counting of surviving neurons after infection and demonstrated that while neither ts756 nor d120 infection resulted in any gross anatomical or behavioral changes of the animals, ts756, but not d120, produced a significant amount of damage in the CA4 cell field and dentate gyrus of the hippocampus. Thus, since crude examination is insufficient to detect subtle but significant degrees of neuron loss, the cytopathic effects of HSV or any vector system must be carefully analyzed. Furthermore, we also observed that uninfected cell lysates damaged neurons, both in vivo and in vitro. This cytotoxicity occurred within the first 24 h post-inoculation and probably arose through the activation of glutamate receptors. For the preparation of HSV vectors, purification of the virus from soluble cellular components by a simple pelleting step can significantly decrease such acute toxicity.

Energy and Glutamate Dependency of 3-Nitropropionic Acid Neurotoxicity in Culture

3-Nitropropionic acid (3-NP) irreversibly inhibits the activity of the mitochondrial enzyme succinate dehydrogenase, leading to selective striatal lesions when administered in vivo. We studied the effects of 3-NP on dissociated cultures of neurons and glia with the following findings: (a) 3-NP killed cultured striatal neurons with a median lethal dose of 2.5 mM after 20 h of incubation in 20.0 mM glucose medium. Despite its selective toxicity in vivo, cultured striatal, hippocampal, septal, and hypothalamic neurons were similarly sensitive to 3-NP incubation. (b) 3-NPs effects were remarkably energy substrate dependent, with the median lethal dose dropping over an order of magnitude when glucose concentrations were lowered to 3.0 mM, a condition that was itself nontoxic. Cultures exposed to 3-NP had a far greater sensitivity to energy availability than those exposed to glutamate. (c) Recent work suggests that 3-NP toxicity may be partially mediated by excitotoxins. Our experiments show that neither kynurenic acid, a nonspecific glutamate receptor antagonist, nor the NMDA-receptor antagonist, DL-2-amino-7-phosphonoheptanoic acid, either in combination or alone, reduced 3-NP toxicity in striatal cultures. However, the noncompetitive NMDA antagonist MK-801 did attenuate 3-NP toxicity.

Neuroprotective effects of an adenoviral vector expressing the glucose transporter: a detailed description of the mediating cellular events

Considerable knowledge exists concerning the events mediating neuron death following a necrotic insult; prompted by this, there have now been successful attempts to use gene therapy approaches to protect neurons from such necrotic injury. In many such studies, however, it is not clear what sequence of cellular events connects the overexpression of the transgene with the enhanced survival. We do so, exploring the effects of overexpressing the Glut-1 glucose transporter with an adenoviral vector in hippocampal cultures challenged with the excitotoxin kainic acid (KA). Such overexpression enhanced glucose transport, attenuated the decline in ATP concentrations, decreased the release of excitatory amino acid neurotransmitters, and decreased the total free cytosolic calcium load. Commensurate with these salutary effects, neuronal survival was enhanced with this gene therapy intervention. Thus, the neuroprotective effects of this particular gene therapy occurs within the known framework of the mechanisms of necrotic neuronal injury.

An adenoviral vector expressing the glucose transporter protects cultured striatal neurons from 3-nitropropionic acid

Considerable interest has focused on the possibility of using gene transfer techniques to introduce protective genes into neurons around the time of necrotic insults. We have previously used herpes simplex virus amplicon vectors to overexpress the rat brain glucose transporter, Glut-1 (GT), and have shown it to protect against a variety of necrotic insults both in vitro and in vivo, as well as to buffer neurons from the steps thought to mediate necrotic injury. It is critical to show the specificity of the effects of any such transgene overexpression, in order to show that protection arises from the transgene delivered, rather than from the vector delivery system itself. As such, we tested the protective potential of GT overexpression driven, in this case, by an adenoviral vector, against a novel insult, namely exposure of primary striatal cultures to the metabolic poison, 3-nitropropionic acid (3NP). We observed that GT overexpression buffered neurons from neurotoxicity induced by 3NP.

Use of Herpes Simplex Virus Vectors for Protection from Necrotic Neuron Death

Publisher Summary This chapter focuses on use of herpes simplex virus vectors for protection from necrotic neuron death. Central to such neuron death are the excitatory amino acids (EAA) such as glutamate and aspartate. These are the most abundant excitatory neurotransmitters in the brain, and they play vital roles in phenomena such as long-term potentiation in the hippocampus. Necrotic insults, such as global ischemia and infarct, seizure, or hypoglycemia, all result in excessive synaptic concentrations of EAAs, leading to excessive mobilization of free cytosolic calcium. The deleterious consequences of such excess include promiscuous overactivation of calcium-dependent proteases, lipases, and nucleases. The greatest pathologic significance is that the calcium excess leads to the generation of damaging radical oxygen species. The most important routes by which this occurs include calcium-dependent activation of xanthine oxidase, nitric oxide synthase, and phospholipase, or indirectly generate oxygen radicals. Energy plays a critical role in determining whether the EAAs and calcium subserve their normal physiologic roles or pathophysiologic ones. Necrotic neurological insults are energetic in nature, and they dramatically compromise the capacity of neurons to contain EAAs and calcium. During a necrotic insult, energy failure leads to depolarization and enhanced release of EAAs. The other topics discussed in the chapter are neuroprotection using monocistronic vectors and bicistronic vectors, additional neuroprotective strategies using HSV vectors, and cytopathic effects of HSV vectors.