Circulating netrin-1 levels are reduced and related to corneal nerve fiber loss in patients with diabetic neuropathy.

AIMS/INTRODUCTION: Deficiency of neurotropic factors is implicated in diabetic neuropathy (DN). Netrin-1 is a neurotropic factor, but its association with DN has not been explored. We have assessed the association between serum netrin-1 levels and early diabetic neuropathy assessed by quantifying corneal nerve fiber loss using corneal confocal microscopy. MATERIALS AND METHODS: A total of 72 participants with type 2 diabetes, without and with corneal nerve fiber loss (DN- n = 42, DN+ n = 30), and 45 healthy controls were studied. Serum netrin-1 levels were measured by enzyme-linked immunosorbent assay, and corneal nerve morphology was assessed using corneal confocal microscopy. RESULTS: Corneal nerve fiber density, branch density, fiber length and serum netrin-1 levels were significantly lower in the DN- and DN+ groups compared with controls (P < 0.001). Netrin-1 levels correlated with corneal nerve fiber length in the DN+ group (r = 0.51; P < 0.01). A receiver operating characteristic curve analysis showed that a netrin-1 cut-off value of 599.6 (pg/mL) had an area under the curve of 0.85, with a sensitivity of 76% and specificity of 74% (P < 0.001; 95% confidence interval 0.76-0.94) for differentiating patients with and without corneal nerve loss. CONCLUSIONS: Serum netrin-1 levels show a progressive decline with increasing severity of small nerve fiber damage in patients with diabetes. Netrin-1 could act as a biomarker for small nerve fiber damage in DN.

Cinnamaldehyde attenuates streptozocin-induced diabetic osteoporosis in a rat model by modulating netrin-1/DCC-UNC5B signal transduction.

Background: Cinnamaldehyde (CMD) is a major functional component of Cinnamomum verum and has shown treatment effects against diverse bone diseases. This study aimed to assess the anti-diabetic osteoporosis (DOP) potential of diabetes mellitus (DM) and to explore the underlying mechanism driving the activity of CMD. Methods: A DOP model was induced via an intraperitoneal injection of streptozocin (STZ) into Sprague-Dawley rats, and then two different doses of CMD were administered to the rats. The effects of CMD on the strength, remodeling activity, and histological structure of the bones were assessed. Changes in the netrin-1 related pathways also were detected to elucidate the mechanism of the anti-DOP activity by CMD. Results: CMD had no significant effect on the body weight or blood glucose level of the model rats. However, the data showed that CMD improved the bone strength and bone remodeling activity as well as attenuating the bone structure destruction in the DOP rats in a dose-dependent manner. The expression of netrin-1, DCC, UNC5B, RANKL, and OPG was suppressed, while the expression of TGF-ß1, cathepsin K, TRAP, and RANK was induced by the STZ injection. CMD administration restored the expression of all of these indicators at both the mRNA and protein levels, indicating that the osteoclast activity was inhibited by CMD. Conclusion: The current study demonstrated that CMD effectively attenuated bone impairments associated with DM in a STZ-induced DOP rat model, and the anti-DOP effects of CMD were associated with the modulation of netrin-1/DCC/UNC5B signal transduction.

Insights from the neural guidance factor Netrin-1 into neurodegeneration and other diseases.

Netrin-1 was initially discovered as a neuronal growth cue for axonal guidance, and its functions have later been identified in inflammation, tumorigenesis, neurodegeneration, and other disorders. We have recently found its alterations in the brains with Alzheimer's disease, which might provide important clues to the mechanisms of some unique pathologies. To provide better understanding of this promising molecule, we here summarize research progresses in genetics, pathology, biochemistry, cell biology and other studies of Netrin-1 about its mechanistic roles and biomarker potentials with an emphasis on clinical neurodegenerative disorders in order to expand understanding of this promising molecular player in human diseases.

Expression of netrin-1 in uterine serous carcinoma and its association with prognosis.

BACKGROUND/OBJECTIVES: At present, there are few biomarkers used to predict the prognosis of uterine serous carcinoma (USC). Netrin-1 may be a promising biomarker candidate. We investigated netrin-1 expression in USC tissues and healthy endometrial tissues to determine its relevance to disease prognosis. MATERIALS AND METHODS: Netrin-1 expression was examined in the tissues of 48 patients with USC and 30 patients with healthy benign endometrial tissues via immunohistochemistry. RESULTS: None of the healthy tissues were stained with netrin-1. In tumor tissues, the overall positivity rate of netrin-1 was 75%, detected as high expression in 17 patients (35%) and low in 19 (40%). Patients who had tumors with no netrin-1 expression (n = 12) had a median overall survival (OS) of 60.0 months (95% confidence interval [CI], 47-98), whereas patients who had tumors with low to strong netrin-1 expression (n = 33) had a lower median OS of 50 months, but the difference was not statistically significant (95% CI, 58-108; P = 0.531). Disease-free survival (DFS) was not statistically significant between the groups (95% CI, 67.7-115.9; P = 0.566). Patients with a tumor diameter ≥2 cm had higher netrin-1 expression than those with a tumor diameter of 2 cm (P = 0.027). We did not find any difference in overall and DFS when age, tumor stage, histology, tumor diameter, p53 status, lymphovascular space invasion, myometrial invasion, and lymph node metastasis were compared according to netrin-1 expression (P > 0.05). CONCLUSION: Netrin-1 was expressed in USC but not in healthy tissues. Its expression was not associated with OS or DFS.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Can Repetitive Transcranial Magnetic Stimulation (rTMS) Promote Neurogenesis and Axonogenesis in Subacute Human Ischemic Stroke?

BACKGROUND: Ischemic stroke may trigger neuroplastic changes via proliferation, migration towards the lesion, and differentiation of neuroprogenitor cells into mature neurons. Repetitive Transcranial Magnetic Stimulation (rTMS) may promote brain plasticity. This study aimed to assess rTMS's effect on post-stroke endogenous neuroplasticity by dosing plasma miRs 17~92, Netrin-1, Sema3A, and BDNF. METHODS: In this case-controlled study, we randomized 19 ischemic stroke patients within five days from symptoms onset (T0) to neuronavigated-rTMS or sham stimulation. Stimulation was applied on the stroke hemisphere daily between the 7th and 14th day from stroke onset. Blood samples were collected at T0, before the first rTMS section (T7), and at the end of the last rTMS session (T14). Five healthy controls were also enrolled in this study. RESULTS: Of 19 patients, 10 received rTMS and 9 sham stimulation. Compared with the sham group, in the rTMS group, plasma levels of miRs17~92 and Ntn-1 significantly increased whereas Sema3A levels tended to decrease. In multivariate linear regression analyses, rTMS was independently related to Ntn-1 and miR-25 levels at T14. CONCLUSIONS: We found an association between rTMS and neurogenesis/axonogenesis biomarker enhancement. Our preliminary data suggest that rTMS may positively interfere with natural endogenous plasticity phenomena of the post-ischemic human brain.

UNC5C: Novel Gene Associated with Psychiatric Disorders Impacts Dysregulation of Axon Guidance Pathways.

The UNC-5 family of netrin receptor genes, predominantly expressed in brain tissues, plays a pivotal role in various neuronal processes. Mutations in genes involved in axon development contribute to a wide spectrum of human diseases, including developmental, neuropsychiatric, and neurodegenerative disorders. The NTN1/DCC signaling pathway, interacting with UNC5C, plays a crucial role in central nervous system axon guidance and has been associated with psychiatric disorders during adolescence in humans. Whole-exome sequencing analysis unveiled two compound heterozygous causative mutations within the UNC5C gene in a patient diagnosed with psychiatric disorders. In silico analysis demonstrated that neither of the observed variants affected the allosteric linkage between UNC5C and NTN1. In fact, these mutations are located within crucial cytoplasmic domains, specifically ZU5 and the region required for the netrin-mediated axon repulsion of neuronal growth cones. These domains play a critical role in forming the supramodular protein structure and directly interact with microtubules, thereby ensuring the functionality of the axon repulsion process. We emphasize that these mutations disrupt the aforementioned processes, thereby associating the UNC5C gene with psychiatric disorders for the first time and expanding the number of genes related to psychiatric disorders. Further research is required to validate the correlation of the UNC5C gene with psychiatric disorders, but we suggest including it in the genetic analysis of patients with psychiatric disorders.

Serum netrin-1 levels are high in Rheumatoid arthritis associated interstitial lung disease.

BACKGROUND: Recent data show that netrin-1 has a role in development of pulmonary fibrosis. This study was aimed to investigate serum netrin-1 level and its relation to interstitial lung disease(ILD) in patients with rheumatoid arthritis (RA). METHOD: 42 RA patients with RA-ILD, 58 RA patients without RA-ILD (RA non-ILD group), and 61 healthy volunteers were included in this study. The modified DAS28-ESR score was used to calculate disease activity in RA patients. Using the quantitative immunoassay method, Serum netrin-1 levels were measured with an ELISA kit (Catalog number: E-EL-H2328; lab science, lot number: GZWTKZ5SWK, Texas, USA). RESULTS: The median value of netrin-1 was found to be significantly higher in the RA-ILD group (82.9 [59.9-124]) compared to both the RA non-ILD group(52.9 [49.5-73.1])(B = -0.006, OR = 0.994, CI 95 %=0.989-0.999, P = 0.018) and the control group(53.5 [49.5-87.5]) (B: -0.005, OR: 0.994, CI 95 %: 0.990-0.999, p: 0.022). A cut-off value of 61.78 for netrin-1 was found to have a sensitivity of 73.8 % and a specificity of 69 % for the diagnosis of RA-ILD (AUC [95 %Cl] = 0.771 [0.679-0.862], p < 0.0001).It was found that high serum netrin-1 level was strongly associated with the RA-usual interstitial pneumonia(UIP) pattern and poorly related to the RA-nonspecific interstitial pneumonia(NSIP) pattern compared to the RA non-ILD group. CONCLUSIONS: Netrin-1 is elevated in the serum of patients with RA-ILD, especially in the UIP pattern. Netrin-1 may be a potential candidate for predicting the development of RA-ILD that should be investigated in the pathophysiological and therapeutic fields..

Netrin-1 mitigates acute lung injury by preventing the activation of the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling.

Acute lung injury (ALI) is one of the most common high-risk diseases associated with a high mortality rate and is still a challenge to treat effectively. Netrin-1 (NT-1) is a novel peptide with a wide range of biological functions, however, its effects on ALI have not been reported before. In this study, an ALI model was constructed using lipopolysaccharide (LPS) and treated with NT-1. Pulmonary function and lung wet to dry weight ratio (W/D) were detected. The expressions of pro-inflammatory cytokines and chemokines interleukin-8 (IL-8), interleukin-1ß (IL-1ß), and chemokine (C-X-C motif) ligand 2 (CXCL2) were measured using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We found that the levels of NT-1 were reduced in the LPS-induced ALI mice model. Administration of NT-1 improved histopathological changes of lung tissues and lung function in LPS-challenged ALI mice. We also report that NT-1 decreased Myeloperoxidase (MPO) activity and ameliorated pulmonary edema. Additionally, treatment with NT-1 reduced the levels of pro-inflammatory cytokines and chemokines such as IL-8, IL-1ß, and CXCL2 in lung tissues of LPS-challenged ALI mice. Importantly, NT-1 reduced cell count in BALF and mitigated oxidative stress (OS) by reducing the levels of MDA and increasing the levels of GSH. Mechanistically, it is shown that NT-1 reduced the levels of Toll-like receptor 4 (TLR4) and prevented nuclear translocation of nuclear factor-κB (NF-κB) p65. Our findings indicate that NT-1 is a promising agent for the treatment of ALI through inhibiting TLR4/NF-κB signaling.

Adhesion-clutch between DCC and netrin-1 mediates netrin-1-induced axonal haptotaxis.

