Guidance Receptor Research Articles

Circ_0068087 Silencing Ameliorates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Vascular Endothelial Cells Depending on miR-186-5p-Mediated Regulation of Roundabout Guidance Receptor 1.

Background: Circular RNAs (circRNAs) are endogenous non-coding RNAs involved in the progression of atherosclerosis (AS). We investigated the role of circ_0068087 in AS progression and its associated mechanism.Methods: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) were performed to analyze the viability, apoptosis, and inflammatory response of HUVECs, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the Western blot assay were performed to measure the expression of RNA and protein. Cell oxidative stress was analyzed using commercial kits. The dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify the interaction between microRNA-186-5p (miR-186-5p) and circ_0068087 or roundabout guidance receptor 1 (ROBO1).Results: Oxidized low-density lipoprotein (ox-LDL) exposure upregulated the circ_0068087 level in HUVECs. ox-LDL-induced dysfunction in HUVECs was largely attenuated by the silence of circ_0068087. Circ_0068087 negatively regulated the miR-186-5p level by interacting with it in HUVECs. Circ_0068087 knockdown restrained ox-LDL-induced injury in HUVECs partly by upregulating miR-186-5p. ROBO1 was a downstream target of miR-186-5p in HUVECs. Circ_0068087 positively regulated ROBO1 expression by sponging miR-186-5p in HUVECs. MiR-186-5p overexpression exerted a protective role in ox-LDL-induced HUVECs partly by downregulating ROBO1.Conclusion: Circ_0068087 interference alleviated ox-LDL-induced dysfunction in HUVECs partly by reducing ROBO1 expression via upregulating miR-186-5p.

Circular RNA_0001495 increases Robo1 expression by sponging microRNA-527 to promote the proliferation, migration and invasion of bladder cancer cells.

Bladder cancer (BCa) is a heterogeneous disease that poses great threats on public health. Increasing studies have identified the vital functions of circular RNAs (circRNAs) in BCa treatment. Hence, this current study set out to explore the modulatory role of circ_0001495 in BCa development. First, the expression of circ_0001495 was determined by reverse transcription quantitative polymerase chain reaction. Cell biological processes were then analyzed after altering the circ_0001495 expression in T24 cells. Next, interactions among circ_0001495, microRNA-527 (miR-527) and roundabout guidance receptor 1 (Robo1) were investigated by dual luciferase reporter gene assay, RNA pull down assay and FISH assay. Lastly, xenograft tumors in nude mice were established to explore the effect of circ_0001495 in vivo. It was found that circ_0001495 was highly expressed in BCa tissues and cells, and was further correlated with poor prognosis in BCa patients. In addition, circ_0001495 inhibited the activity of miR-527 by acting as a sponge to sponge miR-527, which further elevated the Robo1 expression. Lastly, circ_0001495 was found to promote the proliferation, migration and invasion of BCa cells in vitro through the miR-527/Robo1 axis and promote the growth and metastasis of BCa tumors in vivo. Altogether, findings in our study highlight the promoting role of circ_0001495 in the progression of BCa by increasing Robo1 via sponging miR-527, representing a promising target for BCa management.

Glycosylation in Axonal Guidance.

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.

One Raft to Guide Them All, and in Axon Regeneration Inhibit Them.

Central nervous system damage caused by traumatic injuries, iatrogenicity due to surgical interventions, stroke and neurodegenerative diseases is one of the most prevalent reasons for physical disability worldwide. During development, axons must elongate from the neuronal cell body to contact their precise target cell and establish functional connections. However, the capacity of the adult nervous system to restore its functionality after injury is limited. Given the inefficacy of the nervous system to heal and regenerate after damage, new therapies are under investigation to enhance axonal regeneration. Axon guidance cues and receptors, as well as the molecular machinery activated after nervous system damage, are organized into lipid raft microdomains, a term typically used to describe nanoscale membrane domains enriched in cholesterol and glycosphingolipids that act as signaling platforms for certain transmembrane proteins. Here, we systematically review the most recent findings that link the stability of lipid rafts and their composition with the capacity of axons to regenerate and rebuild functional neural circuits after damage.

