Ultrastructural and transcriptional profiling of neuropathological misregulation of CREB function.

We compare here the neurodegenerative processes observed in the hippocampus of bitransgenic mice with chronically altered levels of cAMP-response element-binding protein (CREB) function. The combination of genome-wide transcriptional profiling of degenerating hippocampal tissue with microscopy analyses reveals that the sustained inhibition of CREB function in A-CREB mice is associated with dark neuron degeneration, whereas its strong chronic activation in VP16-CREB mice primarily causes excitotoxic cell death and inflammation. Furthermore, the meta-analysis with gene expression profiles available in public databases identifies relevant common markers to other neurodegenerative processes and highlights the importance of the immune response in neurodegeneration. Overall, these analyses define the ultrastructural and transcriptional signatures associated with these two forms of hippocampal neurodegeneration, confirm the importance of fine-tuned regulation of CREB-dependent gene expression for CA1 neuron survival and function, and provide novel insight into the function of CREB in the etiology of neurodegenerative processes.

Integrating synapse proteomics with transcriptional regulation.

The mammalian postsynaptic proteome (PSP) comprises a highly interconnected set of approximately 1,000 proteins. The PSP is organized into macromolecular complexes that have a modular architecture defined by protein interactions and function. Signals initiated by neurotransmitter receptors are integrated by these complexes and their constituent enzymes to orchestrate multiple downstream cellular changes, including transcriptional regulation of genes at the nucleus. Genome wide transcriptome studies are beginning to map the sets of genes regulated by the synapse proteome. Conversely, understanding the transcriptional regulation of genes encoding the synapse proteome will shed light on synapse formation. Mutations that disrupt synapse signalling complexes result in cognitive impairments in mice and humans, and recent evidence indicates that these mutation change gene expression profiles. We discuss the need for global approaches combining genetics, transcriptomics and proteomics in order to understand cognitive function and disruption in diseases.

Activity of the nicotinic acetylcholine receptor alpha5 and alpha7 subunit promoters in muscle cells.

The acetylcholine receptor alpha5 and alpha7 subunits are components of different nicotinic receptor subtypes expressed in the nervous system. However, they are also present in non-neuronal tissues. We have detected alpha5 and alpha7 transcripts in mouse C2C12 muscle cells. Moreover, on differentiation of myoblasts into myotubes, the amount of alpha7 transcripts increased significantly, whereas alpha5 remained unchanged. In order to explore how the expression of these neuronal genes is regulated in muscle, we have characterized their promoter activities. Deletion and mutagenesis analysis with transfected reporter genes showed that transcriptional activity was controlled by regulatory elements also operative in neuronal-like cells. Thus, the activity of the alpha5 subunit core promoter decreased to approximately 50% on alteration of one, two, or three of the five Sp1 binding sites present in this region and was almost abolished when four or five sites were mutated simultaneously. In the case of the alpha7 subunit promoter, the upstream stimulatory factor and the early growth response gene transcription factor were involved in regulating its transcriptional activity. In addition, the alpha7 promoter was activated during the differentiation process, in a mechanism partially dependent on the mentioned factors.

Multiple functional Sp1 domains in the minimal promoter region of the neuronal nicotinic receptor alpha5 subunit gene.

The alpha5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to approximately 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha5 promoter activity by approximately 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.