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Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

Agenesis of the Corpus Callosum (ACC) can result in multiple neurological deficits including social and behavioural issues. However, the underlying aetiology, clinical co-morbidity and the contributing risk factors remain elusive, resulting in inaccurate prognosis and delayed therapy. The main objective of this study was to comprehensively describe the epidemiology and clinical co-morbidity associated with patients diagnosed with ACC. The secondary objective was to identify the factors that contribute towards increased risk for ACC. For this, we analysed 22 years (1998–2020) of clinical data across the whole of Wales, UK collected through the Congenital Anomaly Register & Information Service (CARIS) and Public Health Wales (PHW). Our results demonstrate that complete ACC (84.1%) was the prevalent subtype, in comparison to partial ACC. Further, ventriculomegaly/hydrocephalus (26.37%) and ventricular septal defect (21.92%) were identified to be the most prevalent neural malformation (NM) and congenital heart disorder (CHD) in our cohort. Although 12.7% of subjects with ACC had both an NM and CHD, we found no significant association between them (χ2 (1, n = 220) = 3.84, p = 0.33). We found socioeconomic deprivation and increased maternal age contributed towards an increased risk for ACC. To the best of our knowledge, this study for the first time defines the clinical phenotypes and the factors that contribute to ACC within the Welsh population. These findings will be of value to both patients and healthcare professionals, who may take preventative or remedial measures.

Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca

Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density, and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

Patients diagnosed with chromosome microdeletions or duplications, known as copy number variants (CNVs), present a unique opportunity to investigate the relationship between patient genotype and cell phenotype. CNVs have high genetic penetrance and give a good correlation between gene locus and patient clinical phenotype. This is especially effective for the study of patients with neurodevelopmental disorders (NDD), including those falling within the autism spectrum disorders (ASD). A key question is whether this correlation between genetics and clinical presentation at the level of the patient can be translated to the cell phenotypes arising from the neurodevelopment of patient induced pluripotent stem cells (iPSCs).Here, we examine how iPSCs derived from ASD patients with an associated CNV inform our understanding of the genetic and biological mechanisms underlying the aetiology of ASD. We consider selection of genetically characterised patient iPSCs; use of appropriate control lines; aspects of human neurocellular biology that can capture in vitro the patient clinical phenotype; and current limitations of patient iPSC-based studies. Finally, we consider how future research may be enhanced to maximise the utility of CNV patients for research of pathological mechanisms or therapeutic targets.

Copy number variant (CNV) syndromes are often associated with both neurocognitive deficits (NCDs) and congenital heart defects (CHDs). Children and adults with cardiac developmental defects likely to have NCDs leading to increased risk of hospitalisation and reduced level of independence. To date, the association between these two phenotypes have not been explored in relation to CNV syndromes. In order to address this question, we systematically reviewed the prevalence of CHDs in a range of CNV syndromes associated with NCDs. A meta-analysis showed a relationship with the size of CNV and its association with both NCDs and CHDs, and also inheritance pattern. To our knowledge, this is the first review to establish association between NCD and CHDs in CNV patients, specifically in relation to the severity of NCD. Importantly, we also found specific types of CHDs were associated with severe neurocognitive deficits. Finally, we discuss the implications of these results for patients in the clinical setting which warrants further exploration of this association in order to lead an improvement in the quality of patient's life.

Autism Spectrum Disorders (ASD) are a group of heterogeneous neurodevelopmental disorders with an estimated worldwide prevalence of 1-2%. Although it is highly heritable, the contribution of environmental factors and risk associated genes on the aberrant brain development is not well understood.In this review, we summarise some of the key risk factors and explore ASD associated cellular pathology from the perspective of the four predominant cells in the brain; neurons, oligodendrocytes, microglia and astrocytes. Further, we discuss the contributions of the associated cellular pathology to the three common hypotheses of ASD.We highlight the major neuro-pathologies underlying ASD, however more research is needed to ensure appropriate and efficient therapies can be directed towards ASD.

