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Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma, often leads to a poor prognosis due to metastasis. The investigation of N6-methyladenosine (m 6 A) methylation, a crucial RNA modification, and its role in ccRCC, particularly through the m 6 A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), revealed significant insights. We found that IGF2BP2 was notably downregulated in ccRCC, which correlated with tumor aggressiveness and poor prognosis. Thus, IGFBP2 has emerged as an independent prognostic factor of ccRCC. Moreover, a strong positive correlation was observed between the expression of IGF2BP2 and Netrin-4. Netrin-4 was also downregulated in ccRCC, and its lower levels were associated with increased malignancy and poor prognosis. Overexpression of IGF2BP2 and Netrin-4 suppressed the invasion and migration of ccRCC cells, while Netrin-4 knockdown reversed these effects in ccRCC cell lines. RNA immunoprecipitation (RIP)-quantitative polymerase chain reaction validated the robust enrichment of Netrin-4 mRNA in anti-IGF2BP2 antibody immunoprecipitates. MeRlP showed significantly increased Netrin4 m 6 A levels after lGF2BP2 overexpression. Moreover, we found that IGF2BP2 recognized and bound to the m 6 A site within the coding sequence of Netrin-4, enhancing its mRNA stability. Collectively, these results showed that IGF2BP2 plays a suppressive role in the invasion and migration of ccRCC cells by targeting Netrin-4 in an m 6 A-dependent manner. These findings underscore the potential of IGF2BP2/Netrin-4 as a promising prognostic biomarker and therapeutic target in patients with ccRCC metastasis., (© 2024 Wiley Periodicals LLC.)

Axon projection is a spatial- and temporal-specific process in which the growth cone receives environmental signals guiding axons to their final destination. However, the mechanisms underlying changes in axonal projection direction without well-defined landmarks remain elusive. Here, we present evidence showcasing the dynamic nature of axonal projections in Drosophila 's small ventral lateral clock neurons (s-LNvs). Our findings reveal that these axons undergo an initial vertical projection in the early larval stage, followed by a subsequent transition to a horizontal projection in the early-to-mid third instar larvae. The vertical projection of s-LNv axons correlates with mushroom body calyx expansion, while the s-LNv-expressed Down syndrome cell adhesion molecule (Dscam1) interacts with Netrins to regulate the horizontal projection. During a specific temporal window, locally newborn dorsal clock neurons secrete Netrins, facilitating the transition of axonal projection direction in s-LNvs. Our study establishes a compelling in vivo model to probe the mechanisms of axonal projection direction switching in the absence of clear landmarks. These findings underscore the significance of dynamic local microenvironments in the complementary regulation of axonal projection direction transitions., Competing Interests: JL, YW, XL, JH, YT No competing interests declared, (© 2024, Liu et al.)

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis., Competing Interests: PP, JM, KZ, DP, SW, YR, MH, RR, BG, RC, TS, FD No competing interests declared, PM Founder of Netris Pharma, (© 2023, Perampalam et al.)

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding-deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.

