Hyponatraemia alters the biophysical properties of neuronal cells independently of osmolarity: a study on Ni(2+) -sensitive current involvement.

What is the central question of this study? Hyponatraemia, an electrolyte disorder encountered in hospitalized patients, can cause neurological symptoms usually attributed to a reduction in plasma osmolarity. Here, we investigated whether low [Na(+) ] per se can cause neuronal changes independent of osmolarity, focusing on involvement of the Na(+) -Ca(2+) exchanger. What is the main finding and its importance? We show that hyponatraemia per se causes alterations of neuronal properties. The novel finding of Na(+) -Ca(2+) exchanger involvement helps us to elucidate the volume regulation following hyponatraemia. This might have relevance in a translational perspective because Na(+) -Ca(2+) exchanger could be a target for novel therapies. Hyponatraemia is the most frequent electrolyte disorder encountered in hospitalized patients, and it can cause a wide variety of neurological symptoms. Most of the negative effects of this condition on neuronal cells are attributed to cell swelling because of the reduction of plasma osmolarity, although in hyponatraemia different membrane proteins are supposed to be involved in the conservation of neuronal volume. We have recently reported detrimental effects of hyponatraemia on two different neuronal cell lines, SK-N-AS and SH-SY5Y, independent of osmotic alterations. In this study we investigated, in the same cell lines, whether hyponatraemic conditions per se can cause electrophysiological alterations and whether these effects vary over time. Accordingly, we carried out experiments in low-sodium medium in either hyposmotic [Osm(-)] or isosmotic [Osm(+)] conditions, for a short (24 h) or long time (7 days). Using a patch pipette in voltage-clamp conditions, we recorded possible modifications of cell capacitance (Cm ) and membrane conductance (Gm ). Our results indicate that in both Osm(-) and Osm(+) medium, Cm and Gm show a similar increase, but such effects are dependent on the time in culture in different ways. Notably, regarding the possible mechanisms involved in the maintenance of Cm , Gm and Gm /Cm in Osm(+) conditions, we observed a greater contribution of the Na(+) -Ca(2+) exchanger compared with Osm(-) and control conditions. Overall, these novel electrophysiological results help us to understand the mechanisms of volume regulation after ionic perturbation. Our results might also have relevance in a translational perspective because the Na(+) -Ca(2+) exchanger can be considered a target for planning novel therapies.

Altered expression of 3-betahydroxysterol delta-24-reductase/selective Alzheimer's disease indicator-1 gene in Huntington's disease models.

INTRODUCTION: 3-betahydroxysterol delta-24-reductase (DHCR24), also called selective Alzheimer's disease indicator-1, is a crucial enzyme in cholesterol biosynthesis with neuroprotective properties that is downregulated in brain areas affected by Alzheimer's disease. AIM: In the present study, we investigated modifications of DHCR24 expression in models of Huntington's disease (HD), a neurodegenerative disorder caused by a polyglutamine expansion in huntingtin (Htt) protein that induces degeneration of cerebral cortex and striatum as well as lateral hypothalamic abnormality. METHODS: Basal expression of DHCR24 and its modulation after oxidative stress were evaluated in rat striatal precursors cells (ST14A) transfected with wild-type (Htt) or mutant Htt (mHtt) and in brain tissue of an HD mouse model (R6/2). RESULTS: The results showed that DHCR24 transcript levels were decreased in ST14A cells expressing mHtt and in the brain of symptomatic R6/2 mice, but were significantly increased in ST14A cells overexpressing wild-type Htt. In addition, we demonstrated that, in the striatal precursors, the decrease of DHCR24 expression in response to oxidative stress was modified according to the presence of Htt or of its mutant form. Preliminary results indicated a modification of DHCR24 expression in post-mortem brain samples of HD patients. CONCLUSIONS: In conclusion, these results support the hypothesis of a possible role of DHCR24 in HD.

Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na(+) channels and amplitude of Ca(++) currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.

