Gene Signature Associated with Nervous System in an Experimental Radiation- and Estrogen-Induced Breast Cancer Model.

Breast cancer is frequently the most diagnosed female cancer in the world. The experimental studies on cancer seldom focus on the relationship between the central nervous system and cancer. Despite extensive research into the treatment of breast cancer, chemotherapy resistance is an important issue limiting the efficacy of treatment. Novel biomarkers to predict prognosis or sensitivity to chemotherapy are urgently needed. This study examined nervous-system-related genes. The profiling of differentially expressed genes indicated that high-LET radiation, such as that emitted by radon progeny, in the presence of estrogen, induced a cascade of events indicative of tumorigenicity in human breast epithelial cells. Bioinformatic tools allowed us to analyze the genes involved in breast cancer and associated with the nervous system. The results indicated that the gene expression of the Ephrin A1 gene (EFNA1), the roundabout guidance receptor 1 (ROBO1), and the kallikrein-related peptidase 6 (KLK6) was greater in T2 and A5 than in the A3 cell line; the LIM domain kinase 2 gene (LIMK2) was greater in T2 than A3 and A5; the kallikrein-related peptidase 7 (KLK7), the neuroligin 4 X-linked gene (NLGN4X), and myelin basic protein (MBP) were greater than A3 only in T2; and the neural precursor cell expressed, developmentally down-regulated 9 gene (NEDD9) was greater in A5 than in the A3 and E cell lines. Concerning the correlation, it was found a positive correlation between ESR1 and EFNA1 in BRCA-LumA patients; with ROBO1 in BRCA-Basal patients, but this correlation was negative with the kallikrein-related peptidase 6 (KLK6) in BRCA-LumA and -LumB, as well as with LIMK2 and ROBO1 in all BRCA. It was also positive with neuroligin 4 X-linked (NLGN4X) in BRCA-Her2 and BRCA-LumB, and with MBP in BRCA-LumA and -LumB, but negative with KLK7 in all BRCA and BRCA-LumA and NEDD9 in BRCA-Her2. The differential gene expression levels between the tumor and adjacent tissue indicated that the ROBO1, KLK6, LIMK2, KLK7, NLGN4X, MBP, and NEDD9 gene expression levels were higher in normal tissues than in tumors; however, EFNA1 was higher in the tumor than the normal ones. EFNA1, LIMK2, ROBO1, KLK6, KLK7, and MBP gene expression had a negative ER status, whereas NEDD9 and NLGN4X were not significant concerning ER status. In conclusion, important markers have been analyzed concerning genes related to the nervous system, opening up a new avenue of studies in breast cancer therapy.

Congenital Zika Virus Infection Impairs Corpus Callosum Development.

Congenital Zika syndrome (CZS) is a set of birth defects caused by Zika virus (ZIKV) infection during pregnancy. Microcephaly is its main feature, but other brain abnormalities are found in CZS patients, such as ventriculomegaly, brain calcifications, and dysgenesis of the corpus callosum. Many studies have focused on microcephaly, but it remains unknown how ZIKV infection leads to callosal malformation. To tackle this issue, we infected mouse embryos in utero with a Brazilian ZIKV isolate and found that they were born with a reduction in callosal area and density of callosal neurons. ZIKV infection also causes a density reduction in PH3+ cells, intermediate progenitor cells, and SATB2+ neurons. Moreover, axonal tracing revealed that callosal axons are reduced and misrouted. Also, ZIKV-infected cultures show a reduction in callosal axon length. GFAP labeling showed that an in utero infection compromises glial cells responsible for midline axon guidance. In sum, we showed that ZIKV infection impairs critical steps of corpus callosum formation by disrupting not only neurogenesis, but also axon guidance and growth across the midline.

Zebrafish Slit2 and Slit3 Act Together to Regulate Retinal Axon Crossing at the Midline.