Netrin G1 (NTNG1) is a member of the Netrin family and plays a crucial role in various human cancers. However, the molecular functions of NTNG1 in HCC and the underlying mechanisms remain unclear. HCC expression data was obtained from the GEO database and analyzed using various bioinformatics tools. The expression of NTNG1 in HCC tissues and liver cancer cells was evaluated through RT-qPCR and western blotting. Cells with stable NTNG1 overexpression and knockdown were established, and CCK-8, colony formation, and flow cytometry assays were conducted in vitro. The xenograft model was utilized to verify the tumorigenesis capacity of NTNG1 in vivo. IHC was employed to analyze the expression of NTNG1 and CD163 proteins. HCC-specific genes were screened, followed by functional enrichment and immune cell infiltration analysis. Finally, the Co-IP was used to detect the interaction between NTNG1 and N-cadherin. NTNG1 was highly expressed in HCC tissues and liver cancer cells, and associated with significantly poorer OS rates. In addition, NTNG1 overexpression in liver cancer cells significantly increased their proliferation, colony growth, invasion, migration, and EMT, while inhibiting apoptosis. Bioinformatics analyses indicated that NTNG1 was closely related to EMT and tumor infiltration. IHC staining revealed a positive correlation between NTNG1 expression and CD163 in HCC tissues. Additionally, an EMT inhibitor attenuated the expression levels of EMT-related markers and counteracted the effects of NTNG1 overexpression in liver cancer cells. This study is the first to identify NTNG1 as a potential therapeutic target in HCC, promoting tumor development and progression by regulating EMT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Endometrial carcinoma (EC) is a common type of uterine cancer in developed countries, originating from the uterine epithelium. The incidence rate of EC in Taiwan has doubled from 2005. Cancer stem cells (CSCs) are a subpopulation of cancer cells that have high tumorigenicity and play a crucial role in the malignant processes of cancer. Targeting molecules associated with CSCs is essential for effective cancer treatments. This study delves into the role of Exosome component 5 (EXOSC5) in EC. Data from The Cancer Genome Atlas suggests a correlation between high EXOSC5 mRNA expression and unfavorable EC prognosis. EXOSC5 knockdown diminished EC-CSC self-renewal and reduced expression of key cancer stemness proteins, including c-MYC and SOX2. Intriguingly, this knockdown significantly curtailed tumorigenicity and CSC frequency in EC tumor spheres. A mechanistic examination revealed a reduction in netrin4 (NTN4) levels in EXOSC5-depleted EC cells. Moreover, NTN4 treatment amplified EC cell CSC activity and, when secreted, NTN4 partnered with integrin β1, subsequently triggering the FAK/SRC axis to elevate c-MYC activity. A clear positive relation between EXOSC5 and NTN4 was evident in 93 EC tissues. In conclusion, EXOSC5 augments NTN4 expression, activating c-MYC via the integrin β1/FAK/SRC pathway, offering potential avenues for EC diagnosis and treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The cyanotoxin cylindrospermopsin (CYN) has been postulated to cause neurotoxicity, although the studies in this concern are very few. In addition, some studies in vitro indicate its possible effects on development. Furthermore, pesticides can be present in the same environmental samples as cyanotoxins. Therefore, chlorpyrifos (CPF) has been one of the most common pesticides used worldwide. The aim of this report was to study the effects of CYN, isolated and in combination with CPF, in a developmental neurotoxicity in vitro model. The human neuroblastoma SH-SY5Y cell line was exposed during 6 days of differentiation to both toxics to study their effects on cell viability and neurite outgrowth. To further evaluate effects of both toxicants on cholinergic signaling, their agonistic and antagonistic activities on the α7 homomeric nicotinic acetylcholine receptor (nAChR) were studied upon acute exposure. Moreover, a transcriptomic analysis by qPCR was performed after 6 days of CYN-exposure during differentiation. The results showed a concentration-dependent decrease on both cell viability and neurite outgrowth for both toxics isolated, leading to effective concentration 20 (EC 20 ) values of 0.35 µM and 0.097 µM for CYN on cell viability and neurite outgrowth, respectively, and 100 µM and 58 µM for CPF, while the combination demonstrated no significant variations. In addition, 95 µM and 285 µM CPF demonstrated to act as an antagonist to nicotine on the nAChR, although CYN up to 2.4 µM had no effect on the efficacy of these receptors. Additionally, the EC 20 for CYN (0.097 µM) on neurite outgrowth downregulated expression of the 5 genes NTNG2 (netrin G2), KCNJ11 (potassium channel), SLC18A3 (vesicular acetylcholine transporter), APOE (apolipoprotein E), and SEMA6B (semaphorin 6B), that are all important for neuronal development. Thus, this study points out the importance of studying the effects of CYN in terms of neurotoxicity and developmental neurotoxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Axon guidance molecules (AGM) are critical regulators of neural development and play a vital role in guiding axons to their target regions during spinal cord development. The correct wiring of neural circuits depends on these molecules' precise expression and function. Defects in axonal pathfinding, growth cone navigation, axonal branching, and synapse formation have far-reaching implications for neuronal circuit construction and function after CNS traumas, such as spinal cord injury (SCI), which affect the expression or activity of AGM. Ascending and descending paths in the spinal cord have been found to include many AGM, including Netrins, Slits, Semaphorins (Sema), Ephrins, and their receptors. In contrast to the repulsive signals like Slits and Semaphorins, which restrict axonal growth and guide axons away from unsuitable locations, Netrins are appealing guidance cues that encourage axonal growth and guidance. Defects in motor function and sensory processing can result from changes in the expression or activity of Ephrins or their receptors, which play an essential role in axonal guidance and synaptic plasticity in the spinal cord. Herein, we highlighted the expressions, functions, and mechanisms of AGM in ascending and descending spinal cord tracts, which can help us identify novel therapeutic targets to improve axonal regeneration and functional recovery after SCI., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

It was recently reported that netrin‑4 (Ntn‑4), a component of the extracellular matrix, when downregulated, is involved in the progression of several types of cancer, including breast cancer, colorectal tumours, neuroblastoma and gastric cancer. In the present study, the level of Ntn‑4 was examined in a public non‑small cell lung cancer (NSCLC) dataset from the Netherlands Cancer Institute. This analysis revealed that the mRNA expression level of Ntn‑4 was lower in the samples of patients with NSCLC compared with that in the control samples. Consistent with the mRNA level, the protein level of Ntn‑4 was also found to be decreased in NSCLC cells. However, both the function of Ntn‑4 and the underlying mechanisms of Ntn‑4 downregulation in NSCLC have yet to be fully elucidated. As was anticipated, the overexpression of Ntn‑4 led to a marked decrease in the proliferation, migration and invasion of NSCLC cells. Notably, RNA‑binding protein quaking 5 (Qki‑5) was found to exhibit antitumor activity in lung cancer, not only by enhancing the level of Ntn‑4 by binding to Ntn‑4 mRNA, but also by suppressing the proliferation, invasion and migration of NSCLC cells. However, Qki‑5 is known to be frequently downregulated in NSCLC. Moreover, the knockdown of Ntn‑4 was found to reverse the suppressive effects of Qki‑5 on NSCLC progression both in vitro and in vivo . Taken together, the findings of the present study demonstrate that Ntn‑4 is able to suppress the progression of NSCLC, and that the level of Ntn‑4 can be regulated by Qki‑5. Therefore, Ntn‑4 may be a novel diagnostic and therapeutic target for the treatment of NSCLC.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Notch-dependent binary fate choice between sister neurons is one of the mechanisms to generate neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well understood. Here, we report that Notch-dependent binary fate choice in Drosophila medulla neurons is required to regulate the Netrin axon guidance pathway, which controls targeting of transmedullary (Tm) neurons to lobula. In medulla neurons of Notch-on hemilineage composed of mostly lobula-targeting neurons, Notch signaling is required to activate the expression of Netrin-B and repress the expression of its repulsive receptor Unc-5. Turning off Unc-5 is necessary for Tm neurons to target lobula. Furthermore, Netrin-B provided by Notch-on medulla neurons is required for correct targeting of Tm axons from later-generated medulla columns. Thus, the coordinate regulation of Netrin pathway components by Notch signaling ensures correct targeting of Tm axons and contributes to the neuropil assembly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

