21 research outputs found
A Mild Form of SLC29A3 Disorder: A Frameshift Deletion Leads to the Paradoxical Translation of an Otherwise Noncoding mRNA Splice Variant
We investigated two siblings with granulomatous histiocytosis prominent in the nasal area, mimicking rhinoscleroma and Rosai-Dorfman syndrome. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous frameshift deletion in SLC29A3, which encodes human equilibrative nucleoside transporter-3 (hENT3). Germline mutations in SLC29A3 have been reported in rare patients with a wide range of overlapping clinical features and inherited disorders including H syndrome, pigmented hypertrichosis with insulin-dependent diabetes, and Faisalabad histiocytosis. With the exception of insulin-dependent diabetes and mild finger and toe contractures in one sibling, the two patients with nasal granulomatous histiocytosis studied here displayed none of the many SLC29A3-associated phenotypes. This mild clinical phenotype probably results from a remarkable genetic mechanism. The SLC29A3 frameshift deletion prevents the expression of the normally coding transcripts. It instead leads to the translation, expression, and function of an otherwise noncoding, out-of-frame mRNA splice variant lacking exon 3 that is eliminated by nonsense-mediated mRNA decay (NMD) in healthy individuals. The mutated isoform differs from the wild-type hENT3 by the modification of 20 residues in exon 2 and the removal of another 28 amino acids in exon 3, which include the second transmembrane domain. As a result, this new isoform displays some functional activity. This mechanism probably accounts for the narrow and mild clinical phenotype of the patients. This study highlights the ‘rescue’ role played by a normally noncoding mRNA splice variant of SLC29A3, uncovering a new mechanism by which frameshift mutations can be hypomorphic
Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes
Background: In the central nervous system, glial cells provide metabolic and trophic support to neurons and respond to protracted stress and insults by up-regulating inflammatory processes. Reactive astrocytes and microglia are associated with the pathophysiology of neuronal injury, neurodegenerative diseases and major depression, in both animal models and human brains. Several studies have reported clear anti-inflammatory effects of anti-depressant treatment on astrocytes, especially in models of neurological disorders. Trazodone (TDZ) is a triazolopyridine derivative that is structurally unrelated to other major classes of antidepressants. Although the molecular mechanisms of TDZ in neurons have been investigated, it is unclear whether astrocytes are also a TDZ target. Methods: The effects of TDZ on human astrocytes were investigated in physiological conditions and following inflammatory insult with lipopolysaccharide (LPS) and tumour necrosis factor-aα (TNF-aα). Astrocytes were assessed for their responses to pro-inflammatory mediators and cytokines, and the receptors and signalling pathways involved in TDZ-mediated effects were evaluated. Results: TDZ had no effect on cell proliferation, but it decreased pro-inflammatory mediator release and modulated trophic and transcription factor mRNA expression. Following TDZ treatment, the AKT pathway was activated, whereas extracellular signal-regulated kinase and c-Jun NH2-terminal kinase were inhibited. Most importantly, a 72-h TDZ pre-treatment before inflammatory insult completely reversed the anti-proliferative effects induced by LPS-TNF-aα. The expression or the activity of inflammatory mediators, including interleukin-6, c-Jun NH2-terminal kinase and nuclear factor ΚB, were also reduced. Furthermore, TDZ affected astrocyte metabolic support to neurons by counteracting the inflammation-mediated lactate decrease. Finally, TDZ protected neuronal-like cells against neurotoxicity mediated by activated astrocytes. These effects mainly involved an activation of 5-HT1A and an antagonism at 5-HT2A/C serotonin receptors. Fluoxetine, used in parallel, showed similar final effects nevertheless it activates different receptors/intracellular pathways. Conclusions: Altogether, our results demonstrated that TDZ directly acts on astrocytes by regulating intracellular signalling pathways and increasing specific astrocyte-derived neurotrophic factor expression and lactate release. TDZ may contribute to neuronal support by normalizing trophic and metabolic support during neuroinflammation, which is associated with neurological diseases, including major depression
Trazodone regulates neurotrophic/growth factors, mitogen-activated protein kinases and lactate release in human primary astrocytes
Autoradiographic localization of 5-HT1A receptors in the post-mortem human brain using [H-3]WAY-100635 and [C-11]WAY-100635
