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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally.Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue.We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks.Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.

Microglia are the resident immune cells of nervous system. In healthy conditions, microglia actively patrol neural tissues in a homeostatic state, which can rapidly change to an activated state in response to local injury/disease. Dysregulated microglia activation is a hallmark of disorders and diseases of the nervous system, including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The elucidation of the roles of microglia in human biology and disease has recently benefitted from the development of human induced pluripotent stem cell (iPSC)-based approaches to generate microglia-like cells. Microglia represent a heterogeneous group of cells with spatial diversity in both health and disease. This situation poses a considerable challenge along the path towards establishing the most pathologically-relevant human iPSC-derived microglia preparations to investigate the complex roles of microglia in ALS and other neurological diseases. The success of these approaches must account for microglia diversity in different regions of the brain and spinal cord. In this study, we compared the transcriptomes of human iPSC-derived microglia generated using different methods to determine whether or not separate strategies can be used to generate microglia with distinct transcriptional signatures in vitro. We show that microglia derived using two different methods display distinct in vitro maturation characteristics. We also reveal that different derivation methods give rise to preparations comprising human microglia with distinct transcriptomic signatures resembling the gene profiles of specific microglia subpopulations in vivo. These findings suggest that a careful, and coordinated, implementation of multiple microglia differentiation methods from human iPSCs can be an effective approach towards the goal of generating multiple microglia subtypes that will offer enhanced model systems to account for microglia heterogeneity in vivo. Spatially-defined human iPSC-derived microglia would represent an enhanced tool to study the multiple levels of involvement of microglia in mechanisms of motor neuron degeneration in ALS, as well as other neurological diseases and disorders.

Human induced pluripotent stem cells (hiPSCs) derived from healthy and diseased individuals can give rise to many cell types, facilitating the study of mechanisms of development, human disease modeling, and early drug target validation. In this context, experimental model systems based on hiPSC-derived motor neurons (MNs) have been used to study MN diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. Modeling MN disease using hiPSC-based approaches requires culture conditions that can recapitulate in a dish the events underlying differentiation, maturation, aging, and death of MNs. Current hiPSC-derived MN-based applications are often hampered by limitations in our ability to monitor MN morphology, survival, and other functional properties over a prolonged timeframe, underscoring the need for improved long-term culture conditions. Here we describe a cytocompatible dendritic polyglycerol amine (dPGA) substrate-based method for prolonged culture of hiPSC-derived MNs. We provide evidence that MNs cultured on dPGA-coated dishes are more amenable to long-term study of cell viability, molecular identity, and spontaneous network electrophysiological activity. The present study has the potential to improve hiPSC-based studies of human MN biology and disease.We describe the use of a new coating substrate providing improved conditions for long-term cultures of human iPSC-derived motor neurons, thus allowing evaluation of cell viability, molecular identity, spontaneous network electrophysiological activity, and single-cell RNA sequencing of mature motor neurons.

Understanding the rules that govern neuronal dynamics throughout the brain to subserve behavior and cognition remains one of the biggest challenges in neuroscience research. Recent technical advances enable the recording of increasingly larger neuronal populations to produce increasingly more sophisticated datasets. Despite bold and important open-science and data-sharing policies, these datasets tend to include unique data acquisition methods, behaviors, and file structures. Discrepancies between experimental protocols present key challenges in comparing data between laboratories and across different brain regions and species. Here, we discuss our recent efforts to create a standardized and high-throughput research platform to address these issues. The McGill-Mouse-Miniscope (M3) platform is an initiative to combine miniscope calcium imaging with standardized touchscreen-based animal behavioral testing. The goal is to curate an open-source and standardized framework for acquiring, analyzing, and accessing high-quality data of the neuronal dynamics that underly cognition throughout the brain in mice, marmosets, and models of disease. We end with a discussion of future developments and a call for users to adopt this standardized approach.

