Neuronal Guidance Research Articles

A Potential Role of Semaphorin 3A during Orthodontic Tooth Movement.

Background: Induced tooth movement during orthodontic therapy requires mechano-induced bone remodeling. Besides various cytokines and growth-factors, neuronal guidance molecules gained attention for their roles in bone homeostasis and thus, potential roles during tooth movement. Several neuronal guidance molecules have been implicated in the regulation of bone remodeling. Amongst them, Semaphorin 3A is particular interesting as it concurrently induces osteoblast differentiation and disturbs osteoclast differentiation. Methods: Mechano-regulation of Sema3A and its receptors PlexinA1 and Neuropilin (RT-qPCR, WB) was evaluated by applying compressive and tension forces to primary human periodontal fibroblasts (hPDLF) and alveolar bone osteoblasts (hOB). The association of the transcription factor Osterix (SP7) and SEMA3A was studied by RT-qPCR. Mechanisms involved in SEMA3A-mediated osteoblast differentiation were assessed by Rac1GTPase pull-downs, β-catenin expression analyses (RT-qPCR) and nuclear translocation assays (IF). Osteogenic markers were analyzed by RT-qPCR. Results: SEMA3A, PLXNA1 and NRP1 were differentially regulated by tension or compressive forces in hPDLF. Osterix (SP7) displayed the same pattern of regulation. Recombinant Sema3A induced the activation of Rac1GTPase, the nuclear translocation of β-catenin and the expression of osteogenic marker genes. Conclusion: Sema3A, its receptors and Osterix are regulated by mechanical forces in hPDLF. SEMA3A upregulation was associated with Osterix (SP7) modulation. Sema3A-enhanced osteogenic marker gene expression in hOB might be dependent on a pathway involving Rac1GTPase and β-catenin. Thus, Semaphorin 3A might contribute to bone remodeling during induced tooth movement.

Abstract LB199: Trafficking of Semaphorin4D is regulated by endoplasmic reticulum chaperone GRP78

Abstract Localization of endoplasmic reticulum (ER) chaperones to the cell surface is an adaptive mechanism for cells to expand the functionality of these proteins in response to stress by activating signaling pathways distinct from their ER functions, thereby impacting cell survival and signaling. In cancer, cell-surface ER chaperones have been reported to regulate oncogenic signaling pathways, proliferation and cell adhesion through interactions with other cell surface proteins. The emerging role of peripheral cell surface GRP78 (csGRP78), traditionally regarded as an ER luminal chaperone, as a multifunctional co-receptor for many different types of cell surface proteins presents novel opportunities for therapeutic interventions. One such cell surface protein is Semaphorin4D (Sema4D). Sema4D was originally discovered as a cell surface cleavable ligand and neuronal axon guidance cue that mobilizes the actin cytoskeleton to facilitate neuronal growth cone homing during development. In cancer, cell surface Sema4D expression is pro-metastatic and pro-angiogenic. Sema4D has been shown to be highly expressed in the breast cancer and epidemiological studies have demonstrated that Sema4D expression in breast cancer patients confers a poor prognosis and high probability of metastatic organotropism. Our recent advances have preliminarily identified a critical role for GRP78 in facilitating Semaphorin4D (Sema4D) trafficking. We show that Sema4D forms a direct protein-protein interaction with GRP78 and that GRP78 may facilitate membrane trafficking of Sema4D to the cell surface, where it exerts pro-angiogenic and metastatic effects. Furthermore, csGRP78 may interact with and facilitate many of the functions of Sema4D. Understanding the conditions by which GRP78 contributes to surface expression of Sema4D and the downstream effects may lead to novel therapeutics that abrogate the effects of Sema4D in cancer and to the elucidation of novel mechanisms by which Sema4D trafficking in cancer is regulated by GRP78. Citation Format: Anthony Carlos, Yuan-Li Tsai, Yan Ma, Amy Lee. Trafficking of Semaphorin4D is regulated by endoplasmic reticulum chaperone GRP78 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB199.