How axon guidance pathways are utilized in coordination with temporal and spatial patterning of neural progenitors to regulate neuropil assembly is not well understood. We study this question in the Drosophila medulla using the transmedullary (Tm) projection neurons that target lobula through the inner optic chiasm (IOC). We demonstrate that the Netrin pathway plays multiple roles in guidance of Tm axons and that temporal patterning of medulla neuroblasts determines pioneer versus follower Tm neurons during this process. Loss of Frazzled (Fra) in early-born pioneer Tm neurons leads to IOC defects, while loss of Fra from follower neurons does not affect the IOC. In the follower projection neurons, Fra is required in other targeting steps including lobula branch extension and layer-specific targeting. Furthermore, different from other identified scenarios of Netrin/Fra involved axon guidance in Drosophila, we demonstrate that diffusible Netrin is required for the correct axon targeting and optic lobe organization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Olfactory sensory neurons that express related odorant receptors specifically target large identifiable neuropils called protoglomeruli when they first reach the olfactory bulb in the zebrafish. This crude odorant receptor-related mapping is further refined as odorant receptor-specific glomeruli segregate from protoglomeruli later in development. Netrins are a prominent class of axon guidance molecules whose contribution to olfactory circuit formation is poorly studied. Morpholino knock down experiments have suggested that Netrin/Dcc signaling is involved in normal protoglomerular targeting. Here we extend these findings with more detailed characterization and modeling of netrin expression, and by examining protoglomerular targeting in mutant lines fornetrin1a (ntn1a), netrin1b (ntn1b), and their receptorsunc5b,dcc, andneo1a. We confirm thatntn1a,ntn1b, anddccare required for normal protoglomerular guidance of a subset of olfactory sensory neurons that are labeled with the Tg(or111-7:IRES:Gal4) transgene. We also observe errors in the targeting of these axons inunc5bmutants, but not inneo1a mutants. Our findings are consistent with ntn1a andntn1bacting primarily as attractants for olfactory sensory neurons targeting the central zone protoglomerulus., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordianted-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production., Results: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks., Conclusion: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages., (© 2022 American Association for Anatomy.)

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Low-threshold mechanoreceptors (LTMRs) and their cutaneous end organs convert light mechanical forces acting on the skin into electrical signals that propagate to the central nervous system. In mouse hairy skin, hair follicle-associated longitudinal lanceolate complexes, which are end organs comprising LTMR axonal endings that intimately associate with terminal Schwann cell (TSC) processes, mediate LTMR responses to hair deflection and skin indentation. Here, we characterized developmental steps leading to the formation of Aβ rapidly adapting (RA)-LTMR and Aδ-LTMR lanceolate complexes. During early postnatal development, Aβ RA-LTMRs and Aδ-LTMRs extend and prune cutaneous axonal branches in close association with nascent TSC processes. Netrin-G1 is expressed in these developing Aβ RA-LTMR and Aδ-LTMR lanceolate endings, and Ntng1 ablation experiments indicate that Netrin-G1 functions in sensory neurons to promote lanceolate ending elaboration around hair follicles. The Netrin-G ligand (NGL-1), encoded by Lrrc4c , is expressed in TSCs, and ablation of Lrrc4c partially phenocopied the lanceolate complex deficits observed in Ntng1 mutants. Moreover, NGL-1-Netrin-G1 signaling is a general mediator of LTMR end organ formation across diverse tissue types demonstrated by the fact that Aβ RA-LTMR endings associated with Meissner corpuscles and Pacinian corpuscles are also compromised in the Ntng1 and Lrrc4c mutant mice. Thus, axon-glia interactions, mediated in part by NGL-1-Netrin-G1 signaling, promote LTMR end organ formation.