Kulasekaran et al. discover a novel GTPase cascade in which Arf5 activates Rab35. Disruption of this pathway increases EGF receptor activation, leading to upregulation of the transcription factor SPOCD1 with enhanced tumor growth and invasion in glioblastoma., Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.

Nuclear factor-kappaB (NF-κB) is activated in neural progenitor cells in the developing murine cerebral cortex during the neurogenic phase, when it acts to prevent premature neuronal differentiation. Here we show that NF-κB activation continues in mouse neocortical neural progenitor cells during the neurogenic-to-gliogenic switch. Blockade of endogenous NF-κB activity during neocortical gliogenesis leads to the formation of supernumerary committed gliogenic progenitors and premature glial cell differentiation. Conversely, forced NF-κB activation during the neocortical neurogenic-to-gliogenic transition causes delayed gliogenic commitment and decreased astroglial gene expression. NF-κB activation continues in neocortical gliogenic progenitors following commitment and is important to inhibit the differentiation of oligodendrocyte precursor cells and to maintain persistent expression of glial fibrillary acidic protein in maturing astrocytes. These results reveal a number of previously uncharacterized roles for NF-κB during different phases of neocortical gliogenesis and identify NF-κB as an inhibitor of early oligodendrocyte development in the cerebral cortex.

Astrocytes play a number of key functions in health and disease. Reactivation of astrocytes occurs in most, if not all, neurological diseases. Most current information on the mechanisms underlying astrogliosis derives from studies using rodent experimental systems, mainly because the ability to study human astrocytes under healthy and pathological conditions has been hampered by the difficulty in obtaining primary human astrocytes. Here we describe robust and reliable derivation of astrocytes from human induced pluripotent stem cells (iPSCs). Phenotypically characterized human iPSC-derived astrocytes exhibit typical traits of physiological astrocytes, including spontaneous and induced calcium transients. Moreover, human iPSC-derived astrocytes respond to stimulation with a pro-inflammatory combination of tumor necrosis factor alpha, interleukin 1-alpha, and complement component C1q by undergoing changes in gene expression patterns suggesting acquisition of a reactive astrocyte phenotype. Together, these findings provide evidence suggesting that human iPSC-derived astrocytes are a suitable experimental model system to study astrocyte function and reactivation in healthy and pathological conditions of the human nervous system.

Understanding the rules that govern neuronal dynamics throughout the brain to subserve behavior and cognition remain one of the biggest challenges in neuroscience research. Recent technical advances enable the recording of increasingly larger neuronal populations to produce increasingly more sophisticated datasets. Despite bold and important open-science and data-sharing policies, these datasets tend to include unique data acquisition methods, behavior, and file structures. Discrepancies between experimental protocols present several key challenges including the analysis of the data itself, comparison of data collected between laboratories, and for the comparison of dynamics between brain regions and species. Here, we discuss our recent efforts to create a standardized and high-throughput research platform to address these issues. The McGill-Mouse-Marmoset (M3) platform combines miniscope calcium imaging recording in both mice and marmosets with standardized touchscreen-based behavioral testing. The goal is to curate an open-source and standardized framework for acquiring, analyzing, and accessing high-quality data of the neuronal dynamics that underly cognition throughout the brain in mice, marmosets, and models of disease. We end with a discussion of future developments and a call for users to adopt this standardized approach.

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, with a number of molecularly distinct subpopulations present within this MN group. Roughly 19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of median motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and disease.

