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We sought genetic evidence for involvement of neuronal vascular endothelial growth factor (VEGF) in amyotrophic lateral sclerosis (ALS). Mice expressing human ALS mutant superoxide dismutase-1 (SOD1) were crossed with mice that overexpress VEGF in neurons (VEGF+/+). We report that SOD1G93A/VEGF+/+ double-transgenic mice show delayed motor neuron loss, delayed motor impairment and prolonged survival compared to SOD1G93A single-transgenics. These findings indicate that neuronal VEGF protects against motor neuron degeneration, and may have therapeutic implications for ALS.
Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a devastating degenerative disease of upper and lower motor neurons that affects about 2 people per 100,000 and produces weakness, muscle atrophy, fasciculations, spasticity, hypo- or hyperreflexia and extensor plantar responses(Rowland and Shneider, 2001). Existing treatment provides only marginal benefit and death usually occurs within 5 years of onset. In most patients with ALS, the cause is unknown, but 5-10% of cases are familial. Patients with familial ALS have been found to have mutations in any of several genes, including Cu/Zn superoxide dismutase (SOD1)(Rosen et al., 1993), alsin (Hadano et al., 2001; Yang et al., 2001), senataxin (Chen et al., 2004), vesicle-associated membrane protein B (Nishimura et al., 2004), and angiogenin (Greenway et al., 2006). Expression of ALS-associated human SOD1 mutations (e.g., SOD1G93A) in mice produces a dominantly inherited syndrome with clinical and histopathological features of ALS, including limb weakness, muscle atrophy and loss of motor neurons (Gurney et al., 1994).
Recent studies have implicated vascular endothelial growth factor (VEGF) in ALS, and suggest that it might have therapeutic potential. Mice with deletion of the hypoxia-response element in the VEGF promoter region (VEGFδ/δ) develop adult-onset progressive motor neuron degeneration reminiscent of ALS (Oosthuyse et al., 2001). Crossbreeding of VEGFδ/δ with SOD1G93A mice produces VEGFδ/δ/SOD1G93A double mutants with more severe weakness and earlier death than mice carrying the SOD1G93A gene alone (Lambrechts et al., 2003). Intraperitoneal (Zheng et al., 2004) or intracerebroventricular (Storkebaum et al., 2005) administration of VEGF, or intramuscular delivery of a VEGF-expressing lentiviral vector (Azzouz et al., 2004), delays the onset of paralysis, improves motor performance, and prolongs survival in SOD1G93A mice or rats. These findings may have direct clinical relevance, because variations in the human VEGF promoter/leader sequence are associated with reduced levels of circulating VEGF, and confer increased risk for ALS in some populations (Lambrechts et al., 2003).
Experiments were performed according to NIH guidelines and were approved by the Institutional Animal Care and Use Committee. Male mice expressing wild-type human SOD1 (TgN(SOD1)2Gur) and human ALS mutant SOD1 (TgN(SOD1-G93A)1Gur) (Gurney et al., 1994) on a C57BL/6 genetic background were purchased from the Jackson Laboratory and maintained as SOD1G93A hemizygotes. Mice overexpressing human VEGF165 (TgN(NSEVEGF)1651–1653) driven by the rat neuron-specific enolase promoter (VEGF+/+, strain V1) from Department of Neurology, University of Zurich, Switzerland were maintain on a C57BL/6 background (Wang et al., 2005). SOD1G93A and VEGF+/+ mice were cross-bred and genotyped by PCR of tail-tip DNA, using 5′-AGGAGAGATGAGCTTCCTACAG-3′ and 5′-GATGGCTGGCAACTAGAAGGCAC-3′ as primers for VEGF, and 5′-CATCAgCCCTAATCCATCTgA-3′ and 5′-CgCgACTAACAATCAAAgTgA-3′ as primers for SOD1. Aged matched male litter-mates (n=6) were used as controls in all experiments.
Western blots were performed to determine the expression levels of VEGF and mutant SOD1 in the spinal cord of wild-type and transgenic mice using rabbit anti-VEGF antibody (Santa Cruz, CA; 1:500), and rabbit anti-human SOD1 (Chemicon #SOD-100; 1:200), and rabbit anti-mouse/rat SOD1 (Chemicon #SOD-101; 1:200) species-preferring antibodies (Shinder et al., 2001), and a standard protocol as previously described (Jin et al). Antibodies were removed with stripping buffer, and the membrances were reprobed with anti-actin antibody for control of protein loading.
For the rotarod testing, mice were trained for 1 wk to perform on an accelerating rotarod (Ugo Basile, Comerio, Italy). Baseline perfromance was measured at age 60 d, before disease onset, and repeated every 10 d. Each test consisted of three trials, and the best performance (longest duration on the rod without falling, up to a maximum of 180 sec) was recorded.
