Neurobiology of Aging 31 (2010) 2185–2191

Brief communication

VEGF protects motor neurons against excitotoxicity

by upregulation of GluR2
Elke Bogaert a,c,1 , Philip Van Damme a,c,1 , Koen Poesen b,c , Joke Dhondt b,c , Nicole Hersmus a,c ,
Dora Kiraly a,c , Wendy Scheveneels a,c , Wim Robberecht a,c , Ludo Van Den Bosch a,c,∗
a Laboratory of Neurobiology, Experimental Neurology, K.U. Leuven, Leuven, Belgium
b Vesalius Research Center, K.U. Leuven, Leuven, Belgium
c Vesalius Research Center, VIB, Leuven, Belgium

Received 15 September 2008; received in revised form 27 November 2008; accepted 2 December 2008
Available online 29 January 2009

Abstract
Influx of Ca2+ ions through the ␣-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.
© 2008 Elsevier Inc. All rights reserved.

Keywords: Neurodegeneration; Amyotrophic lateral sclerosis; Glutamate receptor; Calcium

1. Introduction excitotoxicity) is an important pathogenetic mechanism (Van

Ca2+ influx through glutamate receptors is an impor-
tant 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 mul-
tiple 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 lat-
eral sclerosis (ALS), excessive stimulation of AMPA–type
of glutamate receptors (also called AMPA receptor-mediated
∗ Corresponding author at: Neurobiology, Experimental Neurology, Cam-
pus Gasthuisberg O&N2, PB1022, Herestraat 49, B-3000 Leuven, Belgium.
Tel.: +32 16 345785; fax: +32 16 330770.
E-mail address: Ludo.Vandenbosch@vib-kuleuven.be
(L. Van Den Bosch).
1 These authors contributed equally to this work.
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 neu-
rons. Moreover, this characteristic is abolished when mutant
SOD1, a genetic cause of ALS, is over expressed in astro-
cytes (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 expres-

0197-4580/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.neurobiolaging.2008.12.007
2186 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191
sion, 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 (VEGF␦/␦ mouse) 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 excito-
toxicity and shortage of VEGF are important mediators
of the motor neuron death observed in ALS. As a con-
sequence, our aim was to find out whether a link exists
between these two mechanisms involved in motor neuron de-
generation.
2. Material and methods
2.1. 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, Den-
mark), and VEGFR2 (1/50; Santa Cruz Biotechnology, Santa
Cruz, USA) antibodies. As secondary antibodies, Alexa555
labelled antibody (1/500; Molecular Probes, Eugene, USA)
was used for VEGFR2 and GFAP was detected using
Alexa488 .
2.2. Quantitative real-time PCR
For quantitative real-time PCR of GluR2, the forward
primer was 5 TTG GGT ACT GGA GTG AAG TGG AT
3 , the reverse primer was 5 GCC CAG ACG TGT CAT
TTC CT 3 and the probe was FAM-CCT AAC TGA GCT
CCC. The primers and probe used for VEGFR2 real-time:
forward primer 5 GCA-CCA-TGC-AGA-CGC-TGA-C;
reverse primer TTC-TAG-CTG-CCA-GTA-CCA-TTG-GA
and probe FAM-TAT-GCC-AAC-CCT-CCC-CTG-CAC-
CAC. Thermal cycling was performed on a 7300 Sequence
Detection System (Applied Biosystems, Foster City, USA).
Each reaction was performed in triplicate. All samples
were normalized to the level of hypoxanthine–guanine
phosphoribosyltransferase (HPRT) RNA or rat ␤-actin
(Rn00667869 m1 TaqMan gene expression assay, Applied
Biosystems). Primers and probes for HPRT: Forward primer:
5 TGA CAC TGG TAA AAC AAT GCA GAC T 3 , Reverse
primer: 5 AGG-TCC-TTT-TCA-CCA-GCA-AGC 3 and
probe: JOE-TGG TCA AGC AGT ACA GCC CCA AAA
TGG.
2.3. Luciferase assay
The GluR2 promoter-PGL2 luciferase vector was a kind
gift of Dr. Dingledine, Department of Pharmacology, Emory
University School of Medicine (Atlanta, USA) (Myers et
al., 1998). Primary cortical neurons were transfected with
this vector using NucleofectorTM (AMAXA, Gaithersburg,
USA). After 24 h, cells were lysed using ‘Cell culture lysis
buffer’ (Luciferase assay system; Promega, Madison, USA).
In the cell lysate the luciferase activity was measured after
adding ‘bright light’ substrate (Perkin Elmer, Wellesley,
USA) in a VICTOR 2 (Perkin Elmer). Luminescence was
normalized to total protein concentration of the cell lysate
determined using the Micro BCA kit (Pierce, Rockford,
USA).
2.4. Surgical procedures: intracerebroventricular pump
implantation
VEGF was infused into the left lateral ventricle of 80-
day-old rats, using Alzet® osmotic pumps, model 2004,
(Cupertino, USA), connected with a catheter to a brain
infusion cannula as described before (Storkebaum et al.,
2005).
2.5. Radioactive PCR of AMPA receptor subunits
Radioactive PCR experiments were performed on cDNA
samples derived from ventral spinal cord lysates. A first
PCR was performed to amplify a 750 bp fragment from the
four subunits, a second PCR labelled fragments with [␣-
32 P]dCTP (Perkin Elmer Waltham, USA). Labelled PCR
fragments were subjected to a subunit-specific digestion,
separated on a 6% polyacrylamide gel, detected using a Phos-
phorImager (Storm 840, Molecular Dynamics) and quantified
using ImageQuant 5.0. The relative mRNA expression was
determined for each subunit and the sum of four different
subunits approximated 100% in all instances (Van Damme et
al., 2007).
2.6. Western blot
Spinal cords from CSF- or VEGF-treated Wistar rats were
isolated, separated into ventral and dorsal parts and homog-
enized in RIPA buffer. Western blots were performed as
described previously (Van Damme et al., 2007).
 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191 2187