Human induced pluripotent stem cells (iPSCs) offer the opportunity to generate specific cell types from healthy and diseased individuals, allowing the study of mechanisms of early human development, modelling a variety of human diseases, and facilitating the development of new therapeutics. Human iPSC-based applications are often limited by the variability among iPSC lines originating from a single donor, as well as the heterogeneity among specific cell types that can be derived from iPSCs. The ability to deeply phenotype different iPSC-derived cell types is therefore of primary importance to the successful and informative application of this technology. Here we describe a combination of motor neuron (MN) derivation and single-cell RNA sequencing approaches to generate and characterize specific MN subtypes obtained from human iPSCs. Our studies provide evidence for rapid and robust generation of MN progenitor cells that can give rise to a heterogenous population of brainstem and spinal cord MNs. Approximately 58% of human iPSC-derived MNs display molecular characteristics of lateral motor column MNs, ∼19% of induced MNs resemble hypaxial motor column MNs, while ∼6% of induced MNs have features of medial motor column MNs. The present study has the potential to improve our understanding of iPSC-derived MN subtype function and dysfunction, possibly leading to improved iPSC-based applications for the study of human MN biology and diseases.

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem‐like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial–mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome‐wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem‐like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells in vivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

Although inhibition of p16INK4a expression is critical to preserve the proliferative capacity of stem cells, the molecular mechanisms responsible for silencing p16INK4a expression remain poorly characterized. Here, we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16INK4a. In the absence of the HAT activity of MOZ, expression of p16INK4a is upregulated in progenitor and stem cells, inducing an early entrance into replicative senescence. Genetic deletion of p16INK4a reverses the proliferative defect in both MozHAT−/− hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16INK4a expression and to protect stem cells from early entrance into replicative senescence. Stem Cells 2014;32:1591–1601

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Runt-related (Runx) transcription factors play essential roles during development and adult tissue homeostasis and are responsible for several human diseases. They regulate a variety of biological mechanisms in numerous cell lineages. Recent years have seen significant progress in our understanding of the functions performed by Runx proteins in the developing and postnatal mammalian nervous system. In both central and peripheral nervous systems, Runx1 and Runx3 display remarkably specific expression in mostly non-overlapping groups of postmitotic neurons. In the central nervous system, Runx1 is involved in the development of selected motor neurons controlling neural circuits mediating vital functions such as chewing, swallowing, breathing, and locomotion. In the peripheral nervous system, Runx1 and Runx3 play essential roles during the development of sensory neurons involved in circuits mediating pain, itch, thermal sensation and sense of relative position. Runx1 and Runx3 orchestrate complex gene expression programs controlling neuronal subtype specification and axonal connectivity. Runx1 is also important in the olfactory system, where it regulates the progenitor-to-neuron transition in undifferentiated neural progenitor cells in the olfactory epithelium as well as the proliferation and developmental maturation of specific glial cells termed olfactory ensheathing cells. Moreover, upregulated Runx expression is associated with brain injury and disease. Increasing knowledge of the functions of Runx proteins in the developing and postnatal nervous system is therefore expected to improve our understanding of nervous system development, homeostasis and disease.

Background and Purpose: Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4–myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined. Experimental Approach: Binding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4–MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells. Key Results: Both curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of pro-inflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4–MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9. Conclusions and Implications: These results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPS-mediated TLR4–MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS.

Runt-related (Runx) transcription factors play essential roles during development and adult tissue homeostasis and are responsible for several human diseases. They regulate a variety of biological mechanisms in numerous cell lineages. Recent years have seen significant progress in our understanding of the functions performed by Runx proteins in the developing and postnatal mammalian nervous system. In both central and peripheral nervous systems, Runx1 and Runx3 display remarkably specific expression in mostly non-overlapping groups of postmitotic neurons. In the central nervous system, Runx1 is involved in the development of selected motor neurons controlling neural circuits mediating vital functions such as chewing, swallowing, breathing, and locomotion. In the peripheral nervous system, Runx1 and Runx3 play essential roles during the development of sensory neurons involved in circuits mediating pain, itch, thermal sensation and sense of relative position. Runx1 and Runx3 orchestrate complex gene expression programs controlling neuronal subtype specification and axonal connectivity. Runx1 is also important in the olfactory system, where it regulates the progenitor-to-neuron transition in undifferentiated neural progenitor cells in the olfactory epithelium as well as the proliferation and developmental maturation of specific glial cells termed olfactory ensheathing cells. Moreover, upregulated Runx expression is associated with brain injury and disease. Increasing knowledge of the functions of Runx proteins in the developing and postnatal nervous system is therefore expected to improve our understanding of nervous system development, homeostasis and disease.