Slit-Robo signaling regulates midline crossing of commissural axons in different systems. In zebrafish, all retinofugal axons cross at the optic chiasm to innervate the contralateral tectum. Here, the mutant for the Robo2 receptor presents severe axon guidance defects, which were not completely reproduced in a Slit2 ligand null mutant. Since slit3 is also expressed around this area at the stage of axon crossing, we decided to analyze the possibility that it collaborates with Slit2 in this process. We found that the disruption of slit3 expression by sgRNA-Cas9 injection caused similar, albeit slightly milder, defects than those of the slit2 mutant, while the same treatment in the slit2-/-mz background caused much more severe defects, comparable to those observed in robo2 mutants. Tracking analysis of in vivo time-lapse experiments indicated differential but complementary functions of these secreted factors in the correction of axon turn errors around the optic chiasm. Interestingly, RT-qPCR analysis showed a mild increase in slit2 expression in slit3-deficient embryos, but not the opposite. Our observations support the previously proposed "repulsive channel" model for Slit-Robo action at the optic chiasm, with both Slits acting in different manners, most probably relating to their different spatial expression patterns.

Autocrine/Paracrine Slit-Robo Signaling Controls Optic Lobe Development in Drosophila melanogaster.

Cell segregation mechanisms play essential roles during the development of the central nervous system (CNS) to support its organization into distinct compartments. The Slit protein is a secreted signal, classically considered a paracrine repellent for axonal growth through Robo receptors. However, its function in the compartmentalization of CNS is less explored. In this work, we show that Slit and Robo3 are expressed in the same neuronal population of the Drosophila optic lobe, where they are required for the correct compartmentalization of optic lobe neuropils by the action of an autocrine/paracrine mechanism. We characterize the endocytic route followed by the Slit/Robo3 complex and detected genetic interactions with genes involved in endocytosis and actin dynamics. Thus, we report that the Slit-Robo3 pathway regulates the morphogenesis of the optic lobe through an atypical autocrine/paracrine mechanism in addition to its role in axon guidance, and in association with proteins of the endocytic pathway and small GTPases.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

Convergent molecular mechanisms underlying cognitive impairment in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by pathogenic variants in the iduronate-2-sulfatase gene (IDS), responsible for the degradation of glycosaminoglycans (GAGs) heparan and dermatan sulfate. IDS enzyme deficiency results in the accumulation of GAGs within cells and tissues, including the central nervous system (CNS). The progressive neurological outcome in a representative number of MPSII patients (neuronopathic form) involves cognitive impairment, behavioral difficulties, and regression in developmental milestones. In an attempt to dissect part of the influence of axon guidance instability over the cognitive impairment presentation in MPS II, we used brain expression data, network propagation, and clustering algorithm to prioritize in the human interactome a disease module associated with the MPS II context. We identified new candidate genes and pathways that act in focal adhesion, integrin cell surface, laminin interactions, ECM proteoglycans, cytoskeleton, and phagosome that converge into functional mechanisms involved in early neural circuit formation defects and could indicate clues about cognitive impairment in patients with MPSII. Such molecular changes during neurodevelopment may precede the morphological and clinical evidence, emphasizing the importance of an early diagnosis and directing the development of potential drug leads. Furthermore, our data also support previous hypotheses pointing to shared pathogenic mechanisms in some neurodegenerative diseases.

Slit2 is necessary for optic axon organization in the zebrafish ventral midline.

Slit-Robo signaling has been implicated in regulating several steps of retinal ganglion cell axon guidance, with a central role assigned to Slit2. We report here the phenotypical characterization of a CRISPR-Cas9-generated zebrafish null mutant for this gene, along with a detailed analysis of its expression pattern by WM-FISH. All evident defects in the optic axons in slit2-/- mutants were detected outside the retina, coincident with the major sites of expression at the ventral forebrain, around the developing optic nerve and anterior to the optic chiasm/proximal tract. Anterograde axon tracing experiments in zygotic and maternal-zygotic mutants, as well as morphants, showed the occurrence of axon sorting defects, which appeared mild at the optic nerve level, but more severe in the optic chiasm and the proximal tract. A remarkable sorting defect was the usual splitting of one of the optic nerves in two branches that surrounded the contralateral nerve at the chiasm. Although all axons eventually crossed the midline, the retinotopic order appeared lost at the proximal optic tract, to eventually correct distally. Time-lapse analysis demonstrated the sporadic occurrence of axon misrouting at the chiasm level, which could be responsible for the sorting errors. Our results support previous evidence of a channeling role for Slit molecules in retinal ganglion cell axons at the optic nerve, in addition to a function in the segregation of axons coming from each nerve and from different retinal regions at the medio-ventral area of the forebrain.

Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System.

Neurogenesis is achieved through a sequence of steps that include specification and differentiation of progenitors into mature neurons. Frequently, precursors migrate to distinct positions before terminal differentiation. The Slit-Robo pathway, formed by the secreted ligand Slit and its membrane bound receptor Robo, was first discovered as a regulator of axonal growth. However, today, it is accepted that this pathway can regulate different cellular processes even outside the nervous system. Since most of the studies performed in the nervous system have been focused on axonal and dendritic growth, it is less clear how versatile is this signaling pathway in the developing nervous system. Here we describe the participation of the Slit-Robo pathway in the development of motion sensitive neurons of the Drosophila visual system. We show that Slit and Robo receptors are expressed in different stages during the neurogenesis of motion sensitive neurons. Furthermore, we find that Slit and Robo regulate multiple aspects of their development including neuronal precursor migration, cell segregation between neural stem cells and daughter cells and formation of their connectivity pattern. Specifically, loss of function of slit or robo receptors in differentiated motion sensitive neurons impairs dendritic targeting, while knocking down robo receptors in migratory progenitors or neural stem cells leads to structural defects in the adult optic lobe neuropil, caused by migration and cell segregation defects during larval development. Thus, our work reveals the co-option of the Slit-Robo signaling pathway in distinct developmental stages of a neural lineage.

Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres.

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.

Shared Neurodevelopmental Perturbations Can Lead to Intellectual Disability in Individuals with Distinct Rare Chromosome Duplications.

Chromosomal duplications are associated with a large group of human diseases that arise mainly from dosage imbalance of genes within the rearrangements. Phenotypes range widely but are often associated with global development delay, intellectual disability, autism spectrum disorders, and multiple congenital abnormalities. How different contiguous genes from a duplicated genomic region interact and dynamically affect the expression of each other remains unclear in most cases. Here, we report a genomic comparative delineation of genes located in duplicated chromosomal regions 8q24.13q24.3, 18p11.32p11.21, and Xq22.3q27.2 in three patients followed up at our genetics service who has the intellectual disability (ID) as a common phenotype. We integrated several genomic data levels by identification of gene content within the duplications, protein-protein interactions, and functional analysis on specific tissues. We found functional relationships among genes from three different duplicated chromosomal regions, reflecting interactions of protein-coding genes and their involvement in common cellular subnetworks. Furthermore, the sharing of common significant biological processes associated with ID has been demonstrated between proteins from the different chromosomal regions. Finally, we elaborated a shared model of pathways directly or indirectly related to the central nervous system (CNS), which could perturb cognitive function and lead to ID in the three duplication conditions.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

Molecular complexity of visual mapping: a challenge for regenerating therapy.

Investigating the cellular and molecular mechanisms involved in the development of topographically ordered connections in the central nervous system constitutes an important issue in neurobiology because these connections are the base of the central nervous system normal function. The dominant model to study the development of topographic maps is the projection from the retinal ganglion cells to the optic tectum/colliculus. The expression pattern of Eph/ephrin system in opposing gradients both in the retina and the tectum, labels the local addresses on the target and gives specific sensitivities to growth cones according to their topographic origin in the retina. The rigid precision of normal retinotopic mapping has prompted the chemoaffinity hypothesis, positing axonal targeting to be based on fixed biochemical affinities between fibers and targets. However, several lines of evidence have shown that the mapping can adjust to experimentally modified targets with flexibility, demonstrating the robustness of the guidance process. Here we discuss the complex ways the Ephs and ephrins interact allowing to understand how the retinotectal mapping is a precise but also a flexible process.

Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development.

Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.

Complementary expression of EphA7 and SCO-spondin during posterior commissure development.

Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.

Mutual influences between the main olfactory and vomeronasal systems in development and evolution.

The sense of smell plays a crucial role in the sensory world of animals. Two chemosensory systems have been traditionally thought to play-independent roles in mammalian olfaction. According to this, the main olfactory system (MOS) specializes in the detection of environmental odorants, while the vomeronasal system (VNS) senses pheromones and semiochemicals produced by individuals of the same or different species. Although both systems differ in their anatomy and function, recent evidence suggests they act synergistically in the perception of scents. These interactions include similar responses to some ligands, overlap of telencephalic connections and mutual influences in the regulation of olfactory-guided behavior. In the present work, we propose the idea that the relationships between systems observed at the organismic level result from a constant interaction during development and reflects a common history of ecological adaptations in evolution. We review the literature to illustrate examples of developmental and evolutionary processes that evidence these interactions and propose that future research integrating both systems may shed new light on the mechanisms of olfaction.