Background: Physical activity (PA) prior to and during pregnancy may have intergenerational effects on offspring health through placental epigenetic modifications. We are unaware of epidemiologic studies on longitudinal PA and placental DNA methylation., Objectives: We evaluated the association between PA before and during pregnancy and placental DNA methylation., Methods: Placental tissues were obtained at delivery and methylation was measured using HumanMethylation450 Beadchips for participants in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Fetal Growth Studies-Singletons among 298 participants. Using the Pregnancy Physical Activity Questionnaire, women recalled periconception PA (past 12 mo) at 8-13 wk of gestation and PA since last visit at 4 follow-up visits at 16-22, 24-29, 30-33, and 34-37 wk. We conducted linear regression for associations of PA at each visit with methylation controlling for false discovery rate (FDR). Top 100 CpGs were queried for enrichment of functional pathways using Ingenuity Pathway Analysis., Results: Periconception PA was significantly associated with 1 CpG site. PA since last visit for visits 1-4 was associated with 2, 2, 8, and 0 CpGs (log fold changes ranging from -0.0319 to 0.0080, after controlling for FDR). The largest change in methylation occurred at a site in TIMP2 , which is known to encode a protein critical for vasodilation, placentation, and uterine expansion during pregnancy (log fold change: -0.05; 95% CI: -0.06, -0.03 per metabolic equivalent of task-h/wk at 30-33 wk). Most significantly enriched pathways include cardiac hypertrophy signaling, B-cell receptor signaling, and netrin signaling. Significant CpGs and enriched pathways varied by visit., Conclusions: Recreational PA in the year prior and during pregnancy was associated with placental DNA methylation. The associated CpG sites varied based on timing of PA. If replicated, the findings may inform the mechanisms underlying the impacts of PA on placenta health. This study was registered at clinicaltrials.gov as NCT00912132., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Most genome-wide association study (GWAS)-identified breast cancer-associated causal variants remain uncharacterized. To provide a framework of understanding GWAS-identified variants to function, we performed a comprehensive study of noncoding regulatory variants at the NTN4 locus (12q22) and NTN4 gene in breast cancer etiology. We find that rs11836367 is the more likely causal variant, disrupting enhancer activity in both enhancer reporter assays and endogenous genome editing experiments. The protective T allele of rs11837367 increases the binding of GATA3 to the distal enhancer and up-regulates NTN4 expression. In addition, we demonstrate that loss of NTN4 gene in mice leads to tumor earlier onset, progression, and metastasis. We discover that NTN4 , as a tumor suppressor, can attenuate the Wnt signaling pathway by directly binding to Wnt ligands. Our findings bridge the gaps among breast cancer-associated single-nucleotide polymorphisms, transcriptional regulation of NTN4 , and breast cancer biology, which provides previously unidentified insights into breast cancer prediction and prevention.

Axon guidance proteins are essential for axonal pathfinding during development. In adulthood, they have been described as pleiotropic proteins with multiple roles in different organs and tissues. While most studies on the roles of these proteins in the cornea have been performed on the Semaphorin family members, with few reports on Netrins or Ephrins, their function in corneal epithelium wound healing and functional nerve regeneration is largely unknown. Here, we studied the expression of ligands belonging to three distinct axon guidance families (Semaphorins, Ephrins, and Netrins) and their most commonly associated receptors in the cornea and trigeminal ganglia (TG) using immunofluorescence staining and RT-qPCR. We also evaluated how their expression recovers after corneal epithelium injury. We found that all ligands studied (Sema3A, Sema3F, EphrinB1, EphrinB2, Netrin-1, and Netrin-4) are abundantly expressed in both the TG and corneal epithelium. Similarly, their receptors (Neuropilin-1, Neuropilin-2, PlexinA1, PlexinA3, EphB2, EphB4, Neogenin, UNC5H1 and DCC) are also expressed in both tissues. Upon corneal epithelium injury, quick recovery of both ligands and receptors was observed at the protein and gene expression levels. While the timing and expression levels vary among these proteins, in general, most of them remained upregulated for several weeks after injury. We propose that the initial protein expression recovery may be related to corneal epithelium recovery since Sema3A, EphrinB2 and Netrin-4 accelerated corneal epithelial cells wound healing. The sustained high expression levels may be functionally related to nerve regeneration and/or patterning. Whilst further studies are required to test this hypothesis, this work contributes to unraveling their function in normal and injured cornea., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of Caenorhabditis elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP, and UNC-34/Enabled. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Epithelial remodelling plays a crucial role during development. The ability of epithelial sheets to temporarily lose their integrity as they fuse with other epithelial sheets underpins events such as the closure of the neural tube and palate. During fusion, epithelial cells undergo some degree of epithelial-mesenchymal transition (EMT), whereby cells from opposing sheets dissolve existing cell-cell junctions, degrade the basement membrane, extend motile processes to contact each other, and then re-establish cell-cell junctions as they fuse. Similar events occur when an epithelium is wounded. Cells at the edge of the wound undergo a partial EMT and migrate towards each other to close the gap. In this review, we highlight the emerging role of Netrins in these processes, and provide insights into the possible signalling pathways involved. Netrins are secreted, laminin-like proteins that are evolutionarily conserved throughout the animal kingdom. Although best known as axonal chemotropic guidance molecules, Netrins also regulate epithelial cells. For example, Netrins regulate branching morphogenesis of the lung and mammary gland, and promote EMT during Drosophila wing eversion. Netrins also control epithelial fusion during optic fissure closure and inner ear formation, and are strongly implicated in neural tube closure and secondary palate closure. Netrins are also upregulated in response to organ damage and epithelial wounding, and can protect against ischemia-reperfusion injury and speed wound healing in cornea and skin. Since Netrins also have immunomodulatory properties, and can promote angiogenesis and re-innervation, they hold great promise as potential factors in future wound healing therapies., (© 2021 S. Karger AG, Basel.)

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

The adult nervous system has a limited capacity to regenerate after accidental damage. Post-injury functional restoration requires proper targeting of the injured axon to its postsynaptic cell. Although the initial response to axonal injury has been studied in great detail, it is rather unclear what controls the re-establishment of a functional connection. Using the posterior lateral microtubule neuron in Caenorhabditis elegans, we found that after axotomy, the regrowth from the proximal stump towards the ventral side and accumulation of presynaptic machinery along the ventral nerve cord correlated to the functional recovery. We found that the loss of insulin receptor DAF-2 promoted 'ventral targeting' in a DAF-16-dependent manner. We further showed that coordinated activities of DAF-16 in neuron and muscle promoted 'ventral targeting'. In response to axotomy, expression of the Netrin receptor UNC-40 was upregulated in the injured neuron in a DAF-16-dependent manner. In contrast, the DAF-2-DAF-16 axis contributed to the age-related decline in Netrin expression in muscle. Therefore, our study revealed an important role for insulin signaling in regulating the axon guidance molecules during the functional rewiring process., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated β-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neuropathic pain refers to pain caused by lesions or diseases of the somatosensory nervous system that is characteristically different from nociceptive pain. Moreover, neuropathic pain occurs in the maxillofacial region due to various factors and is treated using tricyclic antidepressants and nerve block therapy; however, some cases do not fully recover. Netrin is a secreted protein crucially involved in neural circuit formation during development, including cell migration, cell death, neurite formation, and synapse formation. Recent studies show Netrin-4 expressed in the dorsal horn of the spinal cord is associated with chronic pain. Here we found involvement of Netrin-4 in neuropathic pain in the maxillofacial region. Netrin-4, along with one of its receptors, Unc5B, are expressed in the caudal subnucleus of the trigeminal spinal tract nucleus. Inhibition of its binding by anti-Netrin-4 antibodies not only shows a behavioral analgesic effect but also neuronal activity suppression. There was increased Netrin-4 expression at 14 days after infraorbital nerve injury. Our findings suggest that Netrin-4 induced by peripheral nerve injury causes neuropathic pain via Unc5B., Competing Interests: The authors have declared that no competing interests exist.

Diabetic retinopathy is characterized by dysfunction of the retinal vascular network, combined with a persistent low-grade inflammation that leads to vision-threatening complications. Netrin-4 (NTN4) is a laminin-related secreted protein and guidance cue molecule present in the vascular basal membrane and highly expressed in the retina. A number of studies inferred that the angiogenic abilities of NTN4 could contribute to stabilize vascular networks and modulate inflammation. Analyzing human specimens, we show that NTN4 and netrin receptors are upregulated in the diabetic retina. We further evaluated a knock-out model for NTN4 undergoing experimental diabetes induced by streptozotocin. We investigated retina function and immune cells in vivo and demonstrated that NTN4 provides a protective milieu against inflammation in the diabetic retina and prevents cytokine production.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1 + cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1 + CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230 . This article is highlighted in the In This Issue feature, p. 211 ., (©2020 American Association for Cancer Research.)

Axonal trajectories and neural circuit activities strongly rely on a complex system of molecular cues that finely orchestrate the patterning of neural commissures. Several of these axon guidance molecules undergo continuous recycling during brain development, according to incompletely understood intracellular mechanisms, that in part rely on endocytic and autophagic cascades. Based on their pivotal role in both pathways, lysosomes are emerging as a key hub in the sophisticated regulation of axonal guidance cue delivery, localization, and function. In this review, we will attempt to collect some of the most relevant research on the tight connection between lysosomal function and axon guidance regulation, providing some proof of concepts that may be helpful to understanding the relation between lysosomal storage disorders and neurodegenerative diseases.

Frequently reported neurotoxic sequelae of cancer treatment include cognitive deficits and sensorimotor abnormalities that have long-lasting negative effects on the quality of life of an increasing number of cancer survivors. The underlying mechanisms are not fully understood and there is no effective treatment. We show here that cisplatin treatment of mice not only caused cognitive dysfunction but also impaired sensorimotor function. These functional deficits are associated with reduced myelin density and complexity in the cingulate and sensorimotor cortex. At the ultrastructural level, myelin abnormalities were characterized by decompaction. We used this model to examine the effect of bexarotene, an agonist of the RXR-family of nuclear receptors. Administration of only five daily doses of bexarotene after completion of cisplatin treatment was sufficient to normalize myelin density and fiber coherency and to restore myelin compaction in cingulate and sensorimotor cortex. Functionally, bexarotene normalized performance of cisplatin-treated mice in tests for cognitive and sensorimotor function. RNAseq analysis identified the TR/RXR pathway as one of the top canonical pathways activated by administration of bexarotene to cisplatin-treated mice. Bexarotene also activated neuregulin and netrin pathways that are implicated in myelin formation/maintenance, synaptic function and axonal guidance. In conclusion, short term treatment with bexarotene is sufficient to reverse the adverse effects of cisplatin on white matter structure, cognitive function, and sensorimotor performance. These encouraging findings warrant further studies into potential clinical translation and the underlying mechanisms of bexarotene for chemobrain.