HDAC inhibitor, MS-275, increases vascular permeability by suppressing Robo4 expression in endothelial cells

ABSTRACT Roundabout guidance receptor 4 (Robo4) is an endothelial-specific membrane protein that suppresses pathological angiogenesis and vascular hyperpermeability by stabilizing endothelial cells. Robo4 suppresses severe systemic inflammation induced by pathogens and endotoxins and inhibits tumor growth and metastasis, therefore serving as a potential therapeutic target. Although the regulation of Robo4 expression through transcription factors and epigenetic mechanisms has been studied, the role of histone deacetylases (HDACs) has not been explored. In the present study, we investigated the involvement of HDACs in the regulation of Robo4 expression. An HDAC inhibitor, MS-275, which inhibits HDAC1, HDAC2, and HDAC3, was found to suppress Robo4 expression in endothelial cells. Small interfering RNA (siRNA)-mediated knockdown of HDAC3, but not of HDAC1 and 2, also decreased its expression level. MS-275 downregulated the expression of the transcription factor complex GABP, in addition to suppressing Robo4 promoter activity. GABP expression was also downregulated by the siRNA against HDAC3. MS-275 decreased the transendothelial electrical resistance of a monolayer of mouse endothelial cells and increased the rate of leakage of Evans blue dye in the mouse lungs. In addition, MS-275 accelerated cell migration through the endothelial cell monolayer and augmented cell extravasation in the mouse lungs. Taken together, we demonstrated that MS-275 suppresses Robo4 expression by inhibiting HDAC3 in endothelial cells and enhances endothelial and vascular permeability. Thus, we demonstrated a novel mechanism regulating Robo4 expression and vascular permeability, which is anticipated to contribute to future therapies for infectious and inflammatory diseases.

Robo recruitment of the Wave regulatory complex plays an essential and conserved role in midline repulsion.

The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. Here, we show that the heteropentameric Scar/Wave Regulatory Complex (WRC), which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WRC-interacting receptor sequence (WIRS) motif. In Drosophila embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.

PODO: Trial Design: Phase 2 Study of PF-06730512 in Focal Segmental Glomerulosclerosis

IntroductionFocal segmental glomerulosclerosis (FSGS) is characterized by proteinuria and a histologic pattern of glomerular lesions of diverse etiology that share features including glomerular scarring and podocyte foot process effacement. Roundabout guidance receptor 2 (ROBO2)/slit guidance ligand 2 (SLIT2) signaling destabilizes the slit diaphragm and reduces podocyte adhesion to the glomerular basement membrane (GBM). Preclinical studies suggest that inhibition of glomerular ROBO2/SLIT2 signaling can stabilize podocyte adhesion and reduce proteinuria. This clinical trial evaluates the preliminary efficacy and safety of ROBO2/SLIT2 inhibition with the ROBO2 fusion protein PF-06730512 in patients with FSGS.MethodsThe Study to Evaluate PF-06730512 in Adults With FSGS (PODO; ClinicalTrials.gov identifier NCT03448692), an open-label, phase 2a, multicenter trial in adults with FSGS, will enroll patients into 2 cohorts (n = 22 per cohort) to receive either high- or low-dose PF-06730512 (intravenous) every 2 weeks for 12 weeks. Key inclusion criteria include a confirmed biopsy diagnosis of FSGS, an estimated glomerular filtration rate (eGFR) ≥45 ml/min/1.73 m2 based on the Chronic Kidney Disease Epidemiology Collaboration formula (30–45 with a recent biopsy), and urinary protein-to-creatinine ratio (UPCR) >1.5 g/g. Key exclusion criteria include collapsing FSGS, serious/active infection, ≥50% tubulointerstitial fibrosis on biopsy, and organ transplantation. The primary endpoint is change from baseline to week 13 in UPCR; secondary endpoints include safety, changes in eGFR, and PF-06730512 serum concentration.ResultsThis ongoing trial will report the efficacy, safety, pharmacokinetics, and biomarker results of PF-06730512 for patients with FSGS.ConclusionFindings from this proof-of-concept study may support further development and evaluation of PF-06730512 to treat FSGS and warrant assessment in phase 3 clinical trials.

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

SummaryDuring cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a “trimer-of-trimers” super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.

Paxillin Is Required for Proper Spinal Motor Axon Growth into the Limb.