The distribution of 5-HT1A receptors was examined in the post-mortem human brain using whole hemisphere autoradiography and the selective 5-HT1A receptor antagonist [H-3]WAY-100635 ([O-methyl-H-3]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride). The autoradiograms showed very dense binding to hippocampus, raphe nuclei and neocortex. The labeling in neocortex was slightly lower than in the hippocampus and was mainly at superficial layers, although a faintly labeled band could be seen in deeper neocortical layers. Other regions, such as the amygdala, septum and claustrum, showed low densities of [H-3]WAY-100635 binding, reflecting low densities of 5-HT1A receptors. The labeling was very low in basal ganglia, such as nucleus caudatus and putamen, in cerebellum or in structures of the brain stem except in the raphe nuclei. The labeling of human 5-HT1A receptors with [H-3]WAY-100635 was antagonized by the addition of the 5-HT1A receptor ligands, 5-HT, buspirone, pindolol or 8-OH-DPAT (10 mu M) leaving a very low background of non-specific binding. Saturation analysis of semiquantitative data from several human regions indicated that [H-3]WAY-100635 has a K-d of approximately 2.5 nM. The selective labeling of 5-HT1A receptors, with [H-3]WAy-100635 clearly show that this compound is useful for further studies of the human 5-HT1A receptor subtype in vitro. [C-11]WAY-100635 is used for the characterization of 5-HT1A receptors with positron emission tomography (PET). WAY-100635 was also radiolabeled with the short-lived positron-emitting radionuclide carbon-11 (t(1/2)=20 min) and used for in vitro autoradiography on human whole hemisphere cryosections. [C-11]WAY-100635 gave images qualitatively similar to those of [H-3]WAY-100635, although with a lower resolution. Thus, the hippocampal formation was densely labeled, with lower density in the neocortex. Buspirone, pindolol or 8-OH-DPAT (10 mu M), blocked all binding of [C-11]WAY-100635. The in vitro autoradiography of the distribution of 5-HT1A receptors obtained with radiolabeled WAY-100635 provide detailed qualitative and quantitative information on the distribution of 5-HT1A-receptors in the human brain. Moreover, the studies give reference information for the interpretation of previous initial results at much lower resolution in humans with PET and [C-11]WAY-100635. These data provide a strong basis for expecting [C-11]WAY-100635 to behave as a highly selective radioligand in vivo
Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methyl-C-11]WAY-100635, in monkey and human plasma by HPLC: Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET
N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t(1/2) = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-C-11]WAY-lO0635 into humans, radioactivity was found to clear rapidly from blood and plasma, By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-C-11]WAY-lO0635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-C-11]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha(1)-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-C-11]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-ii in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake
Characterization of the radioactive metabolites of the 5-HT1A receptor radioligand, [O-methyl-C-11]WAY-100635, in monkey and human plasma by HPLC:Comparison of the behaviour of an identified radioactive metabolite with parent radioligand in monkey using PET
N-(2-(4-(2-Methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (WAY-100635), labelled in the O-methyl group with carbon-11 (t(1/2) = 20.4 min), is a promising radioligand for application with positron emission tomography (PET) to the study of 5-HT1A receptors in living human brain. An understanding of the metabolism of this new radioligand is crucial to the development of a biomathematical model for the interpretation of the kinetics of radioactivity uptake in brain in terms of receptor-binding parameters. After intravenous injection of [O-methyl-C-11]WAY-lO0635 into humans, radioactivity was found to clear rapidly from blood and plasma, By using established methods for the analysis of radioactivity in plasma, it was found that intravenously injected [O-methyl-C-11]WAY-lO0635 is rapidly metabolised to more polar radioactive compounds in a cynomolgus monkey and in humans. Thus, at 60 min postinjection, parent radioligand represented 40% and 5% of the radioactivity in monkey and human plasma, respectively. In monkey and human, one of the radioactive metabolites was identified as the descyclohexanecarbonyl analogue of the parent radioligand, namely [O-methyl-C-11]WAY-100634. This compound is known to have high affinity for 5-HT1A receptors and alpha(1)-adrenoceptors. In a PET experiment it was demonstrated that, after IV injection of [O-methyl-C-11]WAY-100634 into a cynomolgus monkey, radioactivity was avidly taken up by brain. Uptake of radioactivity was higher in 5-HT1A receptor-rich frontal cortex than in cerebellum, which is devoid of 5-HT1A receptors. Polar radioactive metabolites appeared in plasma. The results suggest that the use of WAY-100635 labelled with carbon-ii in its cyclohexanecarbonyl moiety may provide enhanced signal contrast in PET studies and a possibility to develop a simple biomathematical model for regional brain radioactivity uptake