2.7. Materials and statistics
Media and additives were obtained from Gibco BRL
(Grand Island, NY) and recombinant rat VEGF164 was from
R&D Systems (Minneapolis, USA). All other chemicals,
unless stated otherwise, were obtained from Sigma (St. Louis,
USA). Average data are shown as mean ± SEM and Student’s
t-test was employed to calculate significance. When more
than two groups were compared, a one-way ANOVA with
Bonferroni correction was used.
3. Results and discussion
3.1. 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 cul-
tured on a pre-established glial feeder layer were exposed
to kainic acid, an AMPA-receptor agonist, inducing approx-
imately 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 et
al., 2004), we know that co-administration of VEGF with
AMPA receptor agonists did not modify the vulnerability of
motor neurons to excitotoxicity. This suggests that VEGF
needs time to activate downstream pathways involving pro-
tein synthesis, rather than interfering directly with AMPA
receptor activation. These findings are in line with other stud-
ies describing protective effects of VEGF against excitotoxic
motor neuron death (Tolosa et al., 2008; Tovar et al., 2007).
The neuroprotective properties of VEGF are mostly medi-
ated through VEGFR2 stimulation (Matsuzaki et al., 2001;
Tolosa et al., 2008), but also VEGFR1 stimulation has
recently been reported to protect neurons in various mod-
els of neuronal cell death (Li et al., 2008; Poesen et al.,
2008). Therefore, we tested which type of VEGF receptor was
involved in our model. Administration of SU5416, a receptor
tyrosine kinase inhibitor, blocked the VEGF-induced pro-
tection of motor neurons against excitotoxicity. Moreover
DC101, a blocking antibody specific for VEGFR2, com-
pletely abolished the effect of VEGF (n = 3–16, P < 0.01;
Fig. 1A). These results show that the observed protective
effect of VEGF on excessive AMPA receptor stimulation is
mediated through the kinase activity of the VEGFR2. On the

