Embryonic Neural Precursor Cells Research Articles

Elevated ROS levels during the early development of Angelman syndrome alter the apoptotic capacity of the developing neural precursor cells

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder caused by the maternally inherited loss of function of the UBE3A gene. AS is characterized by a developmental delay, lack of speech, motor dysfunction, epilepsy, autistic features, happy demeanor, and intellectual disability. While the cellular roles of UBE3A are not fully understood, studies suggest that the lack of UBE3A function is associated with elevated levels of reactive oxygen species (ROS). Despite the accumulating evidence emphasizing the importance of ROS during early brain development and its involvement in different neurodevelopmental disorders, up to date, the levels of ROS in AS neural precursor cells (NPCs) and the consequences on AS embryonic neural development have not been elucidated. In this study we show multifaceted mitochondrial aberration in AS brain-derived embryonic NPCs, which exhibit elevated mitochondrial membrane potential (ΔΨm), lower levels of endogenous reduced glutathione, excessive mitochondrial ROS (mROS) levels, and increased apoptosis compared to wild-type (WT) littermates. In addition, we report that glutathione replenishment by glutathione-reduced ethyl ester (GSH-EE) corrects the excessive mROS levels and attenuates the enhanced apoptosis in AS NPCs. Studying the glutathione redox imbalance and mitochondrial abnormalities in embryonic AS NPCs provides an essential insight into the involvement of UBE3A in early neural development, information that can serve as a powerful avenue towards a broader view of AS pathogenesis. Moreover, since mitochondrial dysfunction and elevated ROS levels were associated with other neurodevelopmental disorders, the findings herein suggest some potential shared underlying mechanisms for these disorders as well.

Three-dimensional differentiation of human pluripotent stem cell-derived neural precursor cells using tailored porous polymer scaffolds

This study investigates the utility of a tailored poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This tailored construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs. Statement of significanceThe interconnected porosity of polyHIPE scaffolds exhibits the ability to support three-dimensional neural cell network formation due to limited resistance to cellular migration and re-organisation. The previously developed scaffold material displays mechanical properties similar to that of the mammalian brain. This research also employs the utility of pluripotent stem cell-derived neural cells which are of greater clinical relevance than primary neural cell lines. This scaffold material has future potential in better mimicking three-dimensional neural networks found in the human brain and may result in improved in vitro models for disease modelling and drug screening applications.

Three-Dimensional Differentiation of Human Pluripotent Stem Cell-Derived Neural Precursor Cells Using Tailored Porous Polymer Scaffolds

This study investigates the utility of a novel poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This novel construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs.

Distinctive features of lincRNA gene expression suggest widespread RNA-independent functions.

Eukaryotic genomes produce RNAs lacking protein-coding potential, with enigmatic roles. We integrated three approaches to study large intervening noncoding RNA (lincRNA) gene functions. First, we profiled mouse embryonic stem cells and neural precursor cells at single-cell resolution, revealing lincRNAs expressed in specific cell types, cell subpopulations, or cell cycle stages. Second, we assembled a transcriptome-wide atlas of nuclear lincRNA degradation by identifying targets of the exosome cofactor Mtr4. Third, we developed a reversible depletion system to separate the role of a lincRNA gene from that of its RNA. Our approach distinguished lincRNA loci functioning in trans from those modulating local gene expression. Some genes express stable and/or abundant lincRNAs in single cells, but many prematurely terminate transcription and produce lincRNAs rapidly degraded by the nuclear exosome. This suggests that besides RNA-dependent functions, lincRNA loci act as DNA elements or through transcription. Our integrative approach helps distinguish these mechanisms.

A novel Alzheimer\u2019s disease drug candidate targeting inflammation and fatty acid metabolism

BackgroundCAD-31 is an Alzheimer’s disease (AD) drug candidate that was selected on the basis of its ability to stimulate the replication of human embryonic stem cell-derived neural precursor cells as well as in APPswe/PS1ΔE9 AD mice. To move CAD-31 toward the clinic, experiments were undertaken to determine its neuroprotective and pharmacological properties, as well as to assay its therapeutic efficacy in a rigorous mouse model of AD.ResultsCAD-31 has potent neuroprotective properties in six distinct nerve cell assays that mimic toxicities observed in the old brain. Pharmacological and preliminary toxicological studies show that CAD-31 is brain-penetrant and likely safe. When fed to old, symptomatic APPswe/PS1ΔE9 AD mice starting at 10 months of age for 3 additional months in a therapeutic model of the disease, there was a reduction in the memory deficit and brain inflammation, as well as an increase in the expression of synaptic proteins. Small-molecule metabolic data from the brain and plasma showed that the major effect of CAD-31 is centered on fatty acid metabolism and inflammation. Pathway analysis of gene expression data showed that CAD-31 had major effects on synapse formation and AD energy metabolic pathways.ConclusionsAll of the multiple physiological effects of CAD-31 were favorable in the context of preventing some of the toxic events in old age-associated neurodegenerative diseases.