New insights on the neuroprotective role of sterols and sex steroids: the seladin-1/DHCR24 paradigm.

In 2000 a new gene, i.e. seladin-1 (for selective Alzheimer's disease indicator-1) was identified and found to be down regulated in vulnerable brain regions in Alzheimer's disease. Seladin-1 was considered a novel neuroprotective factor, because of its anti-apoptotic properties. Subsequently, it has been demonstrated that seladin-1 corresponds to the gene that encodes 3-beta-hydroxysterol delta-24-reductase (DHCR24), that catalyzes the synthesis of cholesterol from desmosterol. There is evidence that cholesterol plays a fundamental role in maintaining brain homeostasis. Because of its enzymatic activity, seladin-1/DHCR24 has been considered the human homolog of the plant protein DIMINUTO/DWARF1, that is involved in the synthesis of sterol plant hormones. We have recently demonstrated that seladin-1/DHCR24 is a fundamental mediator of the protective effects of estrogens in the brain. This review describes how this protein interacts with cholesterol and estrogens, thus generating a neuroprotective network, that might open new possibilities in the prevention/treatment of neurodegenerative diseases.

High recovery of mesenchymal progenitor cells with non-density gradient separation of human bone marrow.

BACKGROUND AIMS: Bone marrow (BM) is the most used source of hemopoietic stem cells (HSC) and mesenchymal stromal cells (MSC) in both hematologic settings and regenerative medicine. We compared the feasibility and reproducibility of two gravity separation techniques, with or without the use of a density gradient, in terms of both hematopoietic and mesenchymal human BM progenitors. METHODS: A total of 16 BM samples was processed to obtain mononuclear cells (MNC) and buffy coats (BC). The efficiency of the two procedures was evaluated by recovery of white blood cells (WBC), MNC and CD34(+) cells, clonogenic assays, red blood cell (RBC) depletion, cell viability, expression of embryonic transcriptional regulators and MSC assessment. RESULTS: The two procedures yielded a comparable recovery of HSC. Non-density gradient separation (NDGS) of BM resulted in four times higher MSC recovery and higher expression of embryonic stem cell markers (Nanog and Sox2) compared with density-gradient separation (DGS). MSC derived from both procedures was comparable in terms of phenotype, differentiation and proliferation potential. CONCLUSIONS: NDGS is less time consuming, provides a better MSC enrichment and appears to be a suitable cell preparation method for clinical applications.

Seladin-1 expression is regulated by promoter methylation in adrenal cancer.

BACKGROUND: Seladin-1 overexpression exerts a protective mechanism against apoptosis. Seladin-1 mRNA is variably expressed in normal human tissues. Adrenal glands show the highest levels of seladin-1 expression, which are significantly reduced in adrenal carcinomas (ACC). Since up to now seladin-1 mutations were not described, we investigated whether promoter methylation could account for the down-regulation of seladin-1 expression in ACC. METHODS: A methylation sensitive site was identified in the seladin-1 gene. We treated DNA extracted from two ACC cell lines (H295R and SW13) with the demethylating agent 5-Aza-2-deoxycytidine (5-Aza). Furthermore, to evaluate the presence of an epigenetic regulation also 'in vivo', seladin-1 methylation and its mRNA expression were measured in 9 ACC and in 5 normal adrenal glands. RESULTS: The treatment of cell lines with 5-Aza induced a significant increase of seladin-1 mRNA expression in H295R (fold increase, F.I. = 1.8; p = 0.02) and SW13 (F.I. = 2.9; p = 0.03). In ACC, methylation density of seladin-1 promoter was higher (2682 +/- 686) than in normal adrenal glands (362 +/- 97; p = 0.02). Seladin-1 mRNA expression in ACC (1452 +/- 196) was significantly lower than in normal adrenal glands (3614 +/- 949; p = 0.01). CONCLUSION: On this basis, methylation could be involved in the altered pattern of seladin-1 gene expression in ACC.

Seladin-1 and testicular germ cell tumours: new insights into cisplatin responsiveness.