Transcriptome Analysis of Endothelial Colony-Forming Cells Isolated from Patients with HbSC Hemoglobinopathy and Proliferative Retinopathy

Introduction: Retinopathy is one of the major clinical manifestations of sickle cell disease. Although systemic disease is more severe in sickle cell anemia, ocular disease is apparently more severe in hemoglobin SC disease (HbSC). Sickle cell retinopathy can be classified as non-proliferative or proliferative. In the non-proliferative form, the most common clinical findings are salmon patch hemorrhages, iridescent spots and black sunbursts, which can be observed in the peripheral retina. In the proliferative form, peripheral retinal neovascularization develops after vaso-occlusion and grows anteriorly from perfused to non-perfused retina. These new vessels can lead to vitreous hemorrhage, retinal breaks, tractional or rhegmatogenous retinal detachments, and consequently vision loss can occur. The precise pathogenesis of neovascular formation in sickle cell disease is not fully understood. Endothelial Colony-Forming Cells (ECFC) have been shown to play a role in angiogenesis and could become accessible informers of vascular status and enable the study of vascular disorders. Objective: The aim of this study was to compare differential gene expression profiles of ECFC from HbSC patients with and without retinopathy. Methods: ECFCs were cultured after isolation from peripheral blood samples of three HbSC patients with proliferative retinopathy and five HbSC patients without retinopathy. RNA was extracted from the cell cultures and transcriptome analysis was performed with HiSeq 2500 sequencer (Illumina, Inc., San Diego, CA, USA) with eight RNA-seq libraries per lane (2 × 101-bp paired-end reads). Results: Ten genes were upregulated in cultures from patients with retinopathy when compared with those without retinopathy: ROBO1, SLC38A5, SLC38A5, DDR2, PEG3, PLPP7, TRBJ2-1, PLPP4, APCDD1L and SEMA3B . Discussion and Conclusion: Among these genes, we highlight the most upregulated gene encoding Robo1, a member of the roundabout (Robo) family that serves as a neuronal guidance receptor and has been reported to mediate tumor and retinal angiogenesis. The Robo1-mediated pathway has been proposed as a target for the treatment of several ocular neovascular diseases, so our data support that sickle cell proliferative retinopathy may also benefit from therapeutic strategies blocking this pathway. DisclosuresOzelo:Novo Nordisk: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; CSL Behing: Consultancy; Roche: Consultancy, Speakers Bureau; Biogen: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Grifols: Speakers Bureau. Fertrin:Alexion Pharmaceuticals: Consultancy.

A putative structural mechanism underlying the antithetic effect of homologous RND1 and RhoD GTPases in mammalian plexin regulation.

Plexins are semaphorin receptors that play essential roles in mammalian neuronal axon guidance and in many other important mammalian biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism and is modulated by small GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations, we showed that RND1 reinforces the plexin dimerization interface, whereas RhoD destabilizes it due to their differential interaction with the cell membrane. Upon binding plexin at the Rho-GTPase-binding domain (RBD), RND1 and RhoD interact differently with the inner leaflet of the cell membrane and exert opposite effects on the dimerization interface via an allosteric network involving the RBD, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively charged membrane interface.

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking.

Tunneling nanotubes (TNTs) are actin‐rich structures that connect two or more cells and mediate cargo exchange between spatially separated cells. TNTs transport signaling molecules, vesicles, organelles, and even pathogens. However, the molecular mechanisms regulating TNT formation remain unclear and little is known about the endogenous mechanisms suppressing TNT formation in lung cancer cells. Here, we report that MICAL2PV, a splicing isoform of the neuronal guidance gene MICAL2, is a novel TNT regulator that suppresses TNT formation and modulates mitochondrial distribution. MICAL2PV interacts with mitochondrial Rho GTPase Miro2 and regulates subcellular mitochondrial trafficking. Moreover, down‐regulation of MICAL2PV enhances survival of cells treated with chemotherapeutical drugs. The monooxygenase (MO) domain of MICAL2PV is required for its activity to inhibit TNT formation by depolymerizing F‐actin. Our data demonstrate a previously unrecognized function of MICAL2 in TNT formation and mitochondrial trafficking. Furthermore, our study uncovers a role of the MICAL2PV‐Miro2 axis in mitochondrial trafficking, providing a mechanistic explanation for MICAL2PV activity in suppressing TNT formation and in modulating mitochondrial subcellular distribution.

