A monoamine oxidase B gene variant and short-term antidepressant treatment response

Genetic differences among patients suffering from Major Depression are likely to contribute to interindividual differences in medication treatment response. Thus, the identification of gene variants affecting drug response is needed in order to be able to predict response to psychopharmacological drugs. This study analyzed a possible association of the common A644G single nucleotide polymorphism (SNP) within intron 13 of the monoamine oxidase B (MAOB) gene with antidepressant treatment response. The study population consisted of n = 102 patients with major depression (criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition; DSM-IV) participating in a randomized double-blind controlled clinical trial, conducted at 50 centers in Germany, comparing the efficacy of mirtazapine and paroxetine during 6 weeks of treatment. Overall, female patients homozygous for the A-allele had a significantly faster and more pronounced antidepressant treatment response than AG/GG-carriers. In paroxetine-treated females these differences remained statistically significant. In mirtazapine-treated females homozygous for the A-allele compared to AG/GG-carriers, HAMD-17 scores during the study period were constantly and markedly lower, but not statistically different. In males, we found no association between the MAOB A644G intron 13 SNP and antidepressant treatment response. Our data provide first suggestive evidence that the MAOB A644G SNP is involved in the outcome of treatment with mirtazapine or paroxetine in females with major depression. To confirm the role of the MAOB A644G gene variant in antidepressant treatment response, independent replications are needed. If replicated, the MAOB A644G polymorphism could be considered useful for prospective confirmatory pharmacogenetic trials in patients with major depression.     

Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis

Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study, we show that astroglial cells instruct noncommitted, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell-to-cell contacts and via short-range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30- 60 h after the onset of glial induction. Statistical analyses of time-lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia-derived humoral factors increase their motility. In non-contact co-cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. By contrast, in contact co-cultures with astrocytes, the restricted migration on glial surfaces keeps the cell progenies together, resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes (1) secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells; and (2) provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to noncommitted progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.     

In Vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53-deficient immortalized neuroectodermal progenitor cell line, NE-4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res. 47, 405–416]. Rearrangement of nestin filaments was an early marker of neuron-formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4–5 days compared to the appearence of neurons.Future neurons were sorted out from the substrate-attached population of apparently non-induced cells. The sorting out of future neurons resembled the separation of neural precursorsin vivo. The continuous changes in the shape and also in the position of the cells resulted in the formation of characteristic morphological patterns. On the basis of morphological changes, five characteristic stages of in vitro neural differentiation were distinguished.The analysis of the morphological changes revealed that cell-to-cell interactions played an essential role in the cell fate decision made by induced precursors. Our observations indicate that the NE-4C cell line can serve as an in vitro model to investigate some early steps of neurogenesis.     

Comparison of the Effects of 13-cis Retinoic Acid and Melatonin on the Viabilities of SH-SY5Y Neuroblastoma Cell Line

ObjectiveNeuroblastoma is one of common childhood tumors. Although its mortality is very high, there is no effective treatment yet. The aim of this project is to evaluate cytotoxic effects of melatonin (MLT) an endogen hormone and 13-cis retinoic acid (13-cis-RA) also named as isotretinoin an analogue of vitamin A on neuroblastoma SH-SY5Y cell line.MethodsIn this study, SH-SY5Y cell line was used. After cell culture, the cells were exposed to different doses of MLT and 13-cis-RA. 24 and 48 hours later. While the viabilities was estimated with MTT cell viability assay test, apoptotic indexes were calculated after staining with TUNEL based apoptosis kit.ResultsIt was observed that MLT has very effective cytotoxic potential than 13-cis-RA on neuroblastoma cell line. At the same time, when MLT and 13-cis-RA were combined, this effect was potentiated. On the other hand, it was found that the effect of 13-cis-RA individually on neuroblastoma cells was very slight.ConclusionWe suggest that in the treatment of patient with neuroblastoma, MLT is very effective and also this effect can be augmented by combination with 13-cis-RA.     

An effective inducer of dopaminergic neuron-like differentiation

Rat bone marrow-derived mesenchymal stem cells were cultured and passaged in vitro. After induction with basic fibroblast growth factor for 24 hours, passage 3 bone marrow-derived mesenchymal stem cells were additionally induced into dopaminergic neurons using three different combinations with basic fibroblast growth factor as follows: 20% Xiangdan injection; all-trans retinoic acid + glial-derived neurotrophic factor; or sonic hedgehog + fibroblast growth factor 8. Results suggest that the bone marrow-derived mesenchymal stem cells showed typical neuronal morphological characteristics after induction. In particular, after treatment with sonic hedgehog + fibroblast growth factor 8, the expressions of nestin, neuron-specific enolase, microtubule- associated protein 2, tyrosine hydroxylase and vesicular monoamine transporter-2 in cells were significantly increased. Moreover, the levels of catecholamines in the culture supernatant were significantly increased. These findings indicate that Xiangdan injection, all-trans retinoic acid + glial-derived neurotrophic factor, and sonic hedgehog + fibroblast growth factor 8 can all induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells. In particular, the efficiency of sonic hedgehog + fibroblast growth factor 8 was highest.     