To assemble the functional circuits of the nervous system, the neuronal axonal growth cones must be precisely guided to their proper targets, which can be achieved through cell-surface guidance receptor activation by ligand binding in the periphery. We investigated the function of paxillin, a focal adhesion protein, as an essential growth cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show paxillin expression in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Paxillin loss-of-function and gain-of-function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of paxillin in motor axon guidance. In addition, a neuron-specific paxillin deletion in mice led to LMC axon trajectory selection errors. We also show that knocking down paxillin attenuates the growth preference of LMC neurites against ephrins in vitro, and erythropoietin-producing human hepatocellular (Eph)-mediated retargeting of LMC axons in vivo, suggesting paxillin involvement in Eph-mediated LMC motor axon guidance. Finally, both paxillin knockdown and ectopic expression of a nonphosphorylable paxillin mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating paxillin as a Src target in Eph signal relay in this context. In summary, our findings demonstrate that paxillin is required for motor axon guidance and suggest its essential role in the ephrin-Eph signaling pathway resulting in motor axon trajectory selection.SIGNIFICANCE STATEMENT During the development of neural circuits, precise connections need to be established among neurons or between neurons and their muscle targets. A protein family found in neurons, Eph, is essential at different stages of neural circuit formation, including nerve outgrowth and pathfinding, and is proposed to mediate the onset and progression of several neurodegenerative diseases, such as Alzheimer's disease. To investigate how Ephs relay their signals to mediate nerve growth, we investigated the function of a molecule called paxillin and found it important for the development of spinal nerve growth toward their muscle targets, suggesting its role as an effector of Eph signals. Our work could thus provide new information on how neuromuscular connectivity is properly established during embryonic development.

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3.

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3’s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.

High ROBO3 expression predicts poor survival in non-M3 acute myeloid leukemia

Roundabout guidance receptor proteins are crucial components of the SLIT/ROBO signaling pathway. This pathway is important for the nervous system and in embryonic development. Recently, increasing evidence has shown that roundabout guidance receptor proteins and the SLIT/ROBO signaling pathway also participate in tumorigenesis. Here, by analyzing transcriptome data from the TCGA and GEO databases, we found that ROBO3 is highly expressed in non-M3 acute myeloid leukemia. High ROBO3 expression was associated with increased age at diagnosis and poorer risk classification (both P < 0.01). Patients with high ROBO3 expression had higher rates of TP53 and RUNX1 mutations (both P < 0.05). Significantly worse overall survival and event-free survival were observed in high ROBO3 expression patients compared with low ROBO3 expression patients (OS: P = 0.004; EFS: P= 0.012). High ROBO3 expression was also associated with poorer overall survival and event-free survival in a subgroup of patients who received intensive chemotherapy (OS: P = 0.024; EFS: P = 0.040). Moreover, multivariate analysis indicated that high ROBO3 expression was an independent risk factor for poor overall survival in non-M3 acute myeloid leukemia patients who are younger than 60 and received intensive chemotherapy during remission induction. Bioinformatics analysis by Kyoto Encyclopedia of Genes and Genomes and Gene Ontology revealed that high ROBO3 expression significantly altered cell adhesion and extracellular matrix-related pathways (adjusted P < 0.05). Taken together, the data demonstrate that ROBO3 is upregulated in non-M3 acute myeloid leukemia and may be a potent biomarker of inferior prognosis.

Abstract PR009: The functional genomic landscape of recurrent glioblastoma

Abstract Glioblastoma (GBM) is a highly fatal brain cancer where mortality is predominantly caused by disease recurrence and lack of second-line therapies. Here, utilizing genome-wide CRISPR-Cas9 screening, we uncover treatment-based conditional genetic interactions modulating sensitivity to combined radiation therapy (RT) and temozolomide (TMZ), the standard of care treatment administered to over 70% of glioblastoma patients. To investigate therapy-induced changes in the functional landscape of glioblastoma stem cells (GSCs) without confounding patient-to-patient differences, we systematically mapped genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent GSCs. This comparison highlights a remodelling of genes and pathways regulating tumor cell survival at disease relapse, arming recurrent GSCs with newly acquired genetic drivers and further loss of tumor suppressors. Among these genes, we identify PTP4A2, encoding protein tyrosine phosphatase 4A2, as a major regulator of stemness and tumorigenicity in recurrent GSCs. Genetic perturbation or pharmacological inhibition of PTP4A2 influences the phosphorylation status and expression of interacting proteins belonging to axonal guidance pathways, as phosphoproteomic studies showed specific enrichment in axon guidance regulator Roundabout Guidance Receptor 1 (ROBO1) in recurrent GSCs. We therefore designed and developed efficacious Camelid biotinylated single-domain antibody (bio-sdAb) and tetrameric complexes targeting human ROBO1 (MKRo-20). Subsequently, we orthotopically engrafted recurrent GSCs into immunocompromised mice and administered local treatments of tetrameric MKRo-20. All mice treated with tetrameric MKRo-20 were rendered tumor free, suggesting that modulation of ROBO1 with bio-sdAbs presents a new immunotherapeutic strategy to target recurrent glioblastoma. Our work reveals a context-specific dependence on axonal guidance through a PTP4A2-ROBO axis in recurrent glioblastoma, highlighting new avenues of therapeutic intervention for second-line therapy in GBM. This abstract is also being presented as PO025. Citation Format: Chirayu Chokshi, David Tieu, Kevin Brown, Chitra Venugopal, Lina Liu, Laura Kuhlman, Katherine Chan, Amy Tong, Neil Savage, Dillon McKenna, Nikoo Aghaei, Minomi Subapanditha, Joseph M Salamoun, Martin Rossotti, Peter Wipf, Elizabeth Sharlow, John S. Lazo, Thomas Kislinger, Kevin Henry, Yu Lu, Jason Moffat, Sheila K. Singh. The functional genomic landscape of recurrent glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR009.