Loss-of-function variants in SEMA3F and PLXNA3 encoding semaphorin-3F and its receptor plexin-A3 respectively cause idiopathic hypogonadotropic hypogonadism

PurposeIdiopathic hypogonadotropic hypogonadism (IHH) is characterized by absent puberty and subsequent infertility due to gonadotropin-releasing hormone (GnRH) deficiency. IHH can be accompanied by normal or compromised olfaction (Kallmann syndrome). Several semaphorins are known potent modulators of GnRH, olfactory, and vomeronasal system development. In this study, we investigated the role of Semaphorin-3F signaling in the etiology of IHH. MethodsWe screened 216 IHH patients by exome sequencing. We transiently transfected HEK293T cells with plasmids encoding wild type (WT) or corresponding variants to investigate the functional consequences. We performed fluorescent IHC to assess SEMA3F and PLXNA3 expression both in the nasal region and at the nasal/forebrain junction during the early human fetal development. ResultsWe identified ten rare missense variants in SEMA3F and PLXNA3 in 15 patients from 11 independent families. Most of these variants were predicted to be deleterious by functional assays. SEMA3F and PLXNA3 are both expressed along the olfactory nerve and intracranial projection of the vomeronasal nerve/terminal nerve. PLXNA1-A3 are expressed in the early migratory GnRH neurons. ConclusionSEMA3F signaling through PLXNA1-A3 is involved in the guidance of GnRH neurons and of olfactory and vomeronasal nerve fibers in humans. Overall, our findings suggest that Semaphorin-3F signaling insufficiency contributes to the pathogenesis of IHH.

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.

RHOA signaling defects result in impaired axon guidance in iPSC-derived neurons from patients with tuberous sclerosis complex

Patients with Tuberous Sclerosis Complex (TSC) show aberrant wiring of neuronal connections formed during development which may contribute to symptoms of TSC, such as intellectual disabilities, autism, and epilepsy. Yet models examining the molecular basis for axonal guidance defects in developing human neurons have not been developed. Here, we generate human induced pluripotent stem cell (hiPSC) lines from a patient with TSC and genetically engineer counterparts and isogenic controls. By differentiating hiPSCs, we show that control neurons respond to canonical guidance cues as predicted. Conversely, neurons with heterozygous loss of TSC2 exhibit reduced responses to several repulsive cues and defective axon guidance. While TSC2 is a known key negative regulator of MTOR-dependent protein synthesis, we find that TSC2 signaled through MTOR-independent RHOA in growth cones. Our results suggest that neural network connectivity defects in patients with TSC may result from defects in RHOA-mediated regulation of cytoskeletal dynamics during neuronal development.

Missense variants in DPYSL5 cause a neurodevelopmental disorder with corpus callosum agenesis and cerebellar abnormalities

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and βIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and βIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

PMN-derived netrin-1 attenuates cardiac ischemia-reperfusion injury via myeloid ADORA2B signaling.

Previous studies implicated the neuronal guidance molecule netrin-1 in attenuating myocardial ischemia-reperfusion injury. However, the tissue-specific sources and receptor signaling events remain elusive. Neutrophils are among the first cells responding to an ischemic insult and can be associated with tissue injury or rescue. We found netrin-1 levels were elevated in the blood of patients with myocardial infarction, as well as in mice exposed to myocardial ischemia-reperfusion. Selectively increased infarct sizes and troponin levels were found in Ntn1loxP/loxP Lyz2 Cre+ mice, but not in mice with conditional netrin-1 deletion in other tissue compartments. In vivo studies using neutrophil depletion identified neutrophils as the main source for elevated blood netrin-1 during myocardial injury. Finally, pharmacologic studies using treatment with recombinant netrin-1 revealed a functional role for purinergic signaling events through the myeloid adenosine A2b receptor in mediating netrin-1-elicited cardioprotection. These findings suggest an autocrine signaling loop with a functional role for neutrophil-derived netrin-1 in attenuating myocardial ischemia-reperfusion injury through myeloid adenosine A2b signaling.

Role of Neuronal Guidance Cues in the Pathophysiology of Obesity: A Peripheral and Central Overview.