Fig. 1. VEGF protects motor neurons from AMPA receptor-mediated excitotoxicity. (A) Vulnerability of cultured motor neurons to AMPA receptor-mediated
excitotoxicity induced by a short (30 min) exposure to 300 ␮M kainic acid. Cultures were treated for one week with 200 ng/ml VEGF-A (VEGF), 10 ␮M
SU5416 (SU), 50 ␮g/ml DC101 (Ab) or a combination of reagents (n = 3–16, *P < 0.01). (B) Real-time PCR for VEGFR2 normalized to ß-actin RNA on cDNA
prepared from astrocytes or motor neurons (n = 5, *P < 0.01). (C) Coculture of motor neurons (SMI32; red) grown on a pre-established monolayer of astrocytes
(GFAP, green). Scale bar: 50 ␮m. (D) VEGFR2 (red) and DAPI (blue) staining of a coculture of astrocytes and motor neurons. Scale bar: 50 ␮m.
2188 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191

other hand, blockage of VEGF signaling didn’t enhance the
vulnerability of motor neurons to excitotoxicity.
To explore which cell type was the target of VEGF, we
determined VEGFR2 expression in both astrocytes and motor
neurons. In motor neurons, we could readily detect VEGFR2
mRNA expression, while VEGFR2 mRNA levels in RNA
extracts from astrocytes were extremely low (n = 5, P < 0.01;
Fig. 1B). Immunocytochemistry confirmed this finding and
showed clear VEGFR2 staining on SMI32-positive motor
neurons, while GFAP-positive astrocytes were only faintly
stained (Fig. 1C–D). All together, these results indicate that
a direct stimulation of the VEGFR2 on motor neurons is
responsible for the beneficial effects of VEGF on AMPA
receptor-mediated toxicity.

