In our data set, we identified 164 genes that were sig nificantly up regulated after NGF with drawal and the expression of 48 of these genes increased by more than 2 fold. Conversely, 379 genes were down regulated when the significance threshold was set at p 0. 01 and the expression of 86 of these genes decreased by 2 fold or more. We performed Gene Ontology analysis and functional enrichment ana lysis to identify specific annotations that were enriched following NGF withdrawal. Whilst this type of analysis depends upon a controlled vocabulary and therefore has its limitations, it also represents a powerful method for extracting potentially useful biological information from our gene expression data.
In an analysis of transcription dependent neuronal apoptosis proceeding via the mitochondrial pathway, functional Inhibitors,Modulators,Libraries categories such as intracellular signaling cas cades, transcription and mitochondrial changes might be expected to be enriched. Whilst these categories are indeed enriched after NGF withdrawal, other categories Inhibitors,Modulators,Libraries that contain genes which could suggest additional hypotheses about the mechanisms of neuronal death Dacomitinib were also highlighted. The significance of the induction of ER stress associated genes, for example, may offer new insights into the cell death process, especially since a similar response was observed in cerebellar granule neurons undergoing apoptosis and experiments in other systems suggest a role for interactions between the mitochondria and the ER.
On Inhibitors,Modulators,Libraries the other hand, the down regulation of genes associated with cholesterol and fatty acid biosynthesis may be associated with an inhibition of cell growth since cholesterol and fatty acids are required for the synthesis of membranes. Cluster analysis allowed us to group the genes accord ing to their pattern of expression, especially in the pre sence of the MLK inhibitor, CEP 11004. The expression of many of the genes induced after NGF withdrawal is reduced by CEP 11004, suggesting that they may be tar gets of the MLK JNK c Jun pathway. This group includes c jun, dp5 and mkp1 whose promoters contain ATF sites that bind c Jun and which are important for their induction after NGF withdrawal. The induction of a few genes, such as egln3, is not affected by CEP 11004, suggesting that the tran scription of these genes may be regulated by other tran scription factors that are activated after NGF withdrawal, but not regulated by the JNK pathway, for example, FOXO3a or Myb.
Interestingly, CEP 11004 reverses the decrease in the level of expression of some of the genes that Inhibitors,Modulators,Libraries are down regulated after NGF withdrawal. Many of these genes encode proteins involved in fatty acid metabolism and cholesterol meta bolism, e. g. insig1, sqle, hmgcr, and hmgcs1, and their transcription is activated by sterol regulatory element binding proteins.