Plexin-B2 facilitates glioblastoma infiltration by modulating cell biomechanics

Infiltrative growth is a major cause of high lethality of malignant brain tumors such as glioblastoma (GBM). We show here that GBM cells upregulate guidance receptor Plexin-B2 to gain invasiveness. Deletion of Plexin-B2 in GBM stem cells limited tumor spread and shifted invasion paths from axon fiber tracts to perivascular routes. On a cellular level, Plexin-B2 adjusts cell adhesiveness, migratory responses to different matrix stiffness, and actomyosin dynamics, thus empowering GBM cells to leave stiff tumor bulk and infiltrate softer brain parenchyma. Correspondingly, gene signatures affected by Plexin-B2 were associated with locomotor regulation, matrix interactions, and cellular biomechanics. On a molecular level, the intracellular Ras-GAP domain contributed to Plexin-B2 function, while the signaling relationship with downstream effectors Rap1/2 appeared variable between GBM stem cell lines, reflecting intertumoral heterogeneity. Our studies establish Plexin-B2 as a modulator of cell biomechanics that is usurped by GBM cells to gain invasiveness.

Semaphorins and Plexins in central nervous system patterning: the key to it all?

Semaphorins and Plexins constitute one of the largest family of guidance molecules and receptors involved in setting critical biological steps for central nervous system development. The role of these molecules in axonal development has been extensively characterized but Semaphorins and Plexins are also involved in a variety of other developmental processes, spanning from cell polarization to migration, laminar segregation and neuronal maturation. In this review, we aim to gather discoveries carried in the field of neurodevelopment over the last decade, during which Semaphorin/Plexin complexes have emerged as key regulators of neurogenesis, neural cell migration and adult gliogenesis. As well, we report mechanisms that brought a better understanding of axonal midline crossing.

MiR-140-5p regulates vascular smooth muscle cell viability, migration and apoptosis by targeting ROBO4 gene expression in atherosclerosis

MicroRNAs (miRs) are essential regulators of atherosclerosis (AS) development; however, the pathogenic roles of miR-140-5p during AS development are not completely understood. The present study investigated the effects of miR-140-5p on human vascular smooth muscle cells (VSMCs) and its target gene. miR-140-5p and roundabout guidance receptor 4 (ROBO4) mRNA expression levels were determined by performing reverse transcription-quantitative PCR. ROBO4 protein expression levels were analyzed via western blotting. Cell viability, migration, invasion and apoptosis were evaluated by conducting Cell Counting Kit-8, Transwell and flow cytometry assays, respectively. The binding of miR-140-5p to ROBO4 mRNA was verified using the dual-luciferase reporter assay. miR-140-5p was highly expressed in the plaque-containing artery tissues of patients with AS compared with healthy control tissues. Oxidized-low density lipoprotein (ox-LDL) treatment increased miR-140-5p expression and decreased ROBO4 expression in human VSMCs, which promoted VSMC viability, migration and invasion, but suppressed apoptosis compared with the control group. The effects of ox-LDL treatment on VSMCs were attenuated by miR-140-5p inhibitor. miR-140-5p directly bound to the 3′-untranslated region of ROBO4 mRNA. ROBO4 overexpression mitigated the effects of ox-LDL treatment on VSMC viability, migration, invasion and apoptosis. Therefore, the present study suggested that high level miR-140-5p expression promoted VSMC viability, migration, and invasion, and suppressed VSMC apoptosis by reducing ROBO4 gene expression. The present study provided novel insights into AS pathogenesis that may aid the development of new strategies for the treatment and prevention of AS.

New insights into the molecular mechanisms of axon guidance receptor regulation and signaling.