The molecular basis for the exquisite sensitivity of testicular germ cell tumours of adolescents and adults (TGCTs), ie seminomas and non-seminomatous germ cell tumours, to chemo/radiotherapy has not been fully clarified so far. It has been suggested that it may be dependent on factors involved in the regulation of apoptosis. Seladin-1 is a multi-functional protein involved in various biological processes, including apoptosis. The aim of our study was to assess the expression of seladin-1 in different histological types of TGCTs, known to have varying treatment sensitivity, in order to establish whether this protein may influence cisplatin responsiveness in vitro. Seladin-1 expression levels, both at the mRNA and at the protein level, were higher in the adjacent normal parenchyma than in the pathological counterparts. In tumoural tissues, the level of expression differed among TGCT histological types. The highest tumour-expression level was found in teratoma, whereas the lowest was detected in seminoma, corresponding to the different chemo/and radiosensitivities of these tumour types. In common with other cancers, in TGCT-derived cell lines seladin-1 showed anti-apoptotic properties through inhibition of caspase-3 activation. We confirmed our results using a non-seminomatous cell line model (NT2) before and after differentiation with retinoic acid. Significantly higher seladin-1 expression was observed in the differentiated derivatives (teratoma) and an inverse relationship was found between seladin-1 expression and the amount of cleaved caspase-3. Seladin-1 silencing or overexpression in this cell line supports involvement of seladin-1 in cisplatin responsiveness. Seladin-1 silencing was associated with greater cisplatin responsiveness demonstrated by decreased cell viability and increased expression of apoptotic markers. In contrast, overexpression of seladin-1 was associated with a higher survival rate and a clear anti-apoptotic effect. In conclusion, we have demonstrated for the first time an important role for seladin-1 in the biology of TGCTs and provided new insights into cisplatin responsiveness of these tumours.

Low pulse energy Nd:YAG laser irradiation exerts a biostimulative effect on different cells of the oral microenvironment: "an in vitro study".

BACKGROUND AND OBJECTIVES: Dental lasers represent a promising therapeutic tool in the treatment of periodontal and peri-implant diseases. However, their clinical application remains still limited. Here, we investigated the potential biostimulatory effect of low pulse energy neodymium:yttrium-aluminum-garnet (Nd:YAG) laser irradiation on different cells representative of the oral microenvironment and elucidated the underlying molecular mechanisms. MATERIALS AND METHODS: Saos-2 osteoblasts, H-end endothelial cells, and NIH/3T3 fibroblasts pre-treated or not with photosensitizing dye methylene blue (MB), were irradiated with low pulse energy (20 mJ) and high repetition rate (50-70 Hz) Nd:YAG laser, and evaluated for cell viability and proliferation as well as for the expression of specific differentiation markers by confocal immunofluorescence and real-time RT-PCR. Changes in intracellular Ca(2+) levels after laser exposure were also evaluated in living osteoblasts. RESULTS: Nd:YAG laser irradiation did not affect cell viability in all the tested cell types, even when combined with pre-treatment with MB, and efficiently stimulated cell growth in the non-sensitized osteoblasts. Moreover, a significant induction in the expression of osteopontin, ALP, and Runx2 in osteoblasts, type I collagen in fibroblasts, and vinculin in endothelial cells could be observed in the irradiated cells. Pre-treatment with MB negatively affected cell differentiation in the unstimulated and laser-stimulated cells. Notably, laser irradiation also caused an increase in the intracellular Ca(2+) in osteoblasts through the activation of TRPC1 ion channels. Moreover, the pharmacologic or genetic inhibition of these channels strongly attenuated laser-induced osteopontin expression, suggesting a role for the laser-mediated Ca(2+) influx in regulating osteoblast differentiation. CONCLUSION: Low pulse energy and high repetition rate Nd:YAG laser irradiation may exert a biostimulative effect on different cells representative of the oral microenvironment, particularly osteoblasts. Pre-treatment with MB prior to irradiation hampers this effect and limits the potential clinical application of photosensitizing dyes in dental practice.

