Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFdelta/delta mouse model of amyotrophic lateral sclerosis

Abstract

Background: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFdelta/delta mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease.

Results: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFdelta/delta mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFdelta/delta mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFdelta/delta mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFdelta/delta mice, axon outgrowth is significantly reduced compared to wild-type littermates.

Conclusions: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Figure 1

Representative bioanalyser traces of RNA samples pre- and post-amplification at 3 months, 5 months and 14 months.

Figure 2

Histogram of fold change values of significantly differentially expressed genes at 3 months (green), 5 months (blue) and late stage (red). Upregulated genes are plotted on the positive axis, downregulated genes on the negative axis.

Figure 3

Diagrammatic representation of the components of the mitochondrial electron transport chain, and the constituent proteins encoded by genes significantly upregulated in VEGF^δ/δ mice.

Figure 4

Schematic representation of the cholesterol biosynthesis pathway, with genes that are differentially regulated in VEGF^δ/δ mice at 5 months highlighted in red. Cytochrome B5 reductase is an electron carrier for 5-desaturase and methyl sterol oxidase [119]

Figure 5

Schematic representation of genes involved in neuronal migration, neurite outgrowth and formation and maintenance of the neuromuscular junction, which are all downregulated in the VEGF transgenic mouse.

Figure 6

The average length of the longest axonal process observed in primary motor neuron cultures isolated from E12.5VEGF^δ/δ (n = 4), VEGF^δ/wt (n = 14) and VEGF^wt/wt (n = 8) mice (Figure 6a). These motor neurons were grown for 7 days on laminin with BDNF and CNTF. The average length of dendrites isolated from the same mice is also shown (Figure 6b). Asterisk indicates p < 0.05.