Neuronal RNA-binding proteins in health and disease.

In mammalian cells in general and in neurons in particular, mRNA maturation, translation, and degradation are highly complex and dynamic processes. RNA-binding proteins (RBPs) play crucial roles in all these events. First, they participate in the choice of pre-mRNA splice sites and in the selection of the polyadenylation sites, determining which of the possible isoforms is produced from a given precursor mRNA. Then, once in the cytoplasm, the protein composition of the RNP particles determines whether the mature mRNA is transported along the dendrites or the axon of a neuron to the synapses, how efficiently it is translated, and how stable it is. In agreement with their importance for neuronal function, mutations in genes that code for RBPs are associated with various neurological diseases. In this review, we illustrate how individual RBPs determine the fate of an mRNA, and we discuss how mutations in RBPs or perturbations of the mRNA metabolism can cause neurodegenerative disorders.

Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions.

To study promoters we usually use primer extension to map the transcription start site and a panel of PCR generated deletion mutants. This strategy is complex and time-consuming. Therefore, we decided to improve it by using Gateway and FLOE (Fluorescently Labeled Oligonucleotide Extension). In this report we developed the first luciferase reporter "destination vector" (GW luc basic) for the Gateway technology and tested its efficacy, accuracy and background level by transfecting two distant cell lines (THP1 monocytic and SH-SY5Y neural cells). This vector is a real advantage for the cloning of many PCR fragments and sustains reporter activity also in THP1 cells, which are known to be problematic for transfection/expression. FLOE is a straightforward method to map transcription start sites but a bias in the capillary electrophoretic migration pattern of ROX weight markers has been reported: ROX markers migrated as if they were some bp longer. We hypothesized that this could depend on the use of different enzymes for the two principal reactions (DNA polymerase for the dideoxy chain terminated reaction on DNA and reverse transcriptase for the primer extension on RNA). Therefore, we used the same reverse transcriptase enzyme on both reactions, demonstrating that the reported bias is not due to the use of different enzymes but is an intrinsic feature of the ROX markers. The proposed procedure is important not only because of the timeliness but also for the global impact on the study of the first layer of the gene regulation.

Role of acetylcholinesterase inhibitors in pharmacological regulation of amyloid precursor protein processing.

The triggering events leading to the selective neurodegeneration observed in Alzheimer brains are not yet completely understood. They thus create a great challenge for the definition of a resolutive treatment for the causes and symptoms of Alzheimer's Disease (AD). Since the current therapeutic option for AD patients is the use of acetylcholinesterase inhibitors (AChEIs), several authors have examined whether these drugs can also affect the expression and metabolism of the amyloid precursor protein (AbetaPP). The rationale behind these studies was based on the fact that the literature suggests that cholinergic activities are also involved in the regulation of AbetaPP metabolism. Therefore, the characterization of these aspects of AD pharmacology may allow cholinergic drugs to be tested for their ability to intervene at different levels of the pathogenetic chain, other than providing a replacement therapy for lost neurotransmitters. This paper reviews the evidence that many of these drugs, although with different qualitative effects, are able to modulate the metabolism and expression of AbetaPP. This effect is often sustained by an indirect cholinergic mechanism and does not affect the mRNA expression of the precursor, although some other authors have demonstrated an effect on post-transcriptional regulation of AbetaPP expression. In addition to the effect on AbetaPP processing, we recently explored the possibility that these molecules affect a gene expression program beyond the classical pharmacological effects, for insights on possibly unexplored pathways of intervention in AD.