Fig. 2. VEGF induces GluR2 expression in motor neurons. (A) Real-time PCR for GluR2 normalized to copies HPRT RNA (Cop HPRT) on cDNA prepared
from control or motor neuron mono cultures stimulated for 24 h with 200 ng/ml VEGF (n = 3, *P < 0.05). (B) Effect of 200 ng/ml VEGF (added directly after
seeding, for 24 h) on luciferase activity, measured in counts per second (CPS) normalized to mg of protein, in cortical neurons normalized to total protein
concentration of the lysate (n = 6, *P < 0.05). (C) PCa /PNa values of AMPA receptor currents in motor neurons from control and VEGF treated co-cultures
(n = 5, *P < 0.03). (D) Upper panel: Motor neuron with high relative Ca2+ permeability, suggesting a low GluR2 content. Lower panel: Motor neuron with
low relative Ca2+ permeability, suggesting a high GluR2 content. The panels show the I–V relations of the KA-induced current in the Na+ -rich and Ca2+ -rich
solution. The large shift in reversal potential in the motor neuron in the lower panel is indicative for a low Ca2+ permeability. (E) Relative GluR2 mRNA
expression determined by radioactive PCR in the spinal cords of Wistar rats treated for one week ICV with artificial CSF with or without 10 ␮g/kg VEGF (n = 5,
*P < 0.05). (F) Relative GluR2 protein expression determined with Western blot in the spinal cords of Wistar rats treated for one week ICV with artificial CSF
with or without 10 ␮g/kg VEGF (n = 5, *P < 0.01).
 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191
3.2. VEGF induces GluR2 mRNA in motor neurons
As the selective vulnerability of motor neurons to exci-
totoxicity is determined by the presence of GluR2-lacking
AMPA receptors (Feldmeyer et al., 1999; Kuner et al., 2005;
Van Damme et al., 2002), the effect of VEGF on GluR2
expression was investigated. Compared to untreated control
motor neurons, VEGF augmented the GluR2 mRNA level
with 2.9 ± 0.2 fold (n = 3, P < 0.05; Fig. 2A). In order to find
out whether this increased amount of GluR2 mRNA was due
to a direct effect of VEGF on the promoter of the GluR2
gene, we performed a promoter reporter study. Cortical neu-
rons transfected with a GluR2 promoter–reporter construct
showed an enhanced luciferase expression upon VEGF treat-
ment (+54 ± 17%; n = 6, P < 0.05; Fig. 2B), indeed showing
that VEGF signalling stimulates the GluR2 promoter.
3.3. VEGF reduces the Ca2+ permeability of the AMPA
receptor
Incorporation of GluR2 subunits in surface AMPA recep-
tors results in a decreased Ca2+ permeability of these
receptors. Therefore, we examined whether VEGF pretreat-
ment also resulted in an increase of GluR2-containing surface
receptors. To do so, we studied GluR2-dependent proper-
ties of AMPA receptor currents in perforated patch clamp
recordings, as previously described (Van Damme et al.,
2002). Compared to control motor neurons, VEGF-treated
motor neurons had AMPA receptors with a lower relative
Ca2+ permeability (PCa /PNa ; 0.79 ± 0.16 versus 0.31± 0.06,
Fig. 2C–D), a higher rectification index (RI: 0.80 ± 0.02
versus 0.90 ± 0.06, data not shown) and a lower sensitivity
to inhibition by the polyamine 1-naphthyl acetyl spermine
(% NAS inhibition: 48.5 ± 5.4 versus 32.8 ± 4.9; data not
shown). VEGF treatment thus results in an increased incor-
poration of this subunit in the AMPA receptor complex
and this diminishes the vulnerability of motor neurons to
AMPA receptor-mediated excitotoxicity. Therefore, VEGF
could be used as a therapeutic molecule in pathological con-
ditions where AMPA receptor-mediated excitotoxicity plays
a role.
3.4. VEGF increases the expression of the GluR2
subunit in the ventral spinal cord
Both the GluR2 subunit and VEGF are thought to play
an important role in the pathogenesis of ALS (Lambrechts
et al., 2004; Van Den Bosch et al., 2006). Moreover, upreg-
ulation of GluR2 and VEGF both slowed down the motor
neuron degeneration in mutant SOD1 animals (Azzouz et
al., 2004; Storkebaum et al., 2005; Tateno et al., 2004). To