As the nervous system develops, newly differentiated neurons need to extend their axons toward their synaptic targets to form functional neural circuits. During this highly dynamic process of axon pathfinding, guidance receptors expressed at the tips of motile axons interact with soluble guidance cues or membrane tethered molecules present in the environment to be either attracted toward or repelled away from the source of these cues. As competing cues are often present at the same location and during the same developmental period, guidance receptors need to be both spatially and temporally regulated in order for the navigating axons to make appropriate guidance decisions. This regulation is exerted by a diverse array of molecular mechanisms that have come into focus over the past several decades and these mechanisms ensure that the correct complement of surface receptors is present on the growth cone, a fan-shaped expansion at the tip of the axon. This dynamic, highly motile structure is defined by a lamellipodial network lining the periphery of the growth cone interspersed with finger-like filopodial projections that serve to explore the surrounding environment. Once axon guidance receptors are deployed at the right place and time at the growth cone surface, they respond to their respective ligands by initiating a complex set of signaling events that serve to rearrange the growth cone membrane and the actin and microtubule cytoskeleton to affect axon growth and guidance. In this review, we highlight recent advances that shed light on the rich complexity of mechanisms that regulate axon guidance receptor distribution, activation and downstream signaling.

PlexinA4-Semaphorin3A-mediated crosstalk between main cortical interneuron classes is required for superficial interneuron lamination

In the mammalian cerebral cortex, the developmental events governing allocation of different classes of inhibitory interneurons (INs) to distinct cortical layers are poorly understood. Here we report that the guidance receptor PlexinA4 (PLXNA4) is upregulated in serotonin receptor 3a-expressing (HTR3A+) cortical INs (hINs) as they invade the cortical plate, and that it regulates their laminar allocation to superficial cortical layers. We find that the PLXNA4 ligand Semaphorin3A (SEMA3A) acts as a chemorepulsive factor on hINs migrating into the nascent cortex and demonstrate that SEMA3A specifically controls their laminar positioning through PLXNA4. We identify deep-layer INs as a major source of SEMA3A in the developing cortex and demonstrate that targeted genetic deletion of Sema3a in these INs specifically affects laminar allocation of hINs. These data show that, in the neocortex, deep-layer INs control laminar allocation of hINs into superficial layers.

UNC5B Promotes Vascular Endothelial Cell Senescence via the ROS‐Mediated P53 Pathway

Vascular endothelial cell senescence is involved in human aging and age-related vascular disorders. Guidance receptor UNC5B is implicated in oxidative stress and angiogenesis. Nonetheless, little is known about the role of UNC5B in endothelial cell senescence. Here, we cultured primary human umbilical vein endothelial cells to young and senescent phases. Subsequently, the expression of UNC5B was identified in replicative senescent cells, and then, its effect on endothelial cell senescence was confirmed by UNC5B-overexpressing lentiviral vectors and RNA interference. Overexpression of UNC5B in young endothelial cells significantly increased senescence-associated β-galactosidase-positive cells, upregulated the mRNAs expression of the senescence-associated secretory phenotype genes, reduced total cell number, and inhibited the potential for cell proliferation. Furthermore, overexpression of UNC5B promoted the generation of intracellular reactive oxygen species (ROS) and activated the P53 pathway. Besides, overexpression of UNC5B disturbed endothelial function by inhibiting cell migration and tube formation. Nevertheless, silencing UNC5B generated conflicting outcomes. Blocking ROS production or inhibiting the function of P53 rescued endothelial cell senescence induced by UNC5B. These findings suggest that UNC5B promotes endothelial cell senescence, potentially by activating the ROS-P53 pathway. Therefore, inhibiting UNC5B might reduce endothelial cell senescence and hinder age-related vascular disorders.

Cables1 Links Slit/Robo and Wnt/Frizzled Signaling in Commissural Axon Guidance

During neural circuit formation, axons navigate from one intermediate target to the next, until they find their final target. At intermediate targets, axons switch from being attracted to being repelled by changing the guidance receptors on the growth cone surface. For smooth navigation of the intermediate target and the continuation of their journey, the switch in receptor expression has to be orchestrated in a precisely timed manner. As an alternative to changes in expression, receptor function could be regulated by phosphorylation of receptors or components of signaling pathways. We identifed Cables1 as a linker between floor-plate exit of commissural axons regulated by Slit/Robo signaling and the rostral turn of post-crossing axons regulated by Wnt/Frizzled signaling. Cables1 localizes β-Catenin phosphorylated at tyrosine 489 by Abelson kinase to the distal axon, which in turn is necessary for the correct navigation of post-crossing commissural axons in the developing chicken spinal cord.