A Comparative Proteomics Approach to Identify Novel Therapeutic Targets in an Experimental Stroke Model

Abstract

The advent of new technologies that permit the simultaneous study of many thousands of genes or proteins in parallel has transformed our understanding of many disease processes. As the predominant effector molecule in biological systems, the analysis of protein expression likely offers a more direct measure of the cellular response in a disease of energy insufficiency like stroke. To date, there has been little application of proteomics to stroke research and few studies have investigated global changes within the brain or attempted to validate the functional consequences of manipulating the potential biological targets identified. We hypothesised that a systems biology approach combining large-scale proteomics and bioinformatics could elucidate the molecular changes occurring in the days after stroke which are currently not well understood. The first aim of this thesis was to establish a bioinformatics approach to analyse large-scale proteomic data and to validate the method in a known situation. The analysis method was applied to a historical Western array dataset investigating differential protein expression in the somatosensory cortex at 3 hours post stroke and in sham operated controls. Proteins with a known association to stroke pathophysiology (e.g. Bad, Caspase-7 and Fas ligand) as well as novel proteins not previously related to stroke (e.g. RPL22, DHX38, NXF1 and GTF2F2) were identified through this approach. Protein profiles showed both detrimental and self-restorative mechanisms occur in parallel, and that early reperfusion can prevent the consequences of the expressions of detrimental proteins. In addition, the changes in expression profiles following treatment with two neuroprotectants (MK-801 and FR131706) in the acute phase were also investigated. Both neuroprotectants upregulated the expression of proteins associated with energy metabolism and PI3K/AKT signalling pathways despite the distinct pharmacological mechanisms of action of these drugs, suggesting a novel common pathway of neuroprotection exists. While this more stringent analysis approach provided new insight into neuroprotection, the small number of patients that could ultimately benefit limited the value of pursuing these novel targets as therapeutic agents. iTRAQ mass spectrometry in combination with the developed bioinformatics analysis was then employed to investigate the differential expression of proteins in brain tissues harvested from animals 3, 7 and 14 days following experimental stroke. The study showed upregulation of proteins known to be involved in neuroplasticity and neuroregeneration occurred in the same timeframe as impaired cellular trafficking and synaptic transmission and increased inflammation. Expression of proteins such as MAL2, Fsd1 and Mpp2 which have never been associated with stroke pathology before were also identified. These results suggest that an opportunity exists in the days after stroke to manipulate protein expression and tip the balance in favour of beneficial mechanism. To determine whether manipulating key players in these damaging pathways had the potential to improve post stroke recovery, a proof of concept study was conducted to examine the effect of siRNA knockdown of upregulated inflammatory proteins in an ex vivo organotypic brain slices from CSF-1R-EGFP (‘MacGreen’) mice subjected to stroke. EGFP expression, a marker of inflammatory status in this transgenic mouse line, could be reduced by application of the nonsteroidal anti-inflammatory agent, indomethacin. Administration of siRNAs targeted to cathepsin B and MAL2 also produced a temporal reduction in inflammatory signal that was maximal at day 7 post stroke. Whether small molecule inhibitors of these proteins have the potential to reduce brain inflammation and improve functional recovery in vivo requires further investigation. In summary, comparative proteomic technologies combined with stringent bioinformatics analysis can provide a comprehensive profile of the molecular changes in a complex disease like stroke and can fast-track identification of novel proteins which could be potential therapeutic targets. While further optimisation and validation studies are needed, ex vivo organotypic slice cultures from MacGreen mice subjected to stroke provide a unique opportunity in which to investigate subacute inflammatory mechanisms following stroke.