Perfusion-fixed spinal cords were embedded in paraffin, and serial transverse sections (7 μm) through the lumbar spinal cord were cut. Every fifth section was collected, deparaffinized and stained with cresyl violet. Immunostaining for VEGF and choline acetyltransferase (ChAT) was performed using monoclonal anti-VEGF (Chemicon; 1:200) and goat polyclonal ant-ChAT (Chemicon; 1:500), and visualized with diaminobenzidine and hydrogen peroxide.
Quantitative data were expressed as mean ± SEM and ANOVA and Student's t-test were used for statistical analysis, with p<0.05 considered significant.
If functional VEGF deficiency is an important pathogenic factor for ALS, then overexpression of VEGF should attenuate associated deficits. To test this prediction, we crossed SOD1G93A mutant mice with transgenic (VEGF+/+) mice that selectively overexpress hVEGF165 in neurons (Wang et al., 2005). PCR of mouse-tail genomic DNA showed a band corresponding to mutant SOD1 in SOD1G93A mice, and a band representing hVEGF165 in VEGF+/+ mice, which were absent in wild-type littermates (Fig. 1a). VEGF immunohistochemistry and Western blotting demonstrated increased VEGF expression in both ventral horn motor neurons and dorsal horn neurons in the lumbar spinal cord of 60-day-old SOD1G93A/VEGF+/+ compared to 60-day-old (presymptomatic) SOD1G93A mice (Fig 1b and c). Mutant SOD1 protein was detected in both SOD1G93A and SOD1G93A/VEGF+/+ double-transgenics, and the VEGF+/+ mutation did not appear to affect the abundance of either the endogenous (murine) or transgenic (mutant human) form of SOD1 (Fig 1d).
In principle, transgenic overexpression of VEGF could mistakenly appear to affect the course of disease in SOD1G93A mice if it acted during development to increase the baseline number of spinal cord motor neurons. To address this possibility, we stained lumbar spinal cord sections from 4-week-old (presymptomatic) adult mice with an antibody against the motor neuron marker ChAT, and counted cells in wild type, VEGF+/+, SOD1G93A, and SOD1G93A/VEGF+/+ mice. However, no differences were observed among these four groups (Fig. 2a-b).
The most striking histopathological finding in patients with ALS and in SOD1G93A mice is loss of motor neurons in the anterior horns of the spinal cord. Compared to wild-type littermates (or VEGF+/+ mice), SOD1G93A mice show prominent depletion of anterior horn motor neurons (Fig 3a-b). However, motor neuron loss was markedly reduced in SOD1G93A/VEGF+/+ double-transgenics. Motor impairment was delayed in onset in SOD1G93A/VEGF+/+ mice, although its rate of progression, once manifested, was not appreciably altered. Thus, rotarod performance began to decline about 20 days later in SOD1G93A/VEGF+/+ than in SOD1G93A mice, but deteriorated at the same rate thereafter (Fig. 3c). Finally, compared to SOD1G93A mice, SOD1G93A/VEGF+/+ mice showed a delay in the onset of mortality (149 vs. 129 days), and prolonged average (159 ± 3 vs. 136 ± 3 days) and maximal (167 vs. 143 days) survival (Fig. 3d).
The ability of transgenically expressed neuronal VEGF to postpone the onset of motor neuron disease in ALS mice is consistent with prior evidence for a protective effect of VEGF in this disorder, and may shed new light on the mechanisms involved. Because VEGF overexpression in our VEGF+/+ and SOD1G93A/VEGF+/+ mice was confined to neurons, neuronal overexpression of VEGF seems sufficient for protection, consistent with the observation that selective neuronal infection with a VEGF-expressing lentiviral vector moderates the course of disease in SOD1G93A mice (Azzouz et al., 2004). Since VEGF is a secreted protein, this does not necessarily imply that motor neurons are the cellular targets of neuronal VEGF in SOD1G93A/VEGF+/+ mice. However, autocrine (Ogunshola et al., 2002) and direct neuroprotective (Jin et al., 2000) effects of VEGF are well documented, and transgenic neuronal overexpression of the VEGFR2 receptor also prolongs survival in SOD1G93A mice (Storkebaum et al., 2005), so the mechanism of action of VEGF in this murine model of ALS could be exclusively neuronal.
It is also notable that early onset of VEGF therapy, as achieved in our SOD1G93A/VEGF+/+ transgenic mice or in SOD1G93A mice given a VEGF-expressing lentivirus vector beginning at 21 rather than 90 days of age (Azzouz et al., 2004), produces more marked (17-30%) extension of survival than does treatment started at later times (7-8%) (Zheng et al., 2004); (Storkebaum et al., 2005). This could relate to differences in the levels of VEGF achieved in these different models, but it could also imply that VEGF induces early adaptations that reduce the sensitivity of SOD1G93A motor neurons to degeneration in advance of its onset.
Supported by USPHS grants AG21980 to K.J. and NS44921 to D.A.G.