find out whether the protective effect of VEGF on mutant
SOD1 animals can be explained by an induction of the
GluR2 subunit, we examined the effect of VEGF in vivo.
GluR2 levels in the ventral spinal cord from Wistar rats
were studied after intracerebroventricular (ICV) administra-
2189
tion of 2 ␮g/kg/day VEGF for one week. With this dose
of VEGF, no obvious toxicity was observed in treated rats.
Measurement of the relative abundance of GluR2 mRNA
by radioactive PCR showed that in ventral spinal cord of
VEGF-treated rats the levels of GluR2 mRNA were 34 ± 13%
higher compared to control animals treated with artificial
CSF (n = 5, P < 0.05; Fig. 2E). The increased mRNA levels
was reflected in increased GluR2 protein levels: Western blot
analysis on ventral spinal cord homogenates of VEGF-treated
rats showed 1.6 ± 0,1 fold more GluR2 protein relative to
untreated controls (n = 5, *P < 0.01; Fig. 2F). Interestingly,
the largest therapeutic effect of VEGF was seen in the Wistar
rat strain which is a strain known to express low endogenous
GluR2 levels (Storkebaum et al., 2005; Van Damme et al.,
2007). Although the molecular mechanism underlying the
VEGF-induced GluR2 upregulation is currently unknown,
the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway could
be involved. This is indicated by the fact that the protec-
tive effect of VEGF on chronic excitotoxicity in organotypic
spinal cord sections was linked to PI3-K activation (Tolosa et
al., 2008) and by the observation that VEGF clearly induced
Akt activation in vivo (Dewil et al., 2007).
In conclusion, our results show that induction of GluR2
expression by VEGF protects motor neurons against AMPA
receptor-mediated excitotoxicity and suggest that the pro-
tective effect observed by VEGF treatment in rodent ALS
models can at least partially be due to GluR2 induction and
a reduction of AMPA receptor-mediated excitotoxicity.
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 poten-
tial 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 Uni-
versity of Leuven, the Belgian government (Interuniversity
Attraction Poles, program P6/43 of the Belgian Federal Sci-
ence 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 Innova-
tion by Science and Technology in Flanders (IWT). W.R.
is supported through the E. von Behring Chair for Neu-
romuscular and Neurodegenerative Disorders and L.V.D.B.
receives financial support from the ‘Association Belge contre
les Maladies neuro-Musculaires’ (ABMM) and the ‘Associ-
ation Française contre les Myopathies’ (AFM).
2190 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191
References
Azzouz, M., Ralph, G.S., Storkebaum, E., Walmsley, L.E., Mitrophanous,
K.A., Kingsman, S.M., Carmeliet, P., Mazarakis, N.D., 2004. VEGF
delivery with retrogradely transported lentivector prolongs survival in a
mouse ALS model. Nature 429, 413–417.
Boillee, S., Vande Velde, C., Cleveland, D.W., 2006. ALS: a disease of motor
neurons and their nonneuronal neighbors. Neuron 52, 39–59.
Carriedo, S.G., Yin, H.Z., Weiss, J.H., 1996. Motor neurons are selectively
vulnerable to AMPA/kainate receptor-mediated injury in vitro. J. Neu-
rosci. 16, 4069–4079.
Dewil, M., Lambrechts, D., Sciot, R., Shaw, P.J., Ince, P.G., Robberecht, W.,
Van Den Bosch, L., 2007. Vascular endothelial growth factor counteracts
the loss of phospho-Akt preceding motor neurone degeneration in amy-
otrophic lateral sclerosis. Neuropathol. Appl. Neurobiol. 33, 499–509.
Di Giorgio, F.P., Carrasco, M.A., Siao, M.C., Maniatis, T., Eggan, K., 2007.
Non-cell autonomous effect of glia on motor neurons in an embryonic
stem cell-based ALS model. Nat. Neurosci. 10, 608–614.
Feldmeyer, D., Kask, K., Brusa, R., Kornau, H.C., Kolhekar, R., Rozov, A.,
Burnashev, N., Jensen, V., Hvalby, O., Sprengel, R., Seeburg, P.H., 1999.
Neurological dysfunctions in mice expressing different levels of the Q/R
