Human in vitro reporter model of neuronal development and early differentiation processes

<p>Abstract</p> <p>Background</p> <p>During developmental and adult neurogenesis, doublecortin is an early neuronal marker expressed when neural stem cells assume a neuronal cell fate. To understand mechanisms involved in early processes of neuronal fate decision, we investigated cell lines for their capacity to induce expression of doublecortin upon neuronal differentiation and develop <it>in vitro </it>reporter models using doublecortin promoter sequences.</p> <p>Results</p> <p>Among various cell lines investigated, the human teratocarcinoma cell line NTERA-2 was found to fulfill our criteria. Following induction of differentiation using retinoic acid treatment, we observed a 16-fold increase in doublecortin mRNA expression, as well as strong induction of doublecortin polypeptide expression. The acquisition of a neuronal precursor phenotype was also substantiated by the establishment of a multipolar neuronal morphology and expression of additional neuronal markers, such as Map2, βIII-tubulin and neuron-specific enolase. Moreover, stable transfection in NTERA-2 cells of reporter constructs encoding fluorescent or luminescent genes under the control of the doublecortin promoter allowed us to directly detect induction of neuronal differentiation in cell culture, such as following retinoic acid treatment or mouse Ngn2 transient overexpression.</p> <p>Conclusion</p> <p>Induction of doublecortin expression in differentiating NTERA-2 cells suggests that these cells accurately recapitulate some of the very early events of neuronal determination. Hence, the use of reporter genes under the control of the doublecortin promoter in NTERA-2 cells will help us to investigate factors involved early in the course of neuronal differentiation processes. Moreover the ease to detect the induction of a neuronal program in this model will permit to perform high throughput screening for compounds acting on the early neuronal differentiation mechanisms.</p

Cocaine Is Low on the Value Ladder of Rats: Possible Evidence for Resilience to Addiction

International audienceBACKGROUND:Assessing the relative value of cocaine and how it changes with chronic drug use represents a long-standing goal in addiction research. Surprisingly, recent experiments in rats--by far the most frequently used animal model in this field--suggest that the value of cocaine is lower than previously thought.METHODOLOGY/PRINCIPAL FINDINGS:Here we report a series of choice experiments that better define the relative position of cocaine on the value ladder of rats (i.e., preference rank-ordering of different rewards). Rats were allowed to choose either taking cocaine or drinking water sweetened with saccharin--a nondrug alternative that is not biologically essential. By systematically varying the cost and concentration of sweet water, we found that cocaine is low on the value ladder of the large majority of rats, near the lowest concentrations of sweet water. In addition, a retrospective analysis of all experiments over the past 5 years revealed that no matter how heavy was past cocaine use most rats readily give up cocaine use in favor of the nondrug alternative. Only a minority, fewer than 15% at the heaviest level of past cocaine use, continued to take cocaine, even when hungry and offered a natural sugar that could relieve their need of calories.CONCLUSIONS/SIGNIFICANCE:This pattern of results (cocaine abstinence in most rats; cocaine preference in few rats) maps well onto the epidemiology of human cocaine addiction and suggests that only a minority of rats would be vulnerable to cocaine addiction while the large majority would be resilient despite extensive drug use. Resilience to drug addiction has long been suspected in humans but could not be firmly established, mostly because it is difficult to control retrospectively for differences in drug self-exposure and/or availability in human drug users. This conclusion has important implications for preclinical research on the neurobiology of cocaine addiction and for future medication development

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference