A human in vitro neuronal model for studying homeostatic plasticity at the network level

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.The work was supported by funding from the European Community’s Horizon 2020 Programme (H2020/2014–2020) under grant agreement no. 728018 (Eat2beNICE) (to B.F.); ERA-NET NEURON-102 SYNSCHIZ grant (NWO) 013-17-003 4538 (to D.S.); China Scholarship Council 201906100038 (to X.Y.); ISCIII /MINECO (PT17/0009/0019) and FEDER (to A.E.C.); and M.M. was supported by an internal grant from the Donders Centre for Medical Neurosciences of the Radboud University Medical Center

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-β1 and focal adhesion kinase activation

Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin β1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin β1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin β1 and promotes neuroblastoma cell migration.This work was supported by the Comisión Nacional de Investigación Científica y Tecnológica [21130521]; Fondo Nacional de Desarrollo Científico y Tecnológico [1140697]; Fondo Nacional de Desarrollo Científico y Tecnológico [1180495]; Red Temática de Investigación Cooperativa en Cancer [RD12/0036/0027]; SAF [SAF2015-65175-R]; Red Temática de Investigación Cooperativa en Cancer [RD12/0036/0027]; PSG: Ministerio de Ciencia, Innovación y Universidades and FEDER funds (RTI2018-093596); JGC: Ministerio de Economía y Competitividad of Spain (PI17CIII/00013), Consejería de Educación, Juventud y Deporte, Comunidad de Madrid (P2017/BMD-3692), Fundación Oncohematología Infantil, AFANION, and Asociación Pablo Ugarte.S

Neurodevelopment, brain vasculature and schizophrenia

Schizophrenia is a mental disorder that arises from abnormal neurodevelopment, led by genetic and environmental factors. Several lines of evidence suggest the involvement of both the nervous and vascular system in the etiology and pathophysiology of schizophrenia. In this thesis, human-induced pluripotent stem cell-derived neurons were used to study early neurodevelopment in schizophrenia. To investigate physiological adult neurogenesis and the possible contribution of the cerebral vasculature to the pathophysiology of schizophrenia, single-cell gene expression profiling of post-mortem brain tissues was performed. Our findings suggest that impairments in neuronal communication may already be present during early developmental stages in schizophrenia, compromising the ability of the nervous system for fast and efficient reorganization. This could converge in the development of neural circuits that are more sensitive to harmful or stressful external factors. Conversely, we identified few transcriptional changes between schizophrenia and control brain vasculature, which were limited to pericytes and ependymal cells, suggesting no major involvement of the brain vasculature in schizophrenia. Together, our data and observations contribute to the understanding of schizophrenia brain pathology, as well as to our knowledge of human neurodevelopment and brain vasculature heterogeneity

Brain vasculature disturbance in schizophrenia

PURPOSE OF REVIEW: The vascular hypothesis of schizophrenia (SZ) postulates that brain endothelial dysfunction contributes to brain pathophysiology. This review discusses recent evidence for and against this hypothesis, including data related to blood-brain barrier (BBB), brain endothelium, and brain blood supply, to provide a critical weighed update. RECENT FINDINGS: Different studies report a consistent proportion of SZ patients showing increased BBB permeability, reflected by higher levels of albumin in the cerebral spinal fluid. Of note, this was not a result of antipsychotic medication. The high inflammatory profile observed in some SZ patients is strongly associated with increased BBB permeability to circulating immune cells, and with more severe cognitive deficiencies. Also, sex was found to interact with BBB integrity and permeability in SZ. The strongest independent genetic association with SZ has been identified in FZD1, a hypoxia-response gene that is 600-fold higher expressed in early development endothelium as compared to adult brain endothelium. Regarding brain blood supply, there is evidence to suggest alterations in proper brain perfusion in SZ. Nonetheless, ex-vivo experiments suggested that widely used antipsychotics favor vasoconstriction; thus, alterations in cerebral perfusion might be related to the patients' medication. SUMMARY: In some patients with SZ, a vulnerable brain endothelium may be interacting with environmental stressors, such as inflammation or hypoxia, converging into a more severe SZ symptomatology. Gene expression and performance of human brain endothelium could vary along with development and the establishment of the BBB; therefore, we encourage to investigate its possible contribution to SZ considering this dynamic context