Remyelination in multiple sclerosis (MS) lesions often remains incomplete despite the presence of oligodendrocyte progenitor cells (OPCs). Amongst other factors, successful remyelination depends on the phagocytic clearance of myelin debris. However, the proteins in myelin debris that act as potent and selective inhibitors on OPC differentiation and inhibit CNS remyelination remain unknown. Here, we identify the transmembrane signalling protein EphrinB3 as important mediator of this inhibition, using a protein analytical approach in combination with a primary rodent OPC assay. In the presence of EphrinB3, OPCs fail to differentiate. In a rat model of remyelination, infusion of EphrinB3 inhibits remyelination. In contrast, masking EphrinB3 epitopes using antibodies promotes remyelination. Finally, we identify EphrinB3 in MS lesions and demonstrate that MS lesion extracts inhibit OPC differentiation while antibody-mediated masking of EphrinB3 epitopes promotes it. Our findings suggest that EphrinB3 could be a target for therapies aiming at promoting remyelination in demyelinating disease. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1521-1) contains supplementary material, which is available to authorized users.

Oligodendrocytes are the myelinating cells of the central nervous system. The role of oligodendrocytes in health and disease has been considerably enhanced by the development of methods to isolate and culture oligodendrocytes from central nervous system tissue. The cellular and molecular mechanisms involved in oligodendrocyte differentiation can be identified by challenging oligodendrocyte progenitors cells (OPCs) by altering their extracellular environment and intrinsic differentiation pathways. To address these issues, it is imperative to develop an in vitro protocol where pure OPCs are isolated and cultured in the presence of inhibitory developmental and differentiation cues like Semaphorin 3A. In this chapter, we describe methods to isolate and culture OPCs from neonatal rat brain tissue and further characterise their differentiation into oligodendrocytes. The described protocol is relatively simple in comparison to existing protocols and can be used to study the effect of lesion-associated inhibitors like Semaphorin 3A on oligodendrocyte differentiation.

Failure of oligodendrocyte precursor cell (OPC) differentiation has been recognized as the leading cause for the failure of myelin regeneration in diseases such as multiple sclerosis (MS). One explanation for the failure of OPC differentiation in MS is the presence of inhibitory molecules in demyelinated lesions. So far only a few inhibitory substrates have been identified in MS lesions. Semaphorin 3A (Sema3A), a secreted member of the semaphorin family, can act as repulsive guidance cue for neuronal and glial cells in the CNS. Recent studies suggest that Sema3A is also expressed in active MS lesions. However, the implication of Sema3A expression in MS lesions remains unclear as OPCs are commonly present in chronic demyelinated lesions. In the present study we identify Sema3A as a potent, selective, and reversible inhibitor of OPC differentiationin vitro. Furthermore, we show that administration of Sema3A into demyelinating lesions in the rat CNS results in a failure of remyelination. Our results imply an important role for Sema3A in the differentiation block occurring in MS lesions.

The increasing effectiveness of new disease-modifying drugs that suppress\ud disease activity in multiple sclerosis has opened up opportunities for\ud regenerative medicines that enhance remyelination and potentially slow disease\ud progression. Although several new targets for therapeutic enhancement of\ud remyelination have emerged, few lend themselves readily to conventional drug\ud development. Here, we used transcription profiling to identify mitogen-activated\ud protein kinase (Mapk) signalling as an important regulator involved in the\ud differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes.\ud We show in tissue culture that activation of Mapk signalling by elevation of\ud intracellular levels of cyclic adenosine monophosphate (cAMP) using administration\ud of either dibutyryl-cAMP or inhibitors of the cAMP-hydrolysing enzyme\ud phosphodiesterase-4 (Pde4) enhances OPC differentiation. Finally, we demonstrate\ud that systemic delivery of a Pde4 inhibitor leads to enhanced differentiation\ud of OPCs within focal areas of toxin-induced demyelination and a consequent\ud acceleration of remyelination. These data reveal a novel approach to therapeutic\ud enhancement of remyelination amenable to pharmacological intervention and\ud hence with significant potential for translation.

Remyelination in multiple sclerosis (MS) lesions often remains incomplete despite the presence of oligodendrocyte progenitor cells (OPCs). Amongst other factors, successful remyelination depends on the phagocytic clearance of myelin debris. However, the proteins in myelin debris that act as potent and selective inhibitors on OPC differentiation and inhibit CNS remyelination remain unknown. Here, we identify the transmembrane signalling protein EphrinB3 as important mediator of this inhibition, using a protein analytical approach in combination with a primary rodent OPC assay. In the presence of EphrinB3, OPCs fail to differentiate. In a rat model of remyelination, infusion of EphrinB3 inhibits remyelination. In contrast, masking EphrinB3 epitopes using antibodies promotes remyelination. Finally, we identify EphrinB3 in MS lesions and demonstrate that MS lesion extracts inhibit OPC differentiation while antibody-mediated masking of EphrinB3 epitopes promotes it. Our findings suggest that EphrinB3 could be a target for therapies aiming at promoting remyelination in demyelinating disease.