site-unedited AMPAR subunit GluR-B. Nat. Neurosci. 2, 57–64.
Hazell, A.S., 2007. Excitotoxic mechanisms in stroke: an update of concepts
and treatment strategies. Neurochem. Int. 50, 941–953.
Heath, P.R., Tomkins, J., Ince, P.G., Shaw, P.J., 2002. Quantitative assessment
of AMPA receptor mRNA in human spinal motor neurons isolated by
laser capture microdissection. Neuroreport 13, 1753–1757.
Kawahara, Y., Ito, K., Sun, H., Aizawa, H., Kanazawa, I., Kwak, S., 2004.
Glutamate receptors: RNA editing and death of motor neurons. Nature
427, 801.
Kawahara, Y., Kwak, S., Sun, H., Ito, K., Hashida, H., Aizawa, H., Jeong,
S.Y., Kanazawa, I., 2003. Human spinal motoneurons express low rel-
ative abundance of GluR2 mRNA: an implication for excitotoxicity in
ALS. J. Neurochem. 85, 680–689.
Kilic, E., Kilic, U., Wang, Y., Bassetti, C.L., Marti, H.H., Hermann, D.M.,
2006. The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s
neuroprotective activity and induces blood brain barrier permeability
after focal cerebral ischemia. FASEB J. 20, 1185–1187.
Kim, Y.S., Martinez, T., Deshpande, D.M., Drummond, J., Provost-Javier,
K., Williams, A., McGurk, J., Maragakis, N., Song, H., Ming, G.L.,
Kerr, D.A., 2006. Correction of humoral derangements from mutant
superoxide dismutase 1 spinal cord. Ann. Neurol. 60, 716–728.
Kuner, R., Groom, A.J., Bresink, I., Kornau, H.C., Stefovska, V., Muller, G.,
Hartmann, B., Tschauner, K., Waibel, S., Ludolph, A.C., Ikonomidou, C.,
Seeburg, P.H., Turski, L., 2005. Late-onset motoneuron disease caused
by a functionally modified AMPA receptor subunit. Proc. Natl. Acad.
Sci. U.S.A. 102, 5826–5831.
Lambrechts, D., Storkebaum, E., Carmeliet, P., 2004. VEGF: necessary to
prevent motoneuron degeneration, sufficient to treat ALS? Trends Mol.
Med. 10, 275–282.
Li, B., Xu, W., Luo, C., Gozal, D., Liu, R., 2003. VEGF-induced activation
of the PI3-K/Akt pathway reduces mutant SOD1-mediated motor neuron
cell death. Brain Res. Mol. Brain Res. 111, 155–164.
Li, Y., Zhang, F., Nagai, N., Tang, Z., Zhang, S., Scotney, P., Lennartsson,
J., Zhu, C., Qu, Y., Fang, C., Hua, J., Matsuo, O., Fong, G.H., Ding, H.,
Cao, Y., Becker, K.G., Nash, A., Heldin, C.H., Li, X., 2008. VEGF-B
inhibits apoptosis via VEGFR-1-mediated suppression of the expres-
sion of BH3-only protein genes in mice and rats. J. Clin. Invest. 118,
913–923.
Lipton, S.A., Rosenberg, P.A., 1994. Excitatory amino acids as a final com-
mon pathway for neurologic disorders. N. Engl. J. Med. 330, 613–622.
Matsuzaki, H., Tamatani, M., Yamaguchi, A., Namikawa, K., Kiyama,
H., Vitek, M.P., Mitsuda, N., Tohyama, M., 2001. Vascular endothe-
lial growth factor rescues hippocampal neurons from glutamate-induced
toxicity: signal transduction cascades. FASEB J. 15, 1218–1220.
Matute, C., Alberdi, E., Domercq, M., Perez-Cerda, F., Perez-Samartin,
A., Sanchez-Gomez, M.V., 2001. The link between excitotoxic oligo-
dendroglial death and demyelinating diseases. Trends Neurosci. 24,
224–230.
Myers, S.J., Peters, J., Huang, Y., Comer, M.B., Barthel, F., Dingledine,
R., 1998. Transcriptional regulation of the GluR2 gene: neural-specific
expression, multiple promoters, and regulatory elements. J. Neurosci.
18, 6723–6739.
Nagai, M., Re, D.B., Nagata, T., Chalazonitis, A., Jessell, T.M., Wichterle,
H., Przedborski, S., 2007. Astrocytes expressing ALS-linked mutated
SOD1 release factors selectively toxic to motor neurons. Nat. Neurosci.
10, 615–622.
Nicoletti, J.N., Shah, S.K., McCloskey, D.P., Goodman, J.H., Elkady, A.,
Atassi, H., Hylton, D., Rudge, J.S., Scharfman, H.E., Croll, S.D., 2008.
Vascular endothelial growth factor is up-regulated after status epilepti-
cus and protects against seizure-induced neuronal loss in hippocampus.
Neuroscience 151, 232–241.
Oosthuyse, B., Moons, L., Storkebaum, E., Beck, H., Nuyens, D., Brus-
selmans, K., Van Dorpe, J., Hellings, P., Gorselink, M., Heymans,
S., Theilmeier, G., Dewerchin, M., Laudenbach, V., Vermylen, P.,