Transducin-like enhancer of split-1 (TLE1) plays a critical role in the regulation of neurogenesis by inhibiting the differentiation of neural progenitor cells into neurons. Although TLE1 is also expressed highly in the postnatal brain and through adulthood, its role in postmitotic neurons is not clear. Using cultures of cerebellar granule neurons, we show that expression of TLE1 is reduced in neurons primed to die. Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas suppression of TLE1 expression in otherwise healthy neurons induces cell death. These results show that TLE1 is necessary for the maintenance of neuronal survival. Experiments using pharmacological inhibitors as well as expression of point mutants indicate that phosphorylation of TLE1 by casein kinase-2 (CK2) at Ser-239 and Ser-253 is necessary for its survival-promoting activity. TLE1-mediated survival is also inhibited by pharmacological inhibition of PI3K-Akt signaling but not by inhibitors of Raf-MEK-ERK signaling or other molecules, including histone deacetylases, calcium calmodulin kinase, or CK1. The survival-promoting activity of TLE1 depends critically on interaction with FoxG1, another protein involved in the regulation of neurogenesis and shown previously to promote survival of postmitotic neurons. Likewise, the ability of FoxG1 to promote neuronal survival depends on TLE1. Taken together, our study demonstrates that TLE1 cooperates with FoxG1 to promote neuronal survival in a CK2- and PI3K-Akt-dependent manner.

International audience; Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS. Cell Death and Differentiation (2012) 19, 871-881; doi:10.1038/cdd.2011.171; published online 25 November 2011

Signaling mechanisms mediated by the Transforming Growth Factor-β (TGF-β) superfamily regulate a variety of developmental processes. Here we show that components of both bone morphogenetic protein/growth differentiation factor and TGF-β/activin/Nodal branches of TGF-β superfamily signaling are expressed in the developing subpallium. Furthermore, Smad proteins, transcriptional effectors of TGF-β signaling, are co-expressed and physically interact in the basal ganglia with Dlx homeodomain transcription factors, which are critical regulators of the differentiation, migration and survival of telencephalic GABAergic neurons. We also show that Dlx and Smad proteins localize to promoters/enhancers of a number of common telencephalic genes in vivo and that Smad proteins co-activate transcription with Dlx family members, except with certain mutated human DLX proteins identified in autistic individuals. In agreement with these observations, expression of dominant-negative Smads in the developing basal ganglia phenocopies the cell migration defects observed in Dlx1/2-deficient mice. Together, these results suggest that TGF-β superfamily signaling plays a role in telencephalic GABAergic neuron development through functional interactions with Dlx transcription factors. Electronic supplementary material The online version of this article (doi:10.1007/s11689-009-9035-6) contains supplementary material, which is available to authorized users.

Runt-related (RUNX) transcription factors are evolutionarily conserved regulators of a number of developmental mechanisms. RUNX proteins often control the balance between proliferation and differentiation and alterations of their functions are associated with different types of cancer and other human pathologies. Moreover, RUNX factors control important steps during the developmental acquisition of mature phenotypes. A number of investigations are beginning to shed light on the involvement of RUNX family members in the development of the nervous system. This review summarizes recent progress in the study of the roles of mammalian RUNX proteins during the differentiation of sensory and motor neurons in the peripheral and central nervous system, respectively. The implications of those findings for RUNX-mediated regulation of hematopoietic development will also be discussed.