A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells

P19 embryonal carcinoma (EC) cells are an invaluable tool for approximating the mechanisms that govern neuronal differentiation but with an enormous degree of simplification and have primarily been used to model the early stages of neurogenesis. However, they are often cultured under conditions that promote unrestricted non-neuronal growth that compromises neuronal viability. In this study we report an improved method to differentiate P19 EC cells that gives rise to high yields of functionally and morphologically mature neurons while significantly reducing the over-growth of non-neuronal cells in the cultures. In this protocol, P19 EC cells are induced in Minimum Essential Medium alpha supplemented with all-trans retinoic acid (RA) and 2.5% serum, and cultured as a monolayer. After RA-induction, cells are cultured on Matrigel coated-plates using defined media comprised of Neurobasal-A medium temporally supplemented with N2 and then B-27 for the remaining culture period. By treating the culture with Cytosine β-d-arabinofuranoside and 2′-Deoxycytidine for five days, the cultures are reliably promoted toward the neuronal differentiation vs non-neuronal differentiation, this accounting for a progressive neuronal enrichment of the cultures reaching 56% after 20 days of culture. P19-derived neural progenitor cells progressively expressed neuronal markers such as NeuN, Calretinin, Calbindin and Synapsin I in close resemblance to that occurring in vivo in the central nervous system (CNS). Furthermore, RA-induced P19 EC cells progressively acquired functional neuronal traits and after approximately 3 weeks in culture revealed mature neurophysiological properties, characteristics of CNS neurons. This protocol allows for a more specific assessment of the neuronal differentiation processes in vitro.     

Astrocyte lineage cells are essential for functional neuronal differentiation and synapse maturation in human iPSC-derived neural networks

Human astrocytes differ dramatically in cell morphology and gene expression from murine astrocytes. The latter are well known to be of major importance in the formation of neuronal networks by promoting synapse maturation. However, whether human astrocyte lineage cells have a similar role in network formation has not been firmly established. Here, we investigated the impact of human astrocyte lineage cells on the functional maturation of neural networks that were derived from human induced pluripotent stem cells (hiPSCs). Initial in vitro differentiation of hiPSC-derived neural progenitor cells and immature neurons (glia+ cultures) resulted in spontaneously active neural networks as indicated by synchronous neuronal Ca²⁺ transients. Depleting proliferating neural progenitors from these cultures by short-term antimitotic treatment resulted in strongly astrocyte lineage cell-depleted neuronal networks (glia− cultures). Strikingly, in contrast to glia+ cultures, glia− cultures did not exhibit spontaneous network activity. Detailed analysis of the morphological and electrophysiological properties of neurons by patch clamp recordings revealed reduced dendritic arborization in glia− cultures. In addition, a reduced action potential frequency upon current injection in pyramidal-like neurons was observed, whereas the electrical excitability of multipolar neurons was unaltered. Furthermore, we found a reduced dendritic density of PSD95-positive excitatory synapses, and more immature properties of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) miniature excitatory postsynaptic currents (mEPSCs) in glia− cultures, suggesting that the maturation of glutamatergic synapses depends on the presence of hiPSC-derived astrocyte lineage cells. Intriguingly, addition of the astrocyte-derived synapse maturation inducer cholesterol increased the dendritic density of PSD95-positive excitatory synapses in glia− cultures.     

Neural stem cell deforestation as the main force driving the age-related decline in adult hippocampal neurogenesis

Newborn neurons derived from radial glia-like stem cells located in the dentate gyrus integrate into the adult hippocampal circuitry and participate in memory formation, spatial learning, pattern separation, fear conditioning, and anxiety. This process takes place throughout the life span of mammals, including humans; however, it follows a sharp declining curve. New neurons are generated abundantly during youth but very scarcely in the aged brain. The absolute number of newly generated neurons, or neurogenic output, is determined at different levels along the neurogenic cascade: the activation of quiescent stem cells; the mitotic potential of proliferating precursors; and the survival of neuronal fate-committed precursors. A continuous depletion of the hippocampal neural stem cell pool has been recently proposed as the main force underlying the age-related decline of neurogenesis, in contrast to the previous view of population of neural stem cells whose number remains constant but loses its ability to bear fruit. Nevertheless, the diminished neurogenic output may be reflecting other phenomena such as decreased mitotic capability of proliferating progenitors, decreased survival or changes in differentiation. We describe herein the most important events in determining the amount of neurogenesis in the dentate gyrus and examine the literature to understand the effects of age throughout the cascade.     

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Background

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation.

Methods

In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined.

Results

Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6.

Conclusions

Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.     

HDAC inhibitors dysregulate neural stem cell activity in the postnatal mouse brain

The mammalian central nervous system (CNS) undergoes significant expansion postnatally, producing astrocytes, oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy, a condition commonly treated with valproate/valproic acid (VPA), a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs, however, may be essential for the differentiation of distinct subpopulations of neurons and glia. Here, we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7–P14, combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG, rostral migratory stream (RMS), and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed, however, that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA, OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity, we found that short term treatment with VPA in vivo reduced neurosphere numbers and size, a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor, Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively, these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered, especially in individuals whose brains are actively undergoing key postnatal time windows of development.