Direct effects of estrogens on cholinergic primary neurons from the human fetal nucleus basalis of Meynert

Epidemiological studies have indicated that Alzheimer’s disease (AD) is more common in females and that post-menopausal women are at increased risk than their male counterpart, thus suggesting that estrogens could play a protective role to counteract neurodegenerative processes (1). However, the mechanisms underlying this association remain to be clarified. Since the nucleus basalis of Meynert (nbM) is the major source of cholinergic innervation selectively vulnerable to degeneration in AD, our study is aimed at investigating the effects of estrogens on human cholinergic primary neurons (hfCNs) isolated from the nbM of 12-week old fetuses. The primary culture obtained was immunophenotyped with flow cytometry and resulted almost totally positive (97±2 %) for the neuronal marker MAP2 and for the choline acetyltransferase (ChAT). We demonstrated that hfCNs express receptors for hormones of the reproductive axis (ERs, LHR, GnRHR). In particular, besides to classical estrogen receptors (ERa and ERb), hfCNs express the transmembrane receptor GPR30, which is known to mediate rapid non-genomic estrogen actions. Increasing concentrations of 17-β estradiol (E2, 0.1-100 nM) determined a dose-dependent significant increase in cell number after 24h exposure, which was antagonized by tamoxifen treatment. In addition, E2 exposure determined a significant increase in ChAT expression, thus indicating a direct positive effect of E2 on cholinergic phenotype. Given that substantial evidence now indicates that estrogens exert an anti-inflammatory activity even in the central nervous system (2), we exposed hfCN cells to the proinflammatory cytokine TNFα. E2 treatment (1nM) was able to significantly counteract the TNFα-induced nuclear NF-kB p65 translocation. Interestingly, this effect was mimicked by G1, a GPR30 agonist, and abolished by pretreating cells with the GPR30 antagonist G15, but not by tamoxifen, which usually antagonizes classical ERs. Overall, our results indicate that estrogens exert direct neuroprotective mechanisms on hfCNs through the activation of either classical (trophic) and non-classical (anti-inflammatory) receptors

Primary cell culture from human striatal primordium. Contribution to research on neuronal plasticity

Throughout fetal life, striatal neurons originated in the ganglionic eminence from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of striatal neuronal precursors. In order to identify the regulatory mechanisms controlling striatal cell neurogenesis and differentiation, we have recently isolated and propagated in vitro primary cell cultures from the human fetal striatal primordium (1). These cells express both neuronal and striatal properties, and are responsive to BDNF and FGF2. In this study, we found that human striatal precursor (HSP) cells are a mixed population mainly constituted of neuronal-restricted progenitors and striatal neurons (DARPP32-, GAD1-expressing cells) and neural/stem cells, which under specific in vitro differentiating conditions not only generate neurons, astrocytes and oligodendrocytes, but also possess the ability of osteogenic differentiation. We also observed that BDNF and FGF2 exert different effects on HSP depending on the differentiation state of these cells. In fact, both neurotrophins promote cell proliferation, migration and the expression of neural stem/progenitor markers in undifferentiated HSP cells, while they stimulate neuritogenesis in the neuronal differentiated component as demonstrated after specific neuronal induction. We have previously reported that striatal primordium from human fetus was able to grow into the brain of Huntington’s disease (HD) patients and that this process was associated with metabolic change and some clinical benefit (2,3). Our results add new insight into the developmental processes of human fetal striatal grafts in HD and, in addition, have implications for cell based transplantation approaches in the CNS

FGF2 and ET1 promote human fetal striatal neuroblasts survival in hypoxic conditions