Hairy/Enhancer of split (Hes) 6 is a basic helix-loop-helix protein that interacts with the transcriptional co-repressor, Groucho, and antagonizes the neural functions of the Notch pathway. More specifically, mouse Hes6 regulates cerebral corticogenesis by promoting neurogenesis and suppressing astrocyte differentiation. The molecular mechanisms underlying the anti-astrogenic function of Hes6 are poorly defined. Here we describe studies aimed at testing whether Hes6 inhibits astrocyte differentiation by antagonizing the transcription repression activity of Notch-activated Hes family members like Hes1. It is reported that Hes6 preferentially forms homodimers. Heterodimerization with Hes1 is antagonized in part by a conserved N-terminal patch of negatively charged residues. Mutation of this motif enhances heterodimerization with Hes1 and increases Hes6 ability to antagonize Hes1-mediated transcriptional repression. However, this mutation does not increase, but instead decreases, the anti-astrogenic activity of Hes6. It is shown further that Hes6 harbors a second conserved sequence, a C-terminal SPXXSP motif. This sequence is phosphorylated by the mitogen activated protein kinase pathway and its mutation disrupts the anti-astrogenic activity of Hes6 without affecting its ability to suppress Hes1. Together, these observations suggest that Hes6 homodimers regulate astrocyte differentiation through mechanisms that depend on the phosphorylation of Hes6 C-terminal domain but are independent of its ability to suppress Hes1-mediated transcriptional repression.

Groucho proteins are abundant and broadly expressed nuclear factors that lack intrinsic DNA-binding activity but can interact with a variety of DNA-binding proteins. The recruitment of Groucho to specific gene regulatory sequences results in transcriptional repression. In both invertebrates and vertebrates, Groucho family members act as important regulators of several signaling mechanisms, including the Notch, Wingless/Wnt and Dpp/BMP/TGF-beta signaling pathways. Recent studies of embryonic development in several species point to an important role for Groucho in the regulation of multiple patterning and differentiation events. Moreover, a deregulated expression of human Groucho family members is correlated with several neoplastic conditions. Here we focus on the functions of Groucho proteins during body patterning and their implication in tumorigenesis.

Aristaless-related homeobox gene (ARX) is an important paired-type homeobox gene involved in the development of human brain. The ARX gene mutations are a significant contributor to various forms of X-chromosome-linked mental retardation with and without additional features including epilepsy, lissencephaly with abnormal genitalia, hand dystonia or autism. Here we demonstrate that the human ARX protein is a potent transcriptional repressor, which binds to Groucho/transducin-like enhancer of split (TLE) co-factor proteins and the TLE1 in particular through its octapeptide (Engrailed homology repressor domain (eh-1) homology) domain. We show that the transcription repression activity of ARX is modulated by two strong repression domains, one located within the octapeptide domain and the second in the region of the polyalanine tract 4, and one activator domain, the aristaless domain. Importantly, we show that the transcription repression activity of ARX is affected by various naturally occurring mutations. The introduction of the c.98T>C (p.L33P) mutation results in the lack of binding to TLE1 protein and relaxed transcription repression. The introduction of the two most frequent ARX polyalanine tract expansion mutations increases the repression activity in a manner dependent on the number of extra alanines. Interestingly, deletions of alanine residues within polyalanine tracts 1 and 2 show low or no effect. In summary we demonstrate that the ARX protein is a strong transcription repressor, we identify novel ARX interacting proteins (TLE) and offer an explanation of a molecular pathogenesis of some ARX mutations, including the most frequent ARX mutations, the polyalanine tract expansion mutations, c.304ins(GCG)7 and c.428_451dup.