Single-nucleus RNA sequencing of the midbrain blood-brain barrier cells in schizophrenia

Background: The increased inflammatory state observed in schizophrenia post-mortem brain and midbrain tissue [1-5] may have detrimental consequences on the brain vasculature of patients with schizophrenia. On the other hand, intrinsic alterations in the brain vasculature of schizophrenia patients may impair toxin efflux and favour the ingression of toxic material and immune cells into the central nervous system, leading to neuroinflammation. Evidence derived from blood and cerebrospinal fluid measurements indicated increased levels of adhesion molecules, such as sP-selectin and sICAM, in schizophrenia [6-8], which may favour the ingression of immune cells into the brain, and higher levels of albumin in CSF as compared to controls, suggesting increased BBB permeability in schizophrenia patients [6, 7, 9]. However, the proper functioning of the BBB involves a variety of cell types, and it is not known which cell type(s) of the BBB may be particularly affected in schizophrenia. Methods: We combined a fluorescence activated sorting isolation strategy with single-nucleus RNA sequencing (snRNAseq) to characterize the cells of the BBB in the midbrain of 15 schizophrenia donors and 14 matched controls, from the well-characterized Stanley Brain Collection. The cases used for the present study were previously grouped into high and low inflammation cases, based on the expression of inflammatory cytokines in their cortical grey matter, and 4 out of the 15 schizophrenia cases were assigned to the cortical high-inflammation group [10]. The different cell types of the BBB were identified with unbiased cluster analysis of nuclear transcriptomic profiles and annotated based on the expression of cell type-specific marker genes. Subclustering analysis, gene set enrichment and comparisons with previously published datasets were carried out to identify sub-populations among the main BBB cell types. We compared the transcriptomic profiles of the major BBB cell types between schizophrenia and controls. In addition, the relative abundance of the main BBB cell types and sub-populations was compared between the diagnoses, using generalized linear modeling. Results: We did not identify changes in the relative abundance of the major BBB cell types, nor in the sub-populations, associated with schizophrenia. However, we identified 14 differentially expressed genes in the cells of the BBB in schizophrenia as compared to controls, including genes that have previously been related to schizophrenia, such as FOXP2 and PDE4D. These transcriptional changes associated with schizophrenia were limited to the ependymal cells and pericytes. Additionally, we detected a sub-population of protoplasmic astrocytes enriched in the high inflammation schizophrenia subgroup. Genes more abundantly expressed in these schizophrenia-related protoplasmic astrocytes were associated with glutamatergic synaptic function rather than with inflammation. Conclusion: Our overall diagnostic results suggest the relative abundance of the BBB cell types and cellular sub-populations remains unaltered in schizophrenia pathology. Nonetheless, transcriptional changes in the midbrain BBB cells associated with schizophrenia are found, but are limited and are specific to two cell types, the ependymal and pericytes. In addition, the high inflammatory status observed in a subgroup of the patients may be interfering with the normal activities of protoplasmic astrocytes, possibly associated with midbrain dopamine and glutamate neurons

The Netrin-4/Laminin γ1/Neogenin-1 complex mediates migration in SK-N-SH neuroblastoma cells

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It arises during development of the sympathetic nervous system. Netrin-4 (NTN4), a laminin-related protein, has been proposed as a key factor to target NB metastasis, although there is controversy about its function. Here, we show that NTN4 is broadly expressed in tumor, stroma and blood vessels of NB patient samples. Furthermore, NTN4 was shown to act as a cell adhesion molecule required for the migration induced by Neogenin-1 (NEO1) in SK-N-SH neuroblastoma cells. Therefore, we propose that NTN4, by forming a ternary complex with Laminin γ1 (LMγ1) and NEO1, acts as an essential extracellular matrix component, which induces the migration of SK-N-SH cells