Raat, H., Acker, T., Vleminckx, V., Van Den Bosch, L., Cashman,
N., Fujisawa, H., Drost, M.R., Sciot, R., Bruyninckx, F., Hick-
lin, D.J., Ince, C., Gressens, P., Lupu, F., Plate, K.H., Robberecht,
W., Herbert, J.M., Collen, D., Carmeliet, P., 2001. Deletion of the
hypoxia-response element in the vascular endothelial growth factor
promoter causes motor neuron degeneration. Nat. Genet. 28, 131–
138.
Poesen, K., Lambrechts, D., Van Damme, P., Dhondt, J., Bender, F., Frank,
N., Bogaert, E., Claes, B., Heylen, L., Verheyen, A., Raes, K., Tjwa,
M., Eriksson, U., Shibuya, M., Nuydens, R., Van Den Bosch, L., Meert,
T., D’Hooge, R., Sendtner, M., Robberecht, W., Carmeliet, P., 2008.
Novel role for vascular endothelial growth factor (VEGF) receptor-1
and its ligand VEGF-B in motor neuron degeneration. J. Neurosci. 28,
10451–10459.
Storkebaum, E., Lambrechts, D., Dewerchin, M., Moreno-Murciano, M.P.,
Appelmans, S., Oh, H., Van Damme, P., Rutten, B., Man, W.Y., De
Mol, M., Wyns, S., Manka, D., Vermeulen, K., Van Den Bosch, L.,
Mertens, N., Schmitz, C., Robberecht, W., Conway, E.M., Collen, D.,
Moons, L., Carmeliet, P., 2005. Treatment of motoneuron degeneration
by intracerebroventricular delivery of VEGF in a rat model of ALS. Nat.
Neurosci. 8, 85–92.
Svensson, B., Peters, M., Konig, H.G., Poppe, M., Levkau, B., Rothermundt,
M., Arolt, V., Kogel, D., Prehn, J.H., 2002. Vascular endothelial growth
factor protects cultured rat hippocampal neurons against hypoxic injury
via an antiexcitotoxic, caspase-independent mechanism. J. Cereb. Blood
Flow Metab. 22, 1170–1175.
Tateno, M., Sadakata, H., Tanaka, M., Itohara, S., Shin, R.M., Miura, M.,
Masuda, M., Aosaki, T., Urushitani, M., Misawa, H., Takahashi, R., 2004.
Calcium-permeable AMPA receptors promote misfolding of mutant
SOD1 protein and development of amyotrophic lateral sclerosis in a
transgenic mouse model. Hum. Mol. Genet. 13, 2183–2196.
Tolosa, L., Mir, M., Asensio, V.J., Olmos, G., Llado, J., 2008. Vascu-
lar endothelial growth factor protects spinal cord motoneurons against
glutamate-induced excitotoxicity via phosphatidylinositol 3-kinase. J.
Neurochem. 105, 1080–1090.
Tovar, Y.R.L.B., Zepeda, A., Tapia, R., 2007. Vascular endothelial growth
factor prevents paralysis and motoneuron death in a rat model of exci-
totoxic spinal cord neurodegeneration. J. Neuropathol. Exp. Neurol. 66,
913–922.
Van Damme, P., Bogaert, E., Dewil, M., Hersmus, N., Kiraly, D., Scheve-
neels, W., Bockx, I., Braeken, D., Verpoorten, N., Verhoeven, K.,
Timmerman, V., Herijgers, P., Callewaert, G., Carmeliet, P., Van Den
Bosch, L., Robberecht, W., 2007. Astrocytes regulate GluR2 expression
in motor neurons and their vulnerability to excitotoxicity. Proc. Natl.
Acad. Sci. U.S.A. 104, 14825–14830.
Van Damme, P., Van Den Bosch, L., Van Houtte, E., Callewaert, G., Rob-
berecht, W., 2002. GluR2-dependent properties of AMPA receptors
determine the selective vulnerability of motor neurons to excitotoxicity.
J. Neurophysiol. 88, 1279–1287.
 E. Bogaert et al. / Neurobiology of Aging 31 (2010) 2185–2191 2191

Van Den Bosch, L., Storkebaum, E., Vleminckx, V., Moons, L., Vanop-
denbosch, L., Scheveneels, W., Carmeliet, P., Robberecht, W., 2004.
Effects of vascular endothelial growth factor (VEGF) on motor neuron
degeneration. Neurobiol. Dis. 17, 21–28.
Van Den Bosch, L., Van Damme, P., Bogaert, E., Robberecht, W., 2006. The
role of excitotoxicity in the pathogenesis of amyotrophic lateral sclerosis.
Biochim. Biophys. Acta 1762, 1068–1082.
Van Den Bosch, L., Vandenberghe, W., Klaassen, H., Van Houtte, E.,
Robberecht, W., 2000. Ca2+ -permeable AMPA receptors and selective
vulnerability of motor neurons. J. Neurol. Sci. 180, 29–34.
Yamanaka, K., Chun, S.J., Boillee, S., Fujimori-Tonou, N., Yamashita, H.,
Gutmann, D.H., Takahashi, R., Misawa, H., Cleveland, D.W., 2008.
Astrocytes as determinants of disease progression in inherited amy-
otrophic lateral sclerosis. Nat. Neurosci. 11, 251–253.