A new study in Caenorhabditis elegans suggests the ubiquitin-proteasome system promotes degradation of the netrin receptor UNC-40 in a particular neuron only in one sex, leading to sex-specific patterns of synaptic connections., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Breast cancer genome-wide association studies (GWASs) have identified 150 genomic risk regions containing more than 13,000 credible causal variants (CCVs). The CCVs are predominantly noncoding and enriched in regulatory elements. However, the genes underlying breast cancer risk associations are largely unknown. Here, we used genetic colocalization analysis to identify loci at which gene expression could potentially explain breast cancer risk phenotypes. Using data from the Breast Cancer Association Consortium (BCAC) and quantitative trait loci (QTL) from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Project (TCGA), we identify shared genetic relationships and reveal novel associations between cancer phenotypes and effector genes. Seventeen genes, including NTN4, were identified as potential mediators of breast cancer risk. For NTN4, we showed the rs61938093 CCV at this region was located within an enhancer element that physically interacts with the NTN4 promoter, and the risk allele reduced NTN4 promoter activity. Furthermore, knockdown of NTN4 in breast cells increased cell proliferation in vitro and tumor growth in vivo. These data provide evidence linking risk-associated variation to genes that may contribute to breast cancer predisposition., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

The GABAergic neural circuit is involved in the motile activities of both larval and juvenile sea urchins. Therefore, its function is inherited beyond metamorphosis, despite large scale remodeling of larval organs during that period. However, the initial neural circuit formation mechanism is not well understood, including how glutamate decarboxylase-expressing blastocoelar cells (GADCs) construct the neural circuit along the circumoral ciliary band (a ciliary band-associated strand, CBAS) on the larval body surface. In this study, using whole-mount immunohistochemistry and 3D reconstructed imaging, the ontogenic process of CBAS patterning was studied by focusing on Netrin and the interaction with its receptor, Unc-5. During the early 2-arm pluteus stage, a small number of GADCs egress onto the apical surface of the larval ectoderm. Then, they line up on the circumoral side of the ciliary band, and by being inserted by a further number of GADCs, form longer multicellular strands along the Netrin stripe. Application of a synthetic peptide, CRFNMELYKLSGRKSGGVC of Hp-Netrin, that binds to the immunoglobulin domain of Unc-5 during the prism stage, causes stunted CBAS formation due to inhibition of GADC egression. This also results in reduced ciliary beating. Thus, the Netrin/Unc-5 interaction is involved in the construction and function of the CBAS.

Netrin is a remarkably conserved midline landmark, serving as a chemotactic factor that organizes the bilateral neural architecture in the post-gastrula bilaterian embryos. Netrin signal also guides cell migration in many other neural and non-neural organogenesis events in later developmental stages but has never been found to participate in gastrulation - the earliest cell migration in metazoan embryogenesis. Here, we found that the netrin signaling molecules and their receptors are expressed during gastrulation of the leech Helobdella. Intriguingly, Hau-netrin-1 was expressed in the N lineage, which gives rise in part to the ventral midline of ectoderm, at the onset of gastrulation. We demonstrated that the N lineage is required for the entrance of mesoderm into the germinal band and that misexpression of Hau-netrin-1 in early gastrulation prevented mesoderm from entering the germinal band. Together, these results suggested that Hau-netrin-1 secreted by the N lineage guides mesoderm migration during germinal band assembly. Furthermore, ectopic expression of Hau-netrin-1 after the completion of germinal band assembly disrupted the epibolic migration of the germinal bands in a later stage of gastrulation. Thus, Hau-netrin-1 is likely involved in two distinct events in sequential stages of leech gastrulation: the assembly of germinal bands in early gastrulation and their epibolic migration in mid-gastrulation. Given that the leech netrin is expressed in the precursor cells of the ventral midline during gastrulation, we propose that a heterochronic change from the midline netrin expression had taken place in the evolution of a novel mode of gastrulation in the directly developing leech embryos., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Fluoxetine is one of the most promising drugs for improving clinical outcome in patients with ischemic stroke. This in vivo study investigated the hypothesis that fluoxetine may affect HIF-1α-Netrin/VEGF cascade, angiogenesis and neuroprotection using a rat model of transient middle cerebral artery occlusion (tMCAO). The rats were given fluoxetine or saline after tMCAO for 4 weeks. Then, protein expression of HIF-1α-Netrin/VEGF cascade was examined at 1, 2, 4 weeks after tMCAO. In vivo synchrotron radiation were performed to observe microangiography of ischemic brain after 4 weeks of tMCAO. The infarct size and neurobehavioral test were carried out 1 to 4 weeks after tMCAO. Results revealed that HIF-1α expression was upregulated in fluoxetine-treated group. Similarly, fluoxetine increased protein expression of Netrin and its receptor DCC, VEGF and its receptor VEGFR. Synchrotron radiation angiography revealed more branches in fluoxetine-treated rats. We found no difference of infarct volume between fluoxetine and saline treated rats after 1 week of tMCAO, and ischemia-induced brain atrophy volume in fluoxetine-treated group was attenuated after 4 weeks of tMCAO. Neurological deficits were improved in fluoxetine-treated rats at 3 and 4 weeks after tMCAO. Our results indicated that fluoxetine could upregulate protein expression of HIF-1α-Netrin/VEGF cascade, promote angiogenesis, and improve long-term functional recovery after ischemic stroke., Competing Interests: Declaration of Competing Interest The authors have no competing financial interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The anchor cell (AC) in C. elegans secretes an epidermal growth factor (EGF) homolog that induces adjacent vulval precursor cells (VPCs) to differentiate. The EGF receptor in the nearest VPC sequesters the limiting EGF amounts released by the AC to prevent EGF from spreading to distal VPCs. Here, we show that not only EGFR localization in the VPCs but also EGF polarity in the AC is necessary for robust fate specification. The AC secretes EGF in a directional manner towards the nearest VPC. Loss of AC polarity causes signal spreading and, when combined with MAPK pathway hyperactivation, the ectopic induction of distal VPCs. In a screen for genes preventing distal VPC induction, we identified sra-9 and nlp-26 as genes specifically required for polarized EGF secretion. sra-9(lf) and nlp-26(lf) mutants exhibit errors in vulval fate specification, reduced precision in VPC to AC alignment and increased variability in MAPK activation. sra-9 encodes a seven-pass transmembrane receptor acting in the AC and nlp-26 a neuropeptide-like protein expressed in the VPCs. SRA-9 and NLP-26 may transduce a feedback signal to channel EGF secretion towards the nearest VPC., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Microglia, the resident immune cells of the central nervous system, accumulate along subcerebral projection axons and support neuronal survival during the early postnatal period. It remains unknown how microglia follow an axon-specific distribution pattern to maintain neural circuits. Here, we investigated the mechanisms of microglial accumulation along subcerebral projection axons that were necessary for microglial accumulation in the internal capsule. Screening of molecules involved in this accumulation of microglia to axons of layer V cortical neurons identified netrin-G1, a member of the netrin family of axon guidance molecules with a glycosyl-phosphatidylinositol anchor. Deletion or knockdown of the netrin-G1 gene Ntng1 reduced microglial accumulation and caused loss of cortical neurons. Netrin-G1 ligand-Ngl1 knockout-mice-derived microglia showed reduced accumulation along the axons compared with wild-type microglia. Thus, microglia accumulate around the subcerebral projection axons via NGL1-netrin-G1 signaling and support neuronal survival. Our observations unveil bidirectional neurotrophic interactions between neurons and microglia., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent cancer studies have found that the netrin family of proteins plays vital roles in the development of some cancers. However, the functions of the many variants of these proteins in cancer remain incompletely understood. In this work, we used the most comprehensive database available, including more than 10000 samples across more than 30 tumor types, to analyze the six members of the netrin family. We performed comprehensive analysis of genetic change and expression of the netrin genes and analyzed epigenetic and pathway relationships, as well as the correlation of expression of these proteins with drug sensitivity. Although the mutation rate of the netrin family is low in pan-cancer, among the tumor patients with netrin mutations, the highest number are Uterine Corpus Endometrial Carcinoma patients, accounting for 13.6% of cases (54 of 397). Interestingly, the highest mutation rate of a netrin family member is 38% for NTNG1 (152 of 397). Netrin proteins may participate in the development of endocrine-related tumors and sex hormone-targeting organ tumors. Additionally, the participation of NTNG1 and NTNG2 in various cancers shows their potential for use as new tumor markers and therapeutic targets. This analysis provides a broad molecular perspective of this protein family and suggests some new directions for the treatment of cancer.

Astrocytes have critical functions throughout the central nervous system (CNS) and have emerged as regulators of synaptic development and function. With their highly complex morphologies, they are able to interact with thousands of synapses via peripheral astrocytic processes (PAPs), ensheathing neuronal axons and dendrites to form the tripartite synapse. In this way, astrocytes engage in crosstalk with neurons to mediate a variety of CNS processes including the regulation of extracellular matrix protein signaling, formation and maintenance of the blood-brain barrier (BBB), axon growth and guidance, homeostasis of the synaptic microenvironment, synaptogenesis, and the promotion of synaptic diversity. In this review, we discuss several key astrocyte signaling factors (thrombospondins, netrins, apolipoproteins, neuregulins, bone morphogenetic proteins, and neuroligins) in the maintenance and regulation of synapse formation. We also explore how these astrocyte signaling factors are impacted by and contribute to substance abuse, particularly alcohol and cocaine use., Competing Interests: The authors declare no conflicts of interest.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases., (© 2019 Federation of American Societies for Experimental Biology.)

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease. Because the molecular mechanisms of DKD are not fully understood, exploration of hub genes and the mechanisms underlying this disease are essential for elucidating the pathogenesis and progression of DKD. Accordingly, in this study, we performed an analysis of gene expression in DKD. The differentially expressed genes (DEGs) included 39 upregulated genes and 113 downregulated genes in the GSE30528 dataset and 127 upregulated genes and 18 downregulated genes in the GSE30529 dataset. Additionally, functional analyses were performed to determine the roles of DEGs using glomeruli samples from patients with DKD and healthy controls from the GSE30528 dataset and using tubule samples from patients with DKD and healthy controls from the GSE30529 dataset. These DEGs were enriched in pathways such as the Wnt signaling pathway, metabolic pathways, and the mammalian target of rapamycin signaling pathway in the GSE30528 dataset and the longevity regulating pathway and Ras signaling pathway in the GSE30529 dataset. Moreover, a protein-protein interaction network was constructed using the identified DEGs, and hub gene analysis was performed. Furthermore, correlation analyses between key genes and pathological characteristics of DKD indicated that CCR4, NTNG1, HGF and ISL1 are related to DKD, and NTNG1 and HGF may server as diagnostic biomarkers in DKD using the receiver-operator characteristic (ROC) curve. Collectively, our findings established 2 reliable biomarkers for DKD.

Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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

Purpose: Netrin-4 (NTN4) is a protein that plays an important role in the regulation of angiogenesis in the pathological retina. Some evidences show that it can also have a role in inflammation and vascular stability. We will explore these questions in vivo in the mature mouse retina., Methods: We created a NTN4 knockout that expresses EGFP in mononuclear phagocytes (CSFR1-positive cells) to track inflammation in vivo in the retina by scanning laser ophthalmoscopy (SLO). Fundus angiography permitted to study blood vessels. Retinal function was assessed with electroretinography (ERG)., Results: Lack of NTN4 leads to an increased amount of amoeboid mononuclear phagocytes in the adult retina, and blood vessels displayed increased tortuosity when compared with the wildtype. Inner retina function also seemed affected in NTN4 null. Lack of NTN4 resulted in a higher persistence of hyaloid artery and spontaneous leakage in the adult retina. No differences were found regarding vessel bifurcation, vessel width, or vein/artery ratio., Conclusions: These in vivo data show for the first time that lack of NTN4 induces changes in the retinal vascular phenotype in a non-pathological scenario. This evidence widens the role of NTN4 as a guidance cue in vascular remodeling.

Netrin-G ligand-3 (NGL-3) is a postsynaptic adhesion molecule known to directly interact with the excitatory postsynaptic scaffolding protein postsynaptic density-95 (PSD-95) and trans-synaptically with leukocyte common antigen-related (LAR) family receptor tyrosine phosphatases to regulate presynaptic differentiation. Although NGL-3 has been implicated in the regulation of excitatory synapse development by in vitro studies, whether it regulates synapse development or function, or any other features of brain development and function, is not known. Here, we report that mice lacking NGL-3 (Ngl3-/- mice) show markedly suppressed normal brain development and postnatal survival and growth. A change of the genetic background of mice from pure to hybrid minimized these developmental effects but modestly suppressed N-methyl-D-aspartate (NMDA) receptor (NMDAR)-mediated synaptic transmission in the hippocampus without affecting synapse development, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR)-mediated basal transmission, and presynaptic release. Intriguingly, long-term depression (LTD) was near-completely abolished in Ngl3-/- mice, and the Akt/glycogen synthase kinase 3β (GSK3β) signaling pathway, known to suppress LTD, was abnormally enhanced. In addition, pharmacological inhibition of Akt, but not activation of NMDARs, normalized the suppressed LTD in Ngl3-/- mice, suggesting that Akt hyperactivity suppresses LTD. Ngl3-/- mice displayed several behavioral abnormalities, including hyperactivity, anxiolytic-like behavior, impaired spatial memory, and enhanced seizure susceptibility. Among them, the hyperactivity was rapidly improved by pharmacological NMDAR activation. These results suggest that NGL-3 regulates brain development, Akt/GSK3β signaling, LTD, and locomotive and cognitive behaviors., Competing Interests: The authors have declared that no competing interests exist.

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Netrin4 (NTN4) is a chemotropic factor that regulates angiogenesis. We found that endothelial expression of the activated, intracellular domain of Notch1 (NICD1), significantly up-regulated NTN4 mRNA as well as intracellular NTN4 protein in both transgenic mice and cultured human umbilical vein endothelial cells (HUVECs). Notch1 activation also increased NTN4 secretion from HUVECs. We subsequently demonstrated that NICD1 bound to CSL (CBF1, Suppressor of Hairless, Lag-1), a core component of Notch transcription complex, at the -53 element of the human NTN4 gene promoter. Loss of the -53 element compromised NICD1-induced NTN4 expression. Our results suggest a conserved role for Notch signalling in transcriptional regulation of endothelial NTN4., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA., Competing Interests: The authors declare no conflict of interest.