Obesity is associated with an exacerbated synthesis and secretion of several molecules, which culminates in chronic low-grade inflammation and insulin resistance. Such conditions affect molecular and physiological responses of several organs and, if not resolved, predispose the obese patients to other diseases such as Type 2 diabetes, atherosclerosis, cancer, neural injuries, and cognitive impairments. A microenvironment with an excess of pro-inflammatory cytokines released by different cells, including immune and adipose cells lead to metabolic and non-metabolic diseases during obesity. In this context, the role of neuronal guidance cues named netrin, semaphorin and ephrin is novel. Specifically, the available literature indicates that besides their classic role as molecules that guide the axon to its target site, the neuronal guidance cues exhibit immunomodulatory functions from adipose tissue to the neural environment. In the current narrative review, we discuss the participation of the neuronal guidance cues on the physiology and pathophysiology of obesity. We also discuss the feedback loop of obesity on the netrin, semaphorin and ephrin functions that impair the structure and function of the brain. The integrative view of the neuronal guidance cues can be relevant in designing new treatments focus on attenuating metabolic and immune disorders in obese patients and reduce the risk of acquiring diseases such as Type 2 diabetes, atherosclerosis, cancer, and neural injuries.

Hypoxia-inducible factor-dependent induction of myeloid-derived netrin-1 attenuates natural killer cell infiltration during endotoxin-induced lung injury.

Sepsis and sepsis‐associated lung inflammation significantly contribute to the morbidity and mortality of critical illness. Here, we examined the hypothesis that neuronal guidance proteins could orchestrate inflammatory events during endotoxin‐induced lung injury. Through a targeted array, we identified netrin‐1 as the top upregulated neuronal guidance protein in macrophages treated with lipopolysaccharide (LPS). Furthermore, we found that netrin‐1 is highly enriched in infiltrating myeloid cells, particularly in macrophages during LPS‐induced lung injury. Transcriptional studies implicate hypoxia‐inducible factor HIF‐1α in the transcriptional induction of netrin‐1 during LPS treatment. Subsequently, the deletion of netrin‐1 in the myeloid compartment (Ntn1loxp/loxp LysM Cre) resulted in exaggerated mortality and lung inflammation. Surprisingly, further studies revealed enhanced natural killer cells (NK cells) infiltration in Ntn1loxp/loxp LysM Cre mice, and neutralization of NK cell chemoattractant chemokine (C‐C motif) ligand 2 (CCL2) reversed the exaggerated lung inflammation. Together, these studies provide functional insight into myeloid cell‐derived netrin‐1 in controlling lung inflammation through the modulation of CCL2‐dependent infiltration of NK cells.

Targeted molecular profiling of rare olfactory sensory neurons identifies fate, wiring, and functional determinants.

Determining the molecular properties of neurons is essential to understand their development, function and evolution. Using Targeted DamID (TaDa), we characterize RNA polymerase II occupancy and chromatin accessibility in selected Ionotropic receptor (Ir)-expressing olfactory sensory neurons in Drosophila. Although individual populations represent a minute fraction of cells, TaDa is sufficiently sensitive and specific to identify the expected receptor genes. Unique Ir expression is not consistently associated with differences in chromatin accessibility, but rather to distinct transcription factor profiles. Genes that are heterogeneously expressed across populations are enriched for neurodevelopmental factors, and we identify functions for the POU-domain protein Pdm3 as a genetic switch of Ir neuron fate, and the atypical cadherin Flamingo in segregation of neurons into discrete glomeruli. Together this study reveals the effectiveness of TaDa in profiling rare neural populations, identifies new roles for a transcription factor and a neuronal guidance molecule, and provides valuable datasets for future exploration.

Sema7A is crucial for resolution of severe inflammation

Endogenous mediators regulating acute inflammatory responses in both the induction and resolution phases of inflammatory processes are pivotal in host defense and tissue homeostasis. Recent studies have identified neuronal guidance proteins characterized in axonal development that display immunomodulatory functions. Here, we identify the neuroimmune guidance cue Semaphorin 7A (Sema7A), which appears to link macrophage (MΦ) metabolic remodeling to inflammation resolution. Sema7A orchestrated MΦ chemotaxis and chemokinesis, activated MΦ differentiation and polarization toward the proresolving M2 phenotype, and promoted leukocyte clearance. Peritoneal MΦSema7A-/- displayed metabolic reprogramming, characterized by reductions in fatty acid oxidation and oxidative phosphorylation, increases in glycolysis and the pentose phosphate pathway, and truncation of the tricarboxylic acid cycle, which resulted in increased levels of the intermediates succinate and fumarate. The low accumulation of citrate in MΦSema7A-/- correlated with the decreased synthesis of prostaglandins, leading to a reduced impact on lipid-mediator class switching and the generation of specialized pro resolving lipid mediators. Signaling network analysis indicated that Sema7A induced the metabolic reprogramming of MΦ by activating the mTOR- and AKT2-signaling pathways. Administration of Sema7ASL4cd orchestrated the resolution response to tissue homeostasis by shortening the resolution interval, promoting tissue protection in murine peritonitis, and enhancing survival in polymicrobial sepsis.

Enabling the Selective Detection of Endocrine-Disrupting Chemicals via Molecularly Surface-Imprinted "Coffee Rings".

Molecularly imprinted polymers (MIPs) represent an intriguing class of synthetic materials that can selectively recognize and bind chemical or biological molecules in a variety of value-added applications in sensors, catalysis, drug delivery, antibodies, and receptors. In this context, many advanced methods of implementing functional MIP materials have been actively studied. Herein, we report a robust strategy to produce highly ordered arrays of surface-imprinted polymer patterns with unprecedented regularity for MIP-based sensor platform, which involves the controlled evaporative self-assembly process of MIP precursor solution in a confined geometry consisting of a spherical lens on a flat Si substrate (i.e., sphere-on-flat geometry) to synergistically utilize the "coffee-ring" effect and repetitive stick-slip motions of the three-phase contact line simply by solvent evaporation. Highly ordered arrays of the ring-patterned MIP films are then polymerized under UV irradiation to achieve semi-interpenetrating polymer networks. The extraction of templated target molecules from the surface-imprinted ring-patterned MIP films leaves behind copious cavities for the recognizable specific "memory sites" to efficiently detect small molecules. As a result, the elaborated surface structuring effect, sensitivity, and specific selectivity of the coffee-ring-based MIP sensors are scrutinized by capitalizing on an endocrine-disrupting chemical, 2,4-dichlorophenoxyacetic acid (2,4-D), as an example. Clearly, the evaporative self-assembly of nonvolatile solutes in a confined geometry renders the creation of familiar yet ordered coffee-ring-like patterns for a wide range of applications, including sensors, scaffolds for cell motility, templates, substrates for neuron guidance, etc., thereby dispensing with the need of multistep lithography techniques and external fields.

Depression-/Anxiety-Like Behavior Alterations in Adult Slit2 Transgenic Mice.

Background: Slit2 is a member of the Slit family of secreted glycoproteins that plays highly conserved roles in neuronal axon guidance and cellular migration. Our previous experimental results showed Alzheimer's disease-like alterations and increased permeability of the blood–brain barrier in Slit2-overexpressing transgenic (Slit2-Tg) mice aged 8–9 months. Nevertheless, relatively little is known about behavioral alterations in adult Slit2-Tg mice (2–6 months of age). To observe the age-related behavioral effects of Slit2 overexpression in adult mice, we performed a battery of behavioral tests with adult Slit2-Tg mice at 2–6 months of age.Results: The body weight of Slit2-Tg mice was lower than that of the wild-type mice from 15 weeks of age. Compared with the control mice, depression-like behaviors were found in Slit2-Tg mice from 15 to 21 weeks of age in the sucrose preference test, although Slit2-Tg mice were hyperactive in the tail suspension test. The anxiety-like behaviors were found in Slit2-Tg mice in the open field test, as well as increased locomotor activity. The anxiety-like behaviors were also found in adult Slit2-Tg mice in the elevated plus maze. Compared to wild-type mice at 23 weeks old, impairment of the hippocampal neurons were found in Slit2-Tg mice at the same age in hematoxylin–eosin staining (H&E), including some eccentric dispersion and expansion of neuronal bodies. In addition, the messenger RNA (mRNA) expression of TNF-α was elevated in the hippocampus of adult Slit2-Tg mice.Conclusions: Slit2 overexpression causes depression-/anxiety-like behaviors in adult mice that may be related to an increase in inflammatory factors and damage to hippocampal neurons.