Seladin-1 is a fundamental mediator of the neuroprotective effects of estrogen in human neuroblast long-term cell cultures.

Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimer's disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

Androgen receptor regulation of the seladin-1/DHCR24 gene: altered expression in prostate cancer.

Prostate cancer (CaP) represents a major leading cause of morbidity and mortality in the Western world. Elevated cholesterol levels, resulting from altered cholesterol metabolism, have been found in CaP cells. Seladin-1 (SELective Alzheimer Disease INdicator-1)/DHCR24 is a recently described gene involved in cholesterol biosynthesis. Here, we demonstrated the androgen regulation of seladin-1/DHCR24 expression, due to the presence of androgen responsive element sequences in its promoter region. In metastatic androgen receptor-negative CaP cells seladin-1/DHCR24 expression and cholesterol amount were reduced compared to androgen receptor-positive cells. In tumor samples from 61 patients who underwent radical prostatectomy the expression of seladin-1/DHCR24 was significantly higher with respect to normal tissues. In addition, in cancer tissues mRNA levels were positively related to T stage. In tumor specimens from 23 patients who received androgen ablation treatment for 3 months before surgery seladin-1/DHCR24 expression was significantly lower with respect to patients treated by surgery only. In conclusion, our study demonstrated for the first time the androgen regulation of the seladin-1/DHCR24 gene and the presence of a higher level of expression in CaP tissues, compared to the normal prostate. These findings, together with the results previously obtained in metastatic disease, suggest an involvement of this gene in CaP.

The effects of Exendin-4 on bone marrow-derived mesenchymal cells.

PURPOSE: GLP-1 receptor agonists are antidiabetic drugs currently used in the therapy of type 2 diabetes. Despite several in vitro and in vivo animal studies suggesting a beneficial effect of GLP-1 analogues on bone, in humans their skeletal effects are not clear and clinical studies report conflicting results. METHODS: We differentiated human mesenchymal stromal cells (hMSC) toward the adipogenic and the osteoblastic lineages, analysing the effect of Exendin-4 (EXE) before, during and after specific differentiations. RESULTS: We showed EXE ability to act selectively on a sub-population of hMSC characterised by a more stem potential, shifting them from G1 to S/M phase of cell cycle. We observed that EXE pre-treatment promotes both adipogenic and osteoblastic differentiations, possibly determined by an increased number of uncommitted progenitors. In fully differentiated cells, EXE affects mature adipocytes by increasing lipolysis, otherwise not altering osteoblasts metabolic activity. Moreover, the increased expression of osteoprotegerin, a modulator of the RANK/RANKL system, observed during osteogenic induction in presence of EXE, could negatively modulate osteoclastogenesis. CONCLUSIONS: Our data suggest a complex action of EXE on bone, targeting the proliferation of mesenchymal progenitors, the metabolism of mature adipocytes and the modulation of osteoclastogenesis. Thus, an overall positive effect of this molecule on bone quality might be hypothesised.

Low extracellular sodium causes neuronal distress independently of reduced osmolality in an experimental model of chronic hyponatremia.

There is evidence that chronic hyponatremia, even when mild, may cause neurological signs and symptoms. These have been traditionally associated with water movement into nervous cells, as a result of the hypotonic state. The aim of the present study was to determine whether low extracellular sodium directly exerts negative effects on human neuronal cells, independently of reduced osmolality. We exposed neuroblastoma SK-N-AS and SH-SY5Y cells to sustained low extracellular sodium, thus mimicking a condition of chronic hyponatremia, both in the presence of reduced and in the presence of unaltered osmolality. We found that very low sodium (i.e., 115 mmol/L in SK-N-AS and 90 mmol/L in SH-SY5Y) significantly reduced cell viability. However, intermediate low sodium was able to cause cell distress, as assessed by the altered expression of anti-apoptotic genes and the reduced ability to differentiate into a mature neuronal phenotype. Noteworthy, these effects were observed also in the presence of unaltered osmolality. Moreover, we performed a comprehensive microarray analysis in cells maintained in normal sodium or in low sodium and unaltered osmolality, and we found that the most altered pathway included genes involved in "cell death and survival." Among the more than 40 differentially expressed genes, the Heme oxygenase gene, which represents a transcriptional response to oxidative stress, showed the highest increase in the expression level. This study demonstrates that low extracellular sodium causes detrimental effects in neuronal cells that are at least in part independent of reduced osmolality. These findings further support the recommendation to effectively correct hyponatremia, even when mild and chronic.

