Genes Regulated in Neurons Undergoing Transcription-dependent Apoptosis Belong to Signaling Pathways Rather than the Apoptotic Machinery

Abstract

Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis.

Trim17-mediated ubiquitination and degradation of Mcl-1 initiate apoptosis in neurons

“…The minimum level of Mcl-1 (approximately 40% of the control) was reached 4-6 h after KCl and serum deprivation, depending on the experiments. This coincided with cytochrome c release from mitochondria, 14 dephosphorylation (and thus activation) of GSK3 ( Figure 1a) and caspase 3 activation (Figure 1a). The reduction of the Mcl-1 protein level was associated with a similar decrease in the mcl-1 mRNA level: about 35% reduction between K25 and K5 conditions after 4-8 h of deprivation ( Figure 1b).…”

Control of neuronal apoptosis by reciprocal regulation of NFATc3 and Trim17

“…We initially identified Trim17 as one of the most highly upregulated genes in CGN following KCl deprivation. 34 To determine whether NFATc3 could be involved in this transcriptional induction, CGNs were transduced with two different shRNAs, which efficiently reduced endogenous NFATc3 expression (Figure 7c, left panel). Importantly, the increase in Trim17 mRNA levels triggered by serum/KCl deprivation was significantly reduced by these two shRNAs (Figure 7c, right panel).…”

Drug target identification at the crossroad of neuronal apoptosis and survival

“…DNA microarray has proved useful for screening cellular targets that govern apoptosis [18,25]. Our most recent studies, in particular, were the first to extend the analysis to survival and led to the identification of key drug targets deregulated in both apoptosis and growth factor rescue [11,15,26].…”

“…By using a brain-biased array, this study profiled the gene expression changes of cerebellar granule neurons (CGNs) induced by different apoptotic signals, including potassium and serum withdrawal (KS-W) [21]. Important progress in understanding the apoptotic program elicited by KCl deprivation derived from genome scale studies based on oligonucleotide microarrays, which identified a great number of temporally coupled genes that delineate functional pathways mobilized during neuronal PCD [14,18]. The limitations of the first studies in encompassing the entire set of deregulated genes in neuronal apoptosis were overcome with the advent of whole-genome microarrays, which allowed the whole apoptotic program to be analyzed [19].…”

“…Among its major advantages is the ability to have direct access to the RNA sequence, the exon/exon boundaries and, therefore, produce a better picture of alternative splicing isoforms. Following both microarray and RNA-seq analysis (Figure 1), deregulated genes and enriched pathways can be identified by means of a variety of bioinformatic and statistical tools that, once applied to large-scale genomic and gene expression data, help in interpreting their biological role [18,19].…”