Somatic motor neurons in the hypoglossal nucleus innervate tongue muscles controlling vital functions such as chewing, swallowing and respiration. Formation of functional hypoglossal nerve circuits depends on the establishment of precise hypoglossal motor neuron maps correlating with specific tongue muscle innervations. Little is known about the molecular mechanisms controlling mammalian hypoglossal motor neuron topographic map formation. Here we show that combinatorial expression of transcription factors Runx1, SCIP and FoxP1 defines separate mouse hypoglossal motor neuron groups with different topological, neurotransmitter and calcium-buffering phenotypes. Runx1 and SCIP are coexpressed in ventromedial hypoglossal motor neurons involved in control of tongue protrusion whereas FoxP1 is expressed in dorsomedial motor neurons associated with tongue retraction. Establishment of separate hypoglossal motor neuron maps depends in part on Runx1-mediated suppression of ventrolateral and dorsomedial motor neuron phenotypes and regulation of FoxP1 expression pattern. These findings suggest that combinatorial actions of Runx1, SCIP and FoxP1 are important for mouse hypoglossal nucleus somatotopic map formation.

Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling.

// Federica Verginelli 1 , Laura Adesso 1 , Isabelle Limon 2 , Anna Alisi 3 , Marie Gueguen 2 , Nadia Panera 3 , Ezio Giorda 4 , Lavinia Raimondi 1 , Roberta Ciarapica 1 , Antonio F. Campese 5 , Isabella Screpanti 5 , Stefano Stifani 6 , Jan Kitajewski 7 , Lucio Miele 8 , Rossella Rota 1,* and Franco Locatelli 1,9,* 1 Department of Oncohematology, Ospedale Pediatrico Bambino Gesu, IRCCS, Rome, Italy 2 Department of Sorbonne Universites, UPMC University Paris 06, CNRS, UMR, IBPS, Paris, France 3 Liver Research Unit, Ospedale Pediatrico Bambino Gesu, IRCCS, Rome, Italy 4 Department of Unit of Flow Cytometry, Ospedale Pediatrico Bambino Gesu, IRCCS, Rome, Italy 5 Department of Molecular Medicine, Sapienza University, Rome, Italy 6 Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, QC, Canada 7 Departments of Pathology and Ob/Gyn, Columbia University Medical Center, New York, NY, USA 8 Department of Genetics and Stanley Scott Cancer Center, Louisiana State University Health Sciences Center and Louisiana Cancer Research Consortium, New Orleans, LA, USA 9 Dipartimento di Scienze Pediatriche, Universita di Pavia, Pavia PV, Italy * These authors have contributed equally to this work Correspondence to: Franco Locatelli, email: // Rossella Rota, email: // Keywords : Notch1, Notch4, endothelial cells, IL-1β, VCAM1, Pathology Section Received : June 11, 2015 Accepted : November 21, 2015 Published : December 03, 2015 Abstract The Notch1 and Notch4 signaling pathways regulate endothelial cell homeostasis. Inflammatory cytokines induce the expression of endothelial adhesion molecules, including VCAM1, partly by downregulating Notch4 signaling. We investigated the role of endothelial Notch1 in this IL-1β-mediated process. Brief treatment with IL-1β upregulated endothelial VCAM1 and Notch ligand Jagged1. IL-1β decreased Notch1 mRNA levels, but levels of the active Notch1ICD protein remained constant. IL-1β-mediated VCAM1 induction was downregulated in endothelial cells subjected to pretreatment with a pharmacological inhibitor of the γ-secretase, which activates Notch receptors, producing NotchICD. It was also downregulated in cells in which Notch1 and/or Jagged1 were silenced. Conversely, the forced expression of Notch1ICD in naive endothelial cells upregulated VCAM1 per se and amplified IL-1β-mediated VCAM1 induction. Jagged1 levels increased and Notch4 signaling was downregulated in parallel. Finally, Notch1ICD and Jagged1 expression was upregulated in the endothelium of the liver in a model of chronic liver inflammation. In conclusion, we describe here a cell-autonomous, pro-inflammatory endothelial Notch1-Jagged1 circuit (i) triggering the expression of VCAM1 even in the absence of inflammatory cytokines and (ii) enhancing the effects of IL-1β. Thus, IL-1β regulates Notch1 and Notch4 activity in opposite directions, consistent with a selective targeting of Notch1 in inflamed endothelium.