Abstract 3006: Oncogenic activity of H3.3 K27M in a spontaneous DIPG mouse model

Abstract Pediatric diffuse high-grade gliomas (HGG) cause a devastatingly poor outcome. Diffuse intrinsic pontine gliomas (DIPG) are brainstem HGGs found almost exclusively in children and represent approximately 50 percent of all pediatric HGGs. Recurrent, clonal somatic mutations in histone H3 are a molecular hallmark that distinguish the genetic mechanisms underlying pediatric and adult HGG, and indicate a strong link between epigenetic dysfunction and pediatric brain tumorigenesis. H3 K27M mutations found in nearly 80 percent of DIPGs and over half of non-brainstem HGGs occurring in thalamus or other midline structures, induce a dominant loss of genome-wide H3K27me3. In contrast, mutually exclusive H3.3 G34R/V mutations occur in approximately 14 percent of cortical HGG tumors in older children through young adulthood suggesting a distinct developmental origin compared to K27M mutant tumors. DNA methylation and gene expression patterns distinguish G34R/V and K27M tumors, but the oncogenic mechanisms and reasons for brain region selectivity of histone mutations remain uncertain. To study these mutations in the developing mammalian brain and investigate how they contribute to oncogenesis, we generated conditionally activated, epitope-tagged knock-in mice to express K27M, G34R or non-mutated H3.3 proteins from the endogenous H3f3a locus. Untransformed embryonic neural precursor or astrocyte cells expressing H3.3 K27M demonstrated H3K27me3 loss and a growth advantage in vitro with brain region-specific, stage-dependent expression and epigenetic signatures. DIPGs frequently harbor genetic alterations in TP53 and PDGFRα in addition to H3.3 K27M, and mice with induced brain-specific Trp53 deletion plus mutated PDGFRαV544ins expression developed spontaneous HGG in vivo. Induction of H3.3 K27M significantly accelerated PDGFRαV544ins;Trp53 knockout HGG formation involving the brainstem. Importantly, the non-mutated H3.3 epitope-tagged protein did not affect tumor burden or latency. The H3.3 K27M tumors recapitulate characteristics observed in human DIPG including histopathological features, strong nuclear K27M expression and robust loss of H3K27me3 throughout the tumors. We relate the genetic, epigenetic and transcriptional landscapes of these models to primary pediatric DIPG to help identify their developmental origins and oncogenic mechanisms downstream of histone H3.3 K27M. Citation Format: Jon D. Larson, Lawryn H. Kasper, Gang Wu, Yiping Fan, David Finkelstein, Hongjian Jin, Barbara S. Paugh, Andre B. Silveira, Beisi Xu, Xiaoyan Zhu, Junyuan Zhang, Helen R. Russell, Peter J. McKinnon, David W. Ellison, Jinghui Zhang, Suzanne J. Baker. Oncogenic activity of H3.3 K27M in a spontaneous DIPG mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2017-3006

C-jun is differentially expressed in embryonic and adult neural precursor cells.

c-jun, a major component of AP-1 transcription factor, has a wide variety of functions. In the embryonic brain, c-jun mRNA is abundantly expressed in germinal layers around the ventricles. Although the subventricular zone (SVZ) of the adult brain is a derivative of embryonic germinal layers and contains neural precursor cells (NPCs), the c-jun expression pattern is not clear. To study the function of c-jun in adult neurogenesis, we analyzed c-jun expression in the adult SVZ by immunohistochemistry and compared it with that of the embryonic brain. We found that almost all proliferating embryonic NPCs expressed c-jun, but the number of c-jun immunopositive cells among proliferating adult NPCs was about half. In addition, c-jun was hardly expressed in post-mitotic migrating neurons in the embryonic brain, but the majority of c-jun immunopositive cells were tangentially migrating neuroblasts heading toward the olfactory bulb in the adult brain. In addition, status epilepticus is known to enhance the transient proliferation of adult NPCs, but the c-jun expression pattern was not significantly affected. These expression patterns suggest that c-jun has a pivotal role in the proliferation of embryonic NPCs, but it has also other roles in adult neurogenesis.

