Elsevier

Neurobiology of Aging

Volume 31, Issue 12, December 2010, Pages 2185-2191
Neurobiology of Aging

Brief communication
VEGF protects motor neurons against excitotoxicity by upregulation of GluR2

https://doi.org/10.1016/j.neurobiolaging.2008.12.007Get rights and content

Abstract

Influx of Ca2+ ions through the α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptors is toxic to neurons and contributes to motor neuron degeneration observed in amyotrophic lateral sclerosis (ALS). The Ca2+ permeability of the AMPA receptor depends on its subunit composition. If the GluR2 subunit is present in the receptor complex, the AMPA receptor is impermeable to Ca2+. In this study, we identified vascular endothelial growth factor-A (VEGF) as a GluR2 inducing molecule. Cultured motor neurons pretreated with VEGF displayed higher GluR2 levels. This resulted in AMPA receptor currents with a low relative Ca2+ permeability and in motor neurons that were less vulnerable to AMPA receptor-mediated excitotoxicity. This effect of VEGF was mediated through the VEGFR2 present on the motor neurons and was due to stimulation of GluR2 transcription. Intracerebroventricular treatment with VEGF similarly induced GluR2 expression in the ventral spinal cord of rats and this mechanism contributes to the protective effect of VEGF on motor neurons.

Introduction

Ca2+ influx through glutamate receptors is an important trigger of cell death in a broad variety of disorders of the central nervous system. It leads to neuronal death in stroke, neurotrauma, epilepsy, neurodegenerative disorders and contributes to degeneration of oligodendrocytes in multiple sclerosis (Hazell, 2007, Lipton and Rosenberg, 1994, Matute et al., 2001, Van Den Bosch et al., 2006).

In motor neuron degeneration, as seen in amyotrophic lateral sclerosis (ALS), excessive stimulation of AMPA–type of glutamate receptors (also called AMPA receptor-mediated excitotoxicity) is an important pathogenetic mechanism (Van Den Bosch et al., 2006). Motor neurons are more prone to AMPA receptor-mediated excitotoxicity as they display a high number of Ca2+-permeable AMPA receptors (Carriedo et al., 1996, Heath et al., 2002, Kawahara et al., 2004, Kawahara et al., 2003, Van Den Bosch et al., 2000). The Ca2+ permeability of the AMPA receptor is determined by the GluR2 subunit and only AMPA receptors lacking GluR2 are permeable to Ca2+ ions. Motor neurons express low levels of the GluR2 subunit rendering them more vulnerable to AMPA receptor-mediated excitotoxicity (Van Damme et al., 2002). Recently, we showed that astrocytes are able to protect against excitotoxicity by inducing GluR2 expression in motor neurons. Moreover, this characteristic is abolished when mutant SOD1, a genetic cause of ALS, is over expressed in astrocytes (Van Damme et al., 2007), a cell type known to be an important contributor to disease progression (Di Giorgio et al., 2007, Kim et al., 2006, Nagai et al., 2007, Yamanaka et al., 2008). Besides their role as regulators of GluR2 expression, astrocytes also secrete growth factors providing trophic support to neurons (Boillee et al., 2006).

One of the growth factors released by astrocytes and involved in the pathogenesis of ALS is vascular endothelial growth factor-A (VEGF) (Lambrechts et al., 2004). VEGF is known to exert direct trophic and protective effects on different types of neurons (Van Den Bosch et al., 2004, Kilic et al., 2006, Li et al., 2003, Matsuzaki et al., 2001, Nicoletti et al., 2008, Svensson et al., 2002, Tolosa et al., 2008, Tovar et al., 2007). A deletion in the hypoxia responsive element in the promoter region of the VEGF gene (VEGFδ/δmouse) caused motor neuron degeneration reminiscent to ALS (Oosthuyse et al., 2001). Interestingly, lowering VEGF levels in mutant SOD1 mice accelerates the motor neuron degeneration (Lambrechts et al., 2004), whereas treatment of mutant SOD1 animals with VEGF slows the disease (Azzouz et al., 2004, Storkebaum et al., 2005).

It is clear that both AMPA receptor-mediated excitotoxicity and shortage of VEGF are important mediators of the motor neuron death observed in ALS. As a consequence, our aim was to find out whether a link exists between these two mechanisms involved in motor neuron degeneration.

Section snippets

Cell cultures, cell death experiments, immunocytochemistry and perforated-patch clamp recordings

Motor neurons from Wistar rats were cultured on a pre-established feeder layer of astrocytes. Cell death experiments and perforated patch clamp recordings were performed as previously described (Van Damme et al., 2002). Motor neuron cultures and astrocyte cultures were stained with glial fibrillary acidic protein (GFAP; 1/1000; DAKO, Denmark), and VEGFR2 (1/50; Santa Cruz Biotechnology, Santa Cruz, USA) antibodies. As secondary antibodies, Alexa555 labelled antibody (1/500; Molecular Probes,

VEGF protects spinal motor neurons from excitotoxicity

In addition to its well-known effects on neuronal survival, we examined whether VEGF also influenced excitotoxic motor neuron death. Therefore Wistar motor neurons cultured on a pre-established glial feeder layer were exposed to kainic acid, an AMPA-receptor agonist, inducing approximately 50% of motor neuron death. Pretreatment of these cultures with recombinant VEGF decreased AMPA receptor-mediated motor neuron death to 30.2%, a reduction by 40% (Fig. 1A). From a previous study (Van Den Bosch

Disclosure Statement

E.B., K.P., J.D., N.H., D.K., W.S. and L.V.D.B.: no actual or potential conflict of interest. W.R. and P.V.D. collaborate with NeuroNova (Stockholm, Sweden) to explore the potential use of VEGF in human ALS. All animal experiments were approved by the Ethical Committee of the Katholieke Universiteit Leuven (K.U.Leuven).

Acknowledgements

This work was supported by grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen), the University of Leuven, the Belgian government (Interuniversity Attraction Poles, program P6/43 of the Belgian Federal Science Policy Office), the Stem Cell Institute Leuven and the ALS Association. E.B. is a research assistant and P.V.D. is a postdoctoral fellow of the FWO-Vlaanderen. K.P. and J.D. are supported by the Institute for the Promotion of Innovation by Science and Technology in

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