Slit/Robo signaling regulates Leydig cell steroidogenesis

BackgroundFirst identified as a regulator of neuronal axon guidance, Slit/Robo signaling has since been implicated in additional physiologic and pathologic processes, such as angiogenesis, organogenesis and cancer progression. However, its roles in the regulation of testis function have been little explored.MethodsImmunohistochemistry and RT-qPCR analyses were performed to detect the expression of Slit/Robo signaling effectors in the adult mouse testis. To identify the roles and mechanisms of Slit/Robo signaling in the regulation of steroidogenesis, RT-qPCR, immunoblotting and hormone measurements were carried out using Leydig cells (primary cultures and the MA10 cell line) treated with exogenous SLIT ligands, and testes from Robo1-null mice.ResultsSlit1, -2 and -3 and Robo1 and -2 expression was detected in the adult mouse testis, particularly in Leydig cells. In vitro treatment of Leydig cells with exogenous SLIT ligands led to a decrease in the expression of the steroidogenic genes Star, Cyp11a1, and Cyp17a1. SLIT2 treatment decreased the phosphorylation of the key steroidogenic gene regulator CREB, possibly in part by suppressing AKT activity. Furthermore, SLIT2 treatment reduced the responsiveness of MA10 cells to luteinizing hormone by decreasing the expression of Lhcgr. Consistent with these in vitro results, an increase in testicular Star mRNA levels and intra-testicular testosterone concentrations were found in Robo1-null mice. Finally, we showed that the expression of the Slit and Robo genes in Leydig cells is enhanced by testosterone treatment in vitro, by an AR-independent mechanism.ConclusionTaken together, these results suggest that Slit/Robo signaling represents a novel mechanism that regulates Leydig cell steroidogenesis. It may act in an autocrine/paracrine manner to mediate negative feedback by testosterone on its own synthesis.4VdBjKyUK_zz54UEEeomAMVideo

Familiar growth factors have diverse roles in neural network assembly.

The assembly of neuronal circuits during development depends on guidance of axonal growth cones by molecular cues deposited in their environment. While a number of families of axon guidance molecules have been identified and reviewed, important and diverse activities of traditional growth factors are emerging. Besides clear and well recognized roles in the regulation of cell division, differentiation and survival, new research shows later phase roles for a number of growth factors in promoting neuronal migration, axon guidance and synapse formation throughout the nervous system.

Macrophage-derived netrin-1 drives adrenergic nerve-associated lung fibrosis.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1-expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1's receptor, deleted in colorectal carcinoma; and therapeutic α1 adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1+ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α1 adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1-driven adrenergic processes and introduced α1 blockers as a potentially new fibrotic therapy.

Neural Networks Recapitulation by Cancer Cells Promotes Disease Progression: A Novel Role of p73 Isoforms in Cancer-Neuronal Crosstalk

Simple SummaryCancer is initiated by alterations in specific genes. However, at late stages, cancer cells become metastatic not necessarily through continuous accumulation of additional mutations, but by hijacking programs of normal embryonic development and reactivating them in an unusual place, at the wrong time. Here, we applied computational and experimental approaches to show that these malignant reactivations include genes that are crucial for the development and function of the nervous system. We use the paradigm of melanoma transition from less invasive to highly aggressive stages in order to show that major players of metastasis, such as TP73 gene products, are implicated in this process. This work provides evidence for interactions between cancer cells and the neuronal system, which may have important future implications for metastasis prevention and cancer management.Mechanisms governing tumor progression differ from those of initiation. One enigmatic prometastatic process is the recapitulation of pathways of neural plasticity in aggressive stages. Cancer and neuronal cells develop reciprocal interactions via mutual production and secretion of neuronal growth factors, neurothrophins and/or axon guidance molecules in the tumor microenvironment. Understanding cancer types where this process is active, as well as the drivers, markers and underlying mechanisms, has great significance for blocking tumor progression and improving patient survival. By applying computational and systemic approaches, in combination with experimental validations, we provide compelling evidence that genes involved in neuronal development, differentiation and function are reactivated in tumors and predict poor patient outcomes across various cancers. Across cancers, they co-opt genes essential for the development of distinct anatomical parts of the nervous system, with a frequent preference for cerebral cortex and neural crest-derived enteric nerves. Additionally, we show that p73, a transcription factor with a dual role in neuronal development and cancer, simultaneously induces neurodifferentiation and stemness markers during melanoma progression. Our data yield the basis for elucidating driving forces of the nerve–tumor cell crosstalk and highlight p73 as a promising regulator of cancer neurobiology.