Elsevier

Brain Research

Volume 908, Issue 1, 10 July 2001, Pages 49-57
Brain Research

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

https://doi.org/10.1016/S0006-8993(01)02572-0Get rights and content

Abstract

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.

Introduction

Considerable knowledge exists concerning the cascade of excitatory amino acid neurotransmitter (EAA) and calcium excess that mediates necrotic neuron death [2]. With this comes the potential for intervention, including the use of viral vectors to express protective transgenes. Such gene therapy approaches have proven protective by targeting metabolic features of injury (by overexpressing the glucose transporter) (e.g., [28]), the calcium excess (with overexpression of the calcium binding protein calbindin D28K) (e.g., [25], [34]), protein malfolding (with hsp72) (e.g., [47]), oxygen radical production (with superoxide dismutase) [25], apoptotic elements (with apoptosis inhibitors such as Bcl-2 or NAIP) (e.g., [1], [30], [32], [45], [46]), and inflammatory elements [6]. In addition, antisense oligonucleotides against the NMDA receptor have been used to decrease excitotoxic injury [44].

These gene therapy studies have examined whether there has been protection (e.g., fewer dead neurons in vitro, a smaller lesion in vivo). In only a few cases has there been documentation that the genetic manipulation actually had its expected proximal effect. For example, overexpression of the calbindin D28K overexpression lowered free cytosolic calcium concentrations [34], while antisense targeting of the NMDA receptor reduced NMDA binding [44]. In other studies, the most proximal consequence was assumed, or there is insufficient knowledge about what that proximal step is.

Few studies documenting the efficacy of a gene therapy approach against a necrotic neurological insult have traced the steps from the proximal consequence of overexpression through the cellular events thought to mediate injury, to the survival endpoint. We have previously studied the consequences of overexpression of rat Glut-1, the non-neuronal brain glucose transporter (in contrast to Glut-3, which is the transporter preferentially found in neurons). We found that Glut-1 overexpression enhances glucose transport in hippocampal cultures and in the hippocampus itself (i.e., the most proximal effect of overexpression), as well as decreasing the neurotoxic effects of a variety of necrotic insults [13], [17], [21], [28], [29]. In this report, we show than enhanced glucose transport is associated with downstream effects likely to enhance the survival of neurons challenged with the excitotoxin kainic acid (KA).

Section snippets

Cell cultures

Mixed neuronal/glial cultures were prepared from the hippocampus of fetal Sprague–Dawley rats (Harlan, Indianapolis, IN) on day 18 of gestation as described [17]. Cultures were used at 10 to 11 days-of-age, at which time approximately 50–70% of cells were non-neuronal, as assessed immunocytochemically with MAP2 and GFAP [22], [28].

293 cells (ATCC CRL 1573) are grown in MEM (Gibco BRL; Cat. No. 61100-061) supplemented with 10% bovine newborn serum (Gibco BRL). All cells were maintained in a 5% CO

Effects on glucose uptake

As the primary consequence of GLUT-1 overexpression, AdRSVGT significantly increased glucose transport, relative to cultures treated with the control vector expressing βgal (Fig. 1). Under those culturing conditions, approximately one quarter of the cells in the dish were infected (Table 1), and uptake rates (as assessed with a bulk measure encompassing the entire dish) were approximately doubled. This implies a substantial increase in uptake in infected cells. The extent to which this increase

Acknowledgements

We thank Frank Graham (McMaster University, Hamilton, ON, Canada) for providing pDE1sp1A and pJM17, Edward Mocarski (Stanford University, Stanford, CA), for providing pON1 and pON820, Morris Birnbaum (Harvard University, Cambridge, MA) for providing the original Glut-1 clone, and Timothy Meier and Angela Lee for their excellent technical assistance. Funding was provided by RO1 NS 32848 to RMS, and a Stanford URO grant to AG.

References (45)

  • N Auer et al.

    Biological differences between ischemia, hypoglycemia, and epilepsy

    Ann. Neurol.

    (1988)
  • V Balcar et al.

    GABA-mediated inhibition in the epileptogenic focus, a process which may be involved in the mechanism of the cobalt-induced epilepsy

    Brain Res.

    (1978)
  • B Belhage et al.

    [3H]d-Aspartate release from cerebellar granule neurons is differentially regulated by glutamate- and K(+)-stimulation

    J. Neurosci. Res.

    (1992)
  • A Bett et al.

    An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3

    Proc. Natl. Acad. Sci. USA

    (1994)
  • A Betz et al.

    Attenuation of stroke size in rats using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist in brain

    J. Cereb. Blood Flow Metab.

    (1997)
  • M Boutelle et al.

    Clinical microdialysis: The role of on-line measurement and quantitative microdialysis

    Acta Neurochir. (Suppl.)

    (1996)
  • B Cheng et al.

    Staurosporine, K-252a, and K252b stabilize calcium homeostasis and promote survival of CNS neurons in the absence of glucose

    J. Neurochem.

    (1994)
  • B Cheng et al.

    Tumor necrosis factors protect neurons against metabolic-excitotoxic insults and promote maintenance of calcium homeostasis

    Neuron

    (1994)
  • F Dagani et al.

    Relationship among ATP synthesis, potassium gradients, and neurotransmitter amino acid levels in isolated rat brain synaptosomes

    J. Neurochem.

    (1987)
  • J Drejer et al.

    Cellular origin of ischemia-induced glutamate release from brain tissue in vivo and in vitro

    J. Neurochem.

    (1985)
  • J Ferkany et al.

    Kainic acid selectively stimulates the release of endogenous excitatory acidic amino acids

    J. Pharmacol. Exp. Ther.

    (1983)
  • F Graham et al.

    Manipulation of adenovirus vectors

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