Susanna Terzano

Expression of the α3 nicotinic receptor subunit mRNA in aging and Alzheimer's disease

Changes in the number of high-affinity nicotine binding sites have been widely reported in specific regions of the human brain during aging and in degenerative neurological diseases associated with aging, such as Alzheimers disease. Nicotinic receptors are highly diverse and a description of the molecular subtypes affected in such conditions has not been achieved to date. To investigate the status of the α3 subunit-containing subtypes in such conditions, we assessed by in situ hybridisation the α3 mRNA density in the hippocampus, entorhinal cortex and thalamus of Alzheimers patients and age-matched controls. No significant difference in the expression of the α3 mRNA, either qualitative or quantitative, was found between Alzheimers individuals and controls in any of the analysed areas. This result suggests that the nicotine binding changes occurring in these areas in Alzheimers patients are not correlated to a variation of the α3 mRNA in the same regions. Nevertheless, a negative correlation between the α3 mRNA density and the age was observed in the entorhinal cortex of both the Alzheimers and the normal subjects, suggesting a potentially extensive decay of the α3-expressing neurons or loss of α3-containing receptors in intact neurons of the entorhinal cortex in the late elderly.

Expression and Transcriptional Regulation of the Human α3 Neuronal Nicotinic Receptor Subunit in T Lymphocyte Cell Lines

Abstract: The expression of neurotransmitter receptors on the surface of immunocompetent cells is generally accepted as evidence that the nervous system can influence immune responses, even though many aspects of these interactions remain to be elucidated. In this article, we analyzed the expression of the α3 nicotinic receptor subunit in human cell lines of myeloid and lymphoid origin and show that the α3 mRNA and the receptor molecules containing this subunit are specifically expressed in T lymphocyte cell lines. We have previously characterized the structural properties of the human α3 nicotinic subunit gene promoter and defined its functional profile in neuronal cells; in this study, we analyzed the activity of the α3 promoter in T lymphocytes and found that the same minimal promoter located in the 0.16‐kb BglII‐AccIII fragment is responsible for the expression of the α3 mRNA in both neuronal and T lymphocyte cell lines. However, the α3 transcription initiation patterns in the two cell types were both qualitatively and quantitatively different, and the minimal promoter was differentially modulated by downstream and upstream regulatory elements. These findings suggest that distinct transcriptional mechanisms allow the same promoter to be regulated in a tissue‐specific fashion, according to the different functional needs of the two cell types.

Neuronal and Extraneuronal Expression and Regulation of the Human α5 Nicotinic Receptor Subunit Gene

Abstract : The mRNA encoding the human α5 nicotinic subunit was detected in several structures of the nervous system but appeared to be mainly expressed in cerebellum, thalamus, and the autonomic ganglia. For the first time, the α5 transcript was also detected in several non‐neuronal tissues, with maximal expressions being found throughout the gastrointestinal tract, thymus, and testis. Many other extraneuronal sites expressed α5, but there were also nonexpressing organs, such as the liver, spleen, and kidney. To understand the transcriptional mechanisms controlling such a diversified expression of α5 in neuronal and nonneuronal cells, we isolated the 5′‐regulatory region of the human gene and characterized its properties. Here we identify the α5 core promoter and demonstrate that the DNA regions surrounding it contain elements (with positive or negative activities) that work in a tissue‐specific fashion. In particular, the segment specifying the 5′‐untranslated region in neuronal cells has most of the properties of an enhancer because it activates a heterologous promoter in a position‐ and orientation‐independent fashion. We therefore conclude that the expression of α5 relies on a highly complex promoter that uses distinct regulatory elements to comply with the different functional and developmental requirements of the various tissues and organs.

The Minimal Promoter of the Human α3 Nicotinic Receptor Subunit Gene MOLECULAR AND FUNCTIONAL CHARACTERIZATION

The minimal promoter of the human α3 nicotinic receptor subunit gene has been mapped to a region of 60 base pairs and found to contain two Sp1 sites, one of which is essential for promoter activity. DNase footprinting has revealed the presence of another region of interaction with nuclear factors (named F2) immediately downstream of the Sp1 sites. This region has been found to be functional since it is capable of stimulating the minimal promoter. The F2 protection is completely and specifically competed by an AP2 consensus oligonucleotide that has been proved to bind AP2α exclusively. However, the AP2α recombinant protein was unable to bind the F2 region directly, thus suggesting that AP2α may participate in F2 protection by protein-protein interactions with other nuclear factors. The minimal promoter has been shown to be stimulated by two additional regions, one located downstream of F2 and the other upstream of the minimal promoter itself. In neuronal cells, the combined stimulatory activities of these three regions have synergistic effects, whereas in non-neuronal cells, there is a negative interference between the upstream and downstream regions. These opposite transcriptional effects may account for at least part of the neuro-specific expression profile of the α3gene.