Failure of oligodendrocyte precursor cell (OPC) differentiation contributes significantly to failed myelin sheath regeneration\ud (remyelination) in chronic demyelinating diseases. Although the reasons for this failure are not completely understood, several\ud lines of evidence point to factors present following demyelination that specifically inhibit differentiation of cells capable of\ud generating remyelinating oligodendrocytes. We have previously demonstrated that myelin debris generated by demyelination\ud inhibits remyelination by inhibiting OPC differentiation and that the inhibitory effects are associated with myelin proteins. In the\ud present study, we narrow down the spectrum of potential protein candidates by proteomic analysis of inhibitory protein fractions\ud prepared by CM and HighQ column chromatography followed by BN/SDS/SDS–PAGE gel separation using Nano-HPLC-ESIQ-TOF\ud mass spectrometry. We show that the inhibitory effects on OPC differentiation mediated by myelin are regulated by\ud Fyn-RhoA-ROCK signalling as well as by modulation of protein kinase C (PKC) signalling. We demonstrate that pharmacological\ud or siRNA-mediated inhibition of RhoA-ROCK-II and/or PKC signalling can induce OPC differentiation in the presence of myelin.\ud Our results, which provide a mechanistic link between myelin, a mediator of OPC differentiation inhibition associated\ud with demyelinating pathologies and specific signalling pathways amenable to pharmacological manipulation, are therefore of\ud significant potential value for future strategies aimed at enhancing CNS remyelination.

Object Promoting repair of central nervous system (CNS) white matter represents an important approach to easing the course of a number of tragic neurological diseases. For this purpose, strategies are currently being evaluated for transplanting cells capable of generating new oligodendrocytes into areas of demyelination and/or enhancing the potential of endogenous stem/precursor cells to give rise to new oligodendrocytes. Emerging evidence, however, indicates that increasing the presence of cells capable of forming new myelin sheaths is not sufficient to promote repair because of unknown inhibitors that accumulate in lesions as a consequence of myelin degeneration and impair the generation of new oligodendrocytes. The aim of the present study was to characterize the nature of the inhibitory molecules present in myelin. Methods Differentiation of primary rat oligodendrocyte precursor cells (OPCs) in the presence of CNS and peripheral nervous system myelin was assessed by immunocytochemical methods. The authors further characterized the nature of the inhibitors by submitting myelin membrane preparations to biochemical precipitation and digestion. Finally, OPCs were grown on purified Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, the most prominent inhibitors of axon regeneration. Results Myelin membrane preparations induced a differentiation block in OPCs that was associated with down-regulation of expression of the transcription factor Nkx2.2. The inhibitory activity in myelin was restricted to the CNS and was predominantly associated with white matter. Furthermore, the results demonstrate that myelin proteins that are distinct from the most prominent inhibitors of axon outgrowth are specific inhibitors of OPC differentiation. Conclusions The inhibitory effect of unknown myelin-associated proteins should be considered in future treatment strategies aimed at enhancing CNS repair.

Summary Human pluripotent stem cells (hPSCs) could provide an infinite source of clinically relevant cells with potential applications in regenerative medicine. However, hPSC lines vary in their capacity to generate specialized cells, and the development of universal protocols for the production of tissue-specific cells remains a major challenge. Here, we have addressed this limitation for the endodermal lineage by developing a defined culture system to expand and differentiate human foregut stem cells (hFSCs) derived from hPSCs. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. Furthermore, near-homogenous populations of hFSCs can be obtained from hPSC lines which are normally refractory to endodermal differentiation. Therefore, hFSCs provide a unique approach to bypass variability between pluripotent lines in order to obtain a sustainable source of multipotent endoderm stem cells for basic studies and to produce a diversity of endodermal derivatives with a clinical value., Highlights • Multipotent foregut stem cells are derived from pluripotent cells • Foregut stem cells can be expanded and retain their differentiation potential • Foregut stem cells differentiate into liver and pancreatic cells • Foregut stem cells decrease differentiation variability between cell lines, Hannan, Vallier, and colleagues have developed a defined culture system to differentiate and expand human foregut stem cells (hFSCs) from human pluripotent cells. hFSCs can self-renew while maintaining their capacity to differentiate into pancreatic and hepatic cells. hFSCs represent a sustainable source of multipotent endodermal cells for basic studies and can produce a diversity of endodermal derivatives with clinical value.