Exendin-4 induces cell adhesion and differentiation and counteracts the invasive potential of human neuroblastoma cells.

Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

Hormonal modulation of cholesterol: experimental evidence and possible translational impact.

Alzheimer's disease (AD) is still an incurable condition. There is in vitro evidence that estrogens exert neuroprotective effects; however, their role in the treatment of AD is still controversial. Approximately 10 years ago, a new gene, named seladin-1 (for selective AD indicator-1), was identified and found to be downregulated in brain regions affected by AD. Seladin-1 has neuroprotective properties, which have been associated, at least in part, with its anti-apoptotic activity. Estrogens stimulate the expression of the seladin-1 gene. Seladin-1 also has enzymatic activity (3-ß-hydroxysterol Δ-24-reductase), which is involved in the synthesis of cholesterol from desmosterol. The amount of membrane cholesterol appears to play an important role in conferring protection to brain cells. This review focuses on the relationship between estrogens (and IGF-1, another hormone with neuroprotective properties), cholesterol and seladin-1.

Neuronal differentiation of human mesenchymal stromal cells increases their resistance to Aß42 aggregate toxicity.

Cell therapy is a promising approach for the treatment of neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. However, the presence of toxic aggregates in tissue raises the question of whether grafted stem cells are susceptible to amyloid toxicity before they differentiate into mature neurons. To address this question, we investigated the relative vulnerability of human mesenchymal stromal cells and their neuronally differentiated counterparts to Aß(42) oligomers and whether susceptibility correlates with membrane GM1 content, a key player in oligomer toxicity. We found that our cell model was highly susceptible to aggregate toxicity, whereas neuronal differentiation induced resistance to amyloid species. This data correlated well with the content of membrane GM1, levels of which decreased considerably in differentiated cells. These findings extend our knowledge of stem cell vulnerability to amyloid species, which remains a controversial issue, and confirm that amyloid-GM1 interactions play an important role in cell impairment.

Molecular mechanisms underlying the pro-inflammatory synergistic effect of tumor necrosis factor alpha and interferon gamma in human microvascular endothelium.

Tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) are among the most potent cytokines involved in orchestrating the inflammation response. The molecular mechanisms implicated in the synergism between cytokines are still poorly characterized. We demonstrate that both cytokines dose-dependently stimulate IFNgamma-inducible-protein-of-10-kDa (IP-10) secretion in human microvascular endothelial cells (HMEC-1), showing a potent synergism which is not restricted to IP-10, but is also evident for monokine-induced-by-IFNgamma (MIG) and IL-6 secretion. Immunofluorescence analysis reveals that TNFalpha and IFNgamma converge on a rapid phosphorylation of ERK, which however results in a different subcellular compartmentalization of the activated enzyme in response to the two cytokines. Differences in the subcellular recruitment of ERK in response to IFNgamma and TNFalpha are responsible for generating different ERK downstream signaling, which can thus synergize on the secretion of IP-10 as well as of other cytokines/chemokines. The importance of ERK activation in mediating the synergism of the two cytokines is further confirmed by the inhibitory effect of the anti-diabetic drug rosiglitazone and ERK blockers on IP-10, MIG and IL-6 secretion. A further mechanism of synergism involving the reciprocal upregulation of TNFalpha-RII and of IFNgamma-R, in response to IFNgamma and TNFalpha, respectively, was revealed by flow cytometry and quantitative real time RT-PCR analysis.

Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1.

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.