Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation.

International audienceVezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

International audienceMicroglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Abstract: Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options

Prenatal Activation of Microglia Induces Delayed Impairment of Glutamatergic Synaptic Function

BACKGROUND: Epidemiological studies have linked maternal infection during pregnancy to later development of neuropsychiatric disorders in the offspring. In mice, experimental inflammation during embryonic development impairs behavioral and cognitive performances in adulthood. Synaptic dysfunctions may be at the origin of cognitive impairments, however the link between prenatal inflammation and synaptic defects remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we show that prenatal alteration of microglial function, including inflammation, induces delayed synaptic dysfunction in the adult. DAP12 is a microglial signaling protein expressed around birth, mutations of which in the human induces the Nasu-Hakola disease, characterized by early dementia. We presently report that synaptic excitatory currents in mice bearing a loss-of-function mutation in the DAP12 gene (DAP12(KI) mice) display enhanced relative contribution of AMPA. Furthermore, neurons from DAP12(KI) P0 pups cultured without microglia develop similar synaptic alterations, suggesting that a prenatal dysfunction of microglia may impact synaptic function in the adult. As we observed that DAP12(KI) microglia overexpress genes for IL1beta, IL6 and NOS2, which are inflammatory proteins, we analyzed the impact of a pharmacologically-induced prenatal inflammation on synaptic function. Maternal injection of lipopolysaccharides induced activation of microglia at birth and alteration of glutamatergic synapses in the adult offspring. Finally, neurons cultured from neonates born to inflamed mothers and cultured without microglia also displayed altered neuronal activity. CONCLUSION/SIGNIFICANCE: Our results demonstrate that prenatal inflammation is sufficient to induce synaptic alterations with delay. We propose that these alterations triggered by prenatal activation of microglia provide a cellular basis for the neuropsychiatric defects induced by prenatal inflammation

A transcriptomic continuum of differentiation arrest identifies myeloid interface acute leukemias with poor prognosis

Abstract: Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options

Ezrin Is Highly Expressed in Early Thymocytes, but Dispensable for T Cell Development in Mice

Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored.We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/-) mice likely arise as a consequence of nutritional stress.We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin