The Expression of Connexins in Neurodegeneration

Abstract

Changes in connexin expression are thought to play a role in neurological disease and the possible effects of these changes in connexion expression in the disease process are therefore important. The need to screen for connexin expression quickly and cost-effectively has directed attention to microarray technology. The aim of the first section of this study was to design a „boutique‟ microarray chip to screen for mRNA for all the reported connexins in the human. Where possible, the probes were designed to be compatible with the rat connexin mRNA. Twenty 50-base length oligonucleotide custom-designed probes, fourteen of them showing cross-species human/rat alignment, and twenty probes for the human connexin sequences designed by the German company MWG were included in the array. The array was tested on rat and on human tissue and the result validated by the Affymetrix GeneChips. Results confirmed that our array design was reliable, had a consistent target binding pattern and that it could detect differential connexin gene expression in both human and rat tissues. As our goal was to screen human brain tissue for connexin expression, obtaining RNA of sufficiently high quality to be used in microarray experiments became a concern. We investigated whether it was possible to obtain high quality RNA from postmortem human tissue using samples obtained from the New Zealand Neurological Foundation Human Brain Bank. The principle finding was that RNA quality is most strongly affected by the pH of the tissue, with both the pH and the RNA quality being influenced by the mode of death. The best quality RNA samples were then used for the screening of human brain tissue for connexin expression. One control and five disease samples from the secondarily affected regions of Huntington‟s (MC), Parkinson‟s (CN) and Alzheimer‟s disease, as well as hippocampus samples from two mesial temporal lobe epilepsy patients were screened on the custom-designed connexin array and on the Illumina Sentrix arrays to search for changes in the expression of connexins and inflammatory cytokines. It proved possible to obtain consistent data from the two platforms from samples with a wide range of RNA quality, and certain common patterns in the change of connexins and inflammatory markers were identified.