Reciprocal insulation analysis of Hi-C data shows that TADs represent a functionally but not structurally privileged scale in the hierarchical folding of chromosomes.

Understanding how regulatory sequences interact in the context of chromosomal architecture is a central challenge in biology. Chromosome conformation capture revealed that mammalian chromosomes possess a rich hierarchy of structural layers, from multi-megabase compartments to sub-megabase topologically associating domains (TADs) and sub-TAD contact domains. TADs appear to act as regulatory microenvironments by constraining and segregating regulatory interactions across discrete chromosomal regions. However, it is unclear whether other (or all) folding layers share similar properties, or rather TADs constitute a privileged folding scale with maximal impact on the organization of regulatory interactions. Here, we present a novel algorithm named CaTCH that identifies hierarchical trees of chromosomal domains in Hi-C maps, stratified through their reciprocal physical insulation, which is a single and biologically relevant parameter. By applying CaTCH to published Hi-C data sets, we show that previously reported folding layers appear at different insulation levels. We demonstrate that although no structurally privileged folding level exists, TADs emerge as a functionally privileged scale defined by maximal boundary enrichment in CTCF and maximal cell-type conservation. By measuring transcriptional output in embryonic stem cells and neural precursor cells, we show that the likelihood that genes in a domain are coregulated during differentiation is also maximized at the scale of TADs. Finally, we observe that regulatory sequences occur at genomic locations corresponding to optimized mutual interactions at the same scale. Our analysis suggests that the architectural functionality of TADs arises from the interplay between their ability to partition interactions and the specific genomic position of regulatory sequences.

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology

ABSTRACTWe sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small‐diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large‐fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole‐ and cytochalasin‐D–treated neural precursor cells in large‐fiber topography, but was not changed in small‐fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485–3502, 2016. © 2016 Wiley Periodicals, Inc.

A Glo1-Methylglyoxal Pathway that Is Perturbed in Maternal Diabetes Regulates Embryonic and Adult Neural Stem Cell Pools in Murine Offspring

Maternal diabetes is known to adversely influence brain development in offspring. Here, we provide evidence that this involves the circulating metabolite methylglyoxal, which is increased in diabetes, and its detoxifying enzyme, glyoxalase 1 (Glo1), which when mutated is associated with neurodevelopmental disorders. Specifically, when Glo1 levels were decreased in embryonic mouse cortical neural precursor cells (NPCs), this led to premature neurogenesis and NPC depletion embryonically and long-term alterations in cortical neurons postnatally. Increased circulating maternal methylglyoxal caused similar changes in embryonic cortical precursors andneurons and long-lasting changes in cortical neurons and NPCs in adult offspring. Depletion of embryonic and adult NPCs was also observed in murine offspring exposed to a maternal diabetic environment. Thus, the Glo1-methylglyoxal pathway integrates maternal and NPC metabolism to regulateneural development, and perturbations in this pathway lead to long-lasting alterations in adult neurons and NPC pools.

Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells.

Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response.

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.

Knockout of Atg5 delays the maturation and reduces the survival of adult-generated neurons in the hippocampus.

Autophagy is an evolutionarily conserved lysosomal degradation pathway that plays important roles in cell maintenance, expansion and differentiation. Removal of genes essential for autophagy from embryonic neural stem and precursor cells reduces the survival and inhibits neuronal differentiation of adult-generated neurons. No study has modified autophagy within the adult precursor cells, leaving the cell-autonomous role of autophagy in adult neurogenesis unknown. Here we demonstrate that autophagic flux exists in the adult dividing progenitor cells and their progeny in the dentate gyrus. To investigate the role of autophagy in adult hippocampal neurogenesis, we genetically deleted Autophagy-related gene 5 (Atg5) that reduced autophagic flux and the survival of the progeny of dividing progenitor cells. This significant reduction in survival of adult-generated neurons is accompanied by a delay in neuronal maturation, including a transient reduction in spine density in the absence of a change in differentiation. The delay in cell maturation and loss of progeny of the Atg5-null cells was not present in mice that lacked the essential pro-apoptotic protein Bax (Bcl-2-associated X protein), suggesting that Atg5-deficient cells die through a Bax-dependent mechanism. In addition, there was a loss of Atg5-null cells following exposure to running, suggesting that Atg5 is required for running-induced increases in neurogenesis. These findings highlight the cell-autonomous requirement of Atg5 in the survival of adult-generated neurons.

