Vascular endothelial growth factors and Alzheimer's disease

Despite their name, vascular endothelial growth factors (VEGFs) are pleiotropic proteins that are neurotrophic, cytoprotective, and widespread throughout the brain. VEGF has been proposed as a potential biomarker related to stage of cognitive decline in Alzheimer's disease (AD). VEGFs are however alternatively spliced to form functionally opposing isoforms, by a series of splicing factors controlled by splicing factor kinases. The balance of VEGF splice variants rather than total amount determines functional outcomes. This project will test the hypothesis that altered splicing in cortical neurons is associated with risk of progression of AD, and that alternatively spliced VEGF isoforms may be biomarkers for AD risk particularly related to neuronal loss.
Experimental plan
a) Determination of VEGF and SRPK1 splicing and expression respectively in Alzheimers patients and association with low/high progression risk. We will initially interrogate existing available databases (e.g. GeTex and Brainiac) for VEGF-A splice variant expression in frontal cortex and cerebellum. We will use our in house RNASeq data from the Brains for Dementia Research cohort (BDR, 512 patients, http://www.brainsfordementiaresearch.org.uk/) to validate data on splice variant expression from existing databases by RT-PCR, quantified by digital droplet PCR(14).
b) Bioinformatic analysis of splice variants in Alzheimer's disease. Splicing analysis will be carried out on RNA-Seq data from cortex and cerebellum from Alzheimer's patients to identify common changes in splicing that may indicate other targets of SRPK1 activity. We will look for common splicing patterns that are affected (e.g. exon skipping, intron retention, 5' and 3' alternative splice sites). Levels of expression (FPKM) of VEGF, SRPK1 and other important splice factors in this system, SRSF1, SRSF6, and Clk, will be determined, and then this set of markers will be used in network inference algorithms to identify a network of interactions18.
c) Effect of VEGF splicing isoforms and splicing inhibitors on neuronal degeneration in vitro. Human cortical neuronal lines (e.g. HCN-2 available through ATCC19, 20), and mouse primary hippocampal/cortical neurons will be used for determination of the effect of VEGF isoforms and SRPK1 inhibitors/over-expressors on neurodegeneration in vitro. Wildtype mouse or human cells will be treated with amyloid protein as a neurotoxin, and the effect of recombinant VEGF-A165b, VEGF-A165b or antibodies to each isoform in the presence or absence of SRPK1 inhibitors will be determined. The effects of VEGF-A165a, VEGF-A165b, anti-VEGF isoform antibodies and SRPK1 inhibitors will also be studied on cortical neurons.
d) Effect of SRPK1 inhibition on other alternative splicing pathways. We have generated novel highly potent SRPK1 inhibitors as part of Prof Bates and Prof Donaldson's drug development company Exonate Ltd. We will use these inhibitors under license from Exonate to determine the effect of SRPK1 inhibition on overall alternative splicing of human and mouse cortical neurons. Cells will be treated with the inhibitors and RNASeq performed to determine the overall splice changes seen.