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FP6

NFG Sintesi della relazione

Project ID: 503221
Finanziato nell'ambito di: FP6-LIFESCIHEALTH
Paese: Italy

Final Report Summary - NFG (Functional genomics of the adult and developing brain)

The project has obtained a pipeline to study gene expression profiling from a small number of neurons. This includes purification of cells by Laser capture microdissection (LCM) (Zeiss) with laser pressure catapulting (PALM); amplification of RNA with the µMACS SuperAmp Kit and hybridisation to the SISSA-RIKEN in house cDNA platform which contains 14000 clones in triplicates from the FANTOM2 mouse cDNA clone collection. Variations to this pipeline present a pre-step with an immunocytochemical reaction to identify neuronal populations and the use of the harvested RNA for CAGE technology. They have also reported the development of the Cap-analysis gene expression (CAGE) technology applied to deep sequencing, which opens the possibility to the comprehensive analysis of the transcript and promoters in the neural tissue, and applied this to the hippocampus transcriptome and promotome. Another important achievement was a comprehensive mapping of the mammalian (mouse and human) promoters and the specific feature of the brain promoters as published in Nature Genetics (Carninci et al.) and Journal of Physiology (Gustincich et al.). All in all, they show a final breakthrough for the molecular profiling of isolated neurons. One of the major achievements of this project, we have created an impressive datasets of gene expression in single neuronal cell groups that is paving the way for systematic studies in this area.

NFG investigated changes of gene expression using a 23 K 63-mer oligonucleotides microarray platform and real-time PCR in three different preparations from the mouse retina: in isolated photoreceptors, in cultured isolated retinas and in retinas dissected from intact alive animals. In all these preparations, NFG have observed a consistent up-regulation of almost two-fold of at least three genes known to be involved in phototransduction: Arrestin (SAG), Guanylate Cyclase Activator 1a (Guca1a) and Guanylate Cyclase Activator 1b (Guca1b). No detectable up-regulation was observed for light exposures lasting less than 1 hour and a full up-regulation of these genes is observed after 2-3 hours of a light exposure of about 1000 Lux. The up-regulation of these genes is expected to decrease the efficiency of the transduction cascade and therefore to partially reactivate the dark current abolished by light. Therefore, these changes of gene expression could mediate a component of light adaptation occurring after about hours of light exposure. Functional tests made in intact mice by recording Electroretinographic (ERG) responses have shown that, upon prolonged illumination with a steady light that initially caused a substantial suppression of the photoresponse (about 80 %), a partial recovery of the dark current occurred after about 2 hours. This partial recovery of the dark current was associated to the ability to transduce more efficiently light signals also in the presence of bright light background. Taken together, all these experimental observations suggest that changes of expression of some genes coding for proteins involved in the signalling cascade controlling phototransduction underly a late component of light adaptation which can be observed after 1 - 2 hours of continuous light.

NFG were able to consolidate the results, to obtain a further analysis and the preparation of manuscript for publication on the detailed genomic comparison of fast-spiking and regular-spiking cells in the cortex of mouse at the single cell level to assess the feasibility of single-cell transcriptomics. NFG have also completed a study analysing the sampling problems in microarray experiments for low copy numbers of RNA, and of the transcriptional differences among phenotypically identical cells and an investigation of the functions of the Pax6 gene in neocortical stem cell maintenance and priming of neuronal gene expression. Finally, NFG have also obtained a detailed characterisation of the electrophysiological phenotypes of different classes of cortical neurons, and their functions in cortical activity.

Informazioni correlate

Reported by

Scuola Internazionale Superiore di Studi Avanzati
Via Beirut 2/6
34014 Trieste
Italy
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