Intrathecal Transplantation of Embryonic Stem Cell-Derived Spinal GABAergic Neural Precursor Cells Attenuates Neuropathic Pain in a Spinal Cord Injury Rat Model.

Neuropathic pain following spinal cord injury (SCI) is a devastating disease characterized by spontaneous pain such as hyperalgesia and allodynia. In this study, we investigated the therapeutic potential of ESC-derived spinal GABAergic neurons to treat neuropathic pain in a SCI rat model. Mouse embryonic stem cell-derived neural precursor cells (mESC-NPCs) were cultured in media supplemented with sonic hedgehog (SHH) and retinoic acid (RA) and efficiently differentiated into GABAergic neurons. Interestingly, low doses of SHH and RA induced MGE-like progenitors, which expressed low levels of DARPP32 and Nkx2.1 and high levels of Irx3 and Pax6. These cells subsequently generated the majority of the DARPP32(-) GABAergic neurons after in vitro differentiation. The spinal mESC-NPCs were intrathecally transplanted into the lesion area of the spinal cord around T10-T11 at 21 days after SCI. The engrafted spinal GABAergic neurons remarkably increased both the paw withdrawal threshold (PWT) below the level of the lesion and the vocalization threshold (VT) to the level of the lesion (T12, T11, and T10 vertebrae), which indicates attenuation of chronic neuropathic pain by the spinal GABAergic neurons. The transplanted cells were positive for GABA antibody staining in the injured region, and cells migrated to the injured spinal site and survived for more than 7 weeks in L4-L5. The mESC-NPC-derived spinal GABAergic neurons dramatically attenuated the chronic neuropathic pain following SCI, suggesting that the spinal GABAergic mESC-NPCs cultured with low doses of SHH and RA could be alternative cell sources for treatment of SCI neuropathic pain by stem cell-based therapies.

Bradykinin-induced inhibition of proliferation rate during neurosphere differentiation: consequence or cause of neuronal enrichment?

Neural stem cells proliferate and differentiate into neurons and glial cells, being responsible for embryonic and postnatal development of the central nervous system (CNS) as well as for regeneration in the adult brain. These cells also play a key role in maintaining the physiological integrity of the CNS in face of injury or disease. The previous study has demonstrated that bradykinin (BK) treatment simultaneously induces neuronal enrichment (indicating that BK contributes to neurogenesis) and reduced proliferation rates during in vitro differentiation of rat embryonic telencephalon neural precursor cells (NPCs). Here, we provide a mechanism for the unresolved question whether (i) the low rate of proliferation is owed to enhanced neurogenesis or, conversely, (ii) the alteration of the population ratio could result from low proliferation of NPCs and glial cells. In agreement with the previous study, BK promoted neuron-specific β3-tubulin and MAP2 expression in differentiating embryonic mouse neurospheres, whereas glial protein expression and global proliferation rates decreased. Furthermore, BK augmented the global frequency of cells in G0 -phase of cell cycle after differentiation. Heterogeneous cell populations were observed at this stage, including neurons that always remaining a quiescent state (G0 -phase). It is noteworthy that BK did not interfere with proliferation of any particular cell type, evidenced by coimmunostaining for nestin, β3-tubulin, glial fibrillary acidic protein (GFAP), and 5-ethynyl-2'-deoxyuridine (EdU). Thus, we conclude that neuronal enrichment is owing only to the fostering of neurogenesis, and that the low proliferation rate on the seventh day of differentiation is a consequence and not the cause of BK-induced neuronal enrichment.

PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone.

The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis.

Fluoxetine regulates neurogenesis in vitro through modulation of GSK-3β/β-catenin signaling.

Background:It is generally accepted that chronic treatment with antidepressants increases hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. Recently, glycogen synthase kinase-3 beta (GSK-3β)/β-catenin signaling was shown to be involved in the mechanism of how antidepressants might influence hippocampal neurogenesis.Methods:The aim of this study was to determine whether GSK-3β/β-catenin signaling is involved in the alteration of neurogenesis as a result of treatment with fluoxetine, a selective serotonin reuptake inhibitor. The mechanisms involved in fluoxetine’s regulation of GSK-3β/β-catenin signaling pathway were also examined.Results:Our results demonstrated that fluoxetine increased the proliferation of embryonic neural precursor cells (NPCs) by up-regulating the phosphorylation of Ser9 on GSK-3β and increasing the level of nuclear β-catenin. The overexpression of a stabilized β-catenin protein (ΔN89 β-catenin) significantly increased NPC proliferation, while inhibition of β-catenin expression in NPCs led to a significant decrease in the proliferation and reduced the proliferative effects induced by fluoxetine. The effects of fluoxetine-induced up-regulation of both phosphorylation of Ser9 on GSK-3β and nuclear β-catenin were significantly prevented by the 5-hydroxytryptamine-1A (5-HT1A) receptor antagonist WAY-100635.Conclusions:The results demonstrate that fluoxetine may increase neurogenesis via the GSK-3β/β-catenin signaling pathway that links postsynaptic 5-HT1A receptor activation.