Fetal striatal transplantation emerged as a new strategy to promote reparative responses in Huntington’s disease (HD) patients1. Mechanisms that support neuroblasts survival and replenishment of damaged cells within the HD brain in hypoxia remain to be elucidated. This study investigated how human fetal striatal neuroblasts (HSP cells) respond to hypoxia, using the hypoxia-mimetic agent cobalt chloride (CoCl2)2. We analyzed CoCl2 effect on hypoxia-related proteins, such as HIF-1α and VEGF, and on a neuroprotective factor, such as Seladin-1. Moreover, we evaluated FGF2 (50 ng/ml) and ET1 (100 nM) proliferative/survival effects in HSP cells in normoxic and hypoxic conditions. These growth factors could be important mediators under pathological conditions for striatal neuroblasts function and response to hypoxia. Dose-response experiments with increasing concentration of CoCl2 (50-750 um) showed an increase of HSP cell proliferation at 24-48h, with maximal effects observed at 400 um, while cell survival was impaired at 72h. Hypoxia increased protein expressions of HIF-1α and VEGF, whereas decreased Seladin-1 levels. FGF2 and ET1 significantly stimulated HSP cells proliferation both in normoxic and hypoxic condition, counteracting the apoptotic CoCl2 effect at 72h. FGF2 and ET1 neuroprotective effect was abolished by the selective inhibition of their receptors (FGFR1, ETA and ETB). In particular, ET1 stimulated HSP cells survival through ETA receptor in normoxic condition and through ETB receptor during hypoxia. Our results support the idea that FGF2 and ET1 promote neurogenesis and survival of HSP cells, through receptor-mediated mechanisms, when grafted into the hypoxic HD brain

Novel mechanisms of neuroprotective effects of Quercetin on human striatal neuroblasts

Human striatal precursor (HSP) primary cell cultures were isolated from ganglionic eminence of 9-12 week old human fetuses and extensively characterized in vitro (1). Our studies demonstrated that these cultures consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons that express and are responsive to many trophic factors, as BDNF and FGF2, and possess an adaptive response to stress conditions as nutrient deprivation and hypoxia through mechanisms involving different factors and neurotrophins (1,2). In the last decades, several in vitro and in vivo studies have provided evidence for neuroprotective effects by Quercetin, a polyphenol widely present in nature, passively absorbed in the small intestine and able to traverse the blood brain barrier (3). However, the mechanisms through which Quercetin exerts its neuroprotective effects are not fully delucidated. Our study was aimed at investigating the effects of Quercetin on HSP cells and its contribution to cell survival in nutrient deprivation condition, obtained replacing culture medium with Phosphate Buffer Saline (PBS). Quercetin treatment significantly promoted cell survival and strongly decreased apoptosis induced by nutrient deprivation condition, as evaluated by MTT assay, Trypan Blue staining and western blot analysis of cell death and proliferation markers. Moreover, since the adhesive capacities of cells are essential for cell survival, we next analysed the expression of some adhesion molecules such as Pancadherin and Focal Adhesion Kinase; our results interestingly showed that PBS exposure determined a strong decrease in all the analysed adhesion molecules, while in presence of Quercetin the expression was significantly increased. Our results add new mechanicistic insights into the comprehension of neuroprotective action of Quercetin treatment, thus suggesting possible implications in sustaining striatal neuron survival during neurodegenerative disorders, such as Huntington Disease

Physical activity modify skeletal muscle fiber types in an animal model of metabolic syndrome

Metabolic Syndrome (MetS) is a cluster of clinical conditions, associated to an increased cardiovascular risk, as well as to hypogonadism in males. Lifestyle modification (including physical exercise, PhyEx) may be beneficial for the condition. Skeletal muscles (SkM) are some of the most highly plastic tissues, able of remodeling in response to use, disuse and disease. In particular, transformations of fiber type may occur in response to physiological milieu to induce functional adaptations. This study is aimed at investigating in experimental MetS, high fat diet-induced in male rabbits [1], the effect of PhyEx on hormonal and metabolic parameters, as well as on SkM composition. Control and MetS rabbits were exercise-trained to run on a treadmill for 12 weeks. Quadriceps femoris samples were collected for histomorphological and gene expression analyses. We found that exercise resistance was significantly reduced in MetS rabbits, as demonstrated by the significant reduction of both running time and distance, compared to control group. MetS rabbits also exhibited the lowest quadriceps mass. Fiber typing by PAS-staining showed a pronounced shift from slower type I to faster type II fibers in MetS group in response to PhysEx, suggesting that MetS condition addressed SkM function towards anaerobic metabolism. Accordingly, extracellular lactate levels were significantly increased and mitochondrial respiration-related genes reduced in SkM of MetS rabbits respect to controls. Interestingly, PhyEx significantly counteracted MetS-related testosterone deficiency and hypercholesterolemia. In conclusion, our results indicate that dysmetabolic milieu induces a reduced proportion of fatigue-resistant type I fibers in response to PhysEx, which however resulted beneficial for MetS condition