The mechanisms regulating the generation of cell diversity in the mammalian cerebral cortex are beginning to be elucidated. In that regard, Hairy/Enhancer of split (Hes) 1 and 5 are basic helix-loop-helix (bHLH) factors that inhibit the differentiation of pluripotent cortical progenitors into neurons. In contrast, a related Hes family member termed Hes6 promotes neurogenesis. It is shown here that knockdown of endogenous Hes6 causes supernumerary cortical progenitors to differentiate into cells that exhibit an astrocytic morphology and express the astrocyte marker protein GFAP. Conversely, exogenous Hes6 expression in cortical progenitors inhibits astrocyte differentiation. The negative effect of Hes6 on astrocyte differentiation is independent of its ability to promote neuronal differentiation. We also show that neither its proneuronal nor its anti-gliogenic functions appear to depend on Hes6 ability to bind to DNA via the basic arm of its bHLH domain. Both of these activities require Hes6 to be localized to nuclei, but only its anti-gliogenic function depends on two short peptides, LNHLL and WRPW, that are conserved in all Hes6 proteins. These findings suggest that Hes6 is an important regulator of the neurogenic phase of cortical development by promoting the neuronal fate while suppressing astrocyte differentiation. They suggest further that separate molecular mechanisms underlie the proneuronal and anti-gliogenic activities of Hes6 in cortical progenitor cells.

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.

AML1/Runx1 (Runx1) is a mammalian transcription factor that plays critical roles in regulating the differentiation of a number of different cell types. In the present study, we have utilized mice expressing beta-galactosidase (beta-gal) under the control of the Runx1 promoter to characterize the spatiotemporal expression pattern of Runx1 during retinogenesis. Expression of beta-gal was first detected at embryonic day 13.5 in post-mitotic cells located in the inner retina and overlapped with expression of the early amacrine and ganglion cell marker protein Islet1. During subsequent developmental stages, the number of beta-gal-positive cells increased in a central-to-peripheral gradient until late embryogenesis but then decreased in the early post-natal retina. beta-gal-positive cells were located primarily in the ganglion cell layer by late embryonic/early post-natal stages and were identified as a subpopulation of displaced amacrine cells by the continued expression of Islet1, as well as Pax6, and the coexpression of the amacrine cell subtype-specific markers choline acetyltransferase, calretinin and the 65-kDa isoform of glutamic acid decarboxylase. These findings identify Runx1 as a novel marker for a restricted amacrine cell subtype and suggest a role for this gene in regulating the post-mitotic development of these cells.

Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murinerunt-related geneRunx1is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1+ olfactory receptor neuron (ORN) progenitors and NeuroD+ ORN precursors. Disruption ofRunx1functionin vivodoes not cause a change in Mash1 expression but leads to a decrease in the number of NeuroD+ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitorp21Cip1in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.

Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.

Pax proteins are DNA-binding transcription factors that regulate embryonic development through the activation and repression of downstream target genes. The Pax2 gene is absolutely required for kidney development and for patterning specific regions of the nervous system such as the eye, ear and hindbrain. The Pax2/5/8 family of proteins contains both transcription activation and repression domains. The activation domain of Pax2 is phosphorylated by the c-Jun N-terminal kinase (JNK) to enhance Pax2-dependent transcription. In this report, we demonstrate that the Groucho/TLE family protein, Grg4, interacts with Pax2 to suppress transactivation. Grg4 is able to specifically inhibit phosphorylation of the Pax2 activation domain, even in the presence of activated JNK. Furthermore, the Grg4 interaction and suppression of phosphorylation depends on Pax2 binding to its target DNA sequence and is independent of histone deacetylation. These data suggest a new model for Groucho mediated suppression of transcription through the specific inhibition of modifications in the activation domain of a transactivator.