Human Neural Precursor Cells Promote Neurologic Recovery in a Viral Model of Multiple Sclerosis

SummaryUsing a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments.

MYCN gene expression is required for the onset of the differentiation programme in neuroblastoma cells

Neuroblastoma is an embryonic tumour of the sympathetic nervous system and is one of the most common cancers in childhood. A high differentiation stage has been associated with a favourable outcome; however, the mechanisms governing neuroblastoma cell differentiation are not completely understood. The MYCN gene is considered the hallmark of neuroblastoma. Even though it has been reported that MYCN has a role during embryonic development, it is needed its decrease so that differentiation can be completed. We aimed to better define the role of MYCN in the differentiation processes, particularly during the early stages. Considering the ability of MYCN to regulate non-coding RNAs, our hypothesis was that N-Myc protein might be necessary to activate differentiation (mimicking embryonic development events) by regulating miRNAs critical for this process. We show that MYCN expression increased in embryonic cortical neural precursor cells at an early stage after differentiation induction. To investigate our hypothesis, we used human neuroblastoma cell lines. In LAN-5 neuroblastoma cells, MYCN was upregulated after 2 days of differentiation induction before its expected downregulation. Positive modulation of various differentiation markers was associated with the increased MYCN expression. Similarly, MYCN silencing inhibited such differentiation, leading to negative modulation of various differentiation markers. Furthermore, MYCN gene overexpression in the poorly differentiating neuroblastoma cell line SK-N-AS restored the ability of such cells to differentiate. We identified three key miRNAs, which could regulate the onset of differentiation programme in the neuroblastoma cells in which we modulated MYCN. Interestingly, these effects were accompanied by changes in the apoptotic compartment evaluated both as expression of apoptosis-related genes and as fraction of apoptotic cells. Therefore, our idea is that MYCN is necessary during the activation of neuroblastoma differentiation to induce apoptosis in cells that are not committed to differentiate.

Antagonistic Interplay between Necdin and Bmi1 Controls Proliferation of Neural Precursor Cells in the Embryonic Mouse Neocortex

Neural precursor cells (NPCs) in the neocortex exhibit a high proliferation capacity during early embryonic development and give rise to cortical projection neurons after maturation. Necdin, a mammal-specific MAGE (melanoma antigen) family protein that possesses anti-mitotic and pro-survival activities, is expressed abundantly in postmitotic neurons and moderately in tissue-specific stem cells or progenitors. Necdin interacts with E2F transcription factors and suppresses E2F1-dependent transcriptional activation of the cyclin-dependent kinase Cdk1 gene. Here we show that necdin serves as a suppressor of NPC proliferation in the embryonic neocortex. Necdin is moderately expressed in the ventricular zone of mouse embryonic neocortex, in which proliferative cell populations are significantly increased in necdin-null mice. In the neocortex of necdin-null embryos, expression of Cdk1 and Sox2, a stem cell marker, is significantly increased, whereas expression of p16, a cyclin-dependent kinase inhibitor, is markedly diminished. Cdk1 and p16 expression levels are also significantly increased and decreased, respectively, in primary NPCs prepared from necdin-null embryos. Intriguingly, necdin interacts directly with Bmi1, a Polycomb group protein that suppresses p16 expression and promotes NPC proliferation. In HEK293A cells transfected with luciferase reporter constructs, necdin relieves Bmi1-dependent repression of p16 promoter activity, whereas Bmi1 counteracts necdin-mediated repression of E2F1-dependent Cdk1 promoter activity. In lentivirus-infected primary NPCs, necdin overexpression increases p16 expression, suppresses Cdk1 expression, and inhibits NPC proliferation, whereas Bmi1 overexpression suppresses p16 expression, increases Cdk1 expression, and promotes NPC proliferation. Our data suggest that embryonic NPC proliferation in the neocortex is regulated by the antagonistic interplay between necdin and Bmi1.