The growth cone, a motile structure located at the tip of growing axons, senses extracellular guidance cues and translates them into directional forces that drive axon outgrowth and guidance. Axon guidance directed by chemical cues on the extracellular adhesive substrate is termed haptotaxis. Recent studies reported that netrin-1 on the substrate functions as a haptotactic axon guidance cue. However, the mechanism mediating netrin-1-induced axonal haptotaxis remains unclear. Here, we demonstrate that substrate-bound netrin-1 induces axonal haptotaxis by facilitating physical interactions between the netrin-1 receptor, DCC, and the adhesive substrates. DCC serves as an adhesion receptor for netrin-1. The clutch-linker molecule shootin1a interacted with DCC, linking it to actin filament retrograde flow at the growth cone. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip on the adhesive substrate. The grip state was more prevalent on netrin-1-coated substrate compared to the control substrate polylysine, thereby transmitting larger traction force on the netrin-1-coated substrate. Furthermore, disruption of the linkage between actin filament retrograde flow and DCC by shootin1 knockout impaired netrin-1-induced axonal haptotaxis. These results suggest that the directional force for netrin-1-induced haptotaxis is exerted on the substrates through the adhesion-clutch between DCC and netrin-1 which occurs asymmetrically within the growth cone.

Netrin-1 protects blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion by activating the Kruppel-like factor 2 (KLF2)/occludin pathway.

Ischemia/reperfusion (I/R)-induced neural damage and neuroinflammation have been associated with pathological progression during stroke. Netrin-1 is an important member of the family of laminin-related secreted proteins, which plays an important role in governing axon elongation. However, it is unknown whether Netrin-1 possesses a beneficial role in stroke. Here, we employed the middle cerebral artery occlusion (MCAO) model to study the function of Netrin-1 in alleviating brain injuries. Our results demonstrate that Netrin-1 rescued poststroke neurological deficits and inhibited production of the inflammatory cytokines such as interleukin 6 (IL-6) and endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1). Importantly, Netrin-1 protected against MCAO-induced dysfunction of the blood-brain barrier (BBB) in mice and a reduction in the expression of the tight junction (TJ) protein occludin. Additionally, we report that Netrin-1 could ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury and prevent aggravation in endothelial monolayer permeability in bEnd.3 human brain microvascular endothelial cells (HBMVECs). Mechanistically, Netrin-1 ameliorated OGD/R-induced decrease in occludin and Kruppel-like factor 2 (KLF2) in HBMVECs. Notably, silencing of KLF2 abolished the beneficial effects of Netrin-1 in protecting endothelial permeability and occludin expression, suggesting that these effects are mediated by KLF2. In conclusion, our findings suggest that Netrin-1 could constitute a novel therapeutic strategy for ischemic stroke.

Netrin-1 Alleviates Early Brain Injury by Regulating Ferroptosis via the PPARγ/Nrf2/GPX4 Signaling Pathway Following Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a type of stroke with high morbidity and mortality. Netrin-1 (NTN-1) can alleviate early brain injury (EBI) following SAH by enhancing peroxisome proliferator-activated receptor gamma (PPARγ), which is an important transcriptional factor modulating lipid metabolism. Ferroptosis is a newly discovered type of cell death related to lipid metabolism. However, the specific function of ferroptosis in NTN-1-mediated neuroprotection following SAH is still unclear. This study aimed to evaluate the neuroprotective effects and the possible molecular basis of NTN-1 in SAH-induced EBI by modulating neuronal ferroptosis using the filament perforations model of SAH in mice and the hemin-stimulated neuron injury model in HT22 cells. NTN-1 or a vehicle was administered 2 h following SAH. We examined neuronal death, brain water content, neurological score, and mortality. NTN-1 treatment led to elevated survival probability, greater survival of neurons, and increased neurological score, indicating that NTN-1-inhibited ferroptosis ameliorated neuron death in vivo/in vitro in response to SAH. Furthermore, NTN-1 treatment enhanced the expression of PPARγ, nuclear factor erythroid 2-related factor 2 (Nrf2), and glutathione peroxidase 4 (GPX4), which are essential regulators of ferroptosis in EBI after SAH. The findings show that NTN-1 improves neurological outcomes in mice and protects neurons from death caused by neuronal ferroptosis. Furthermore, the mechanism underlying NTN-1 neuroprotection is correlated with the inhibition of ferroptosis, attenuating cell death via the PPARγ/Nrf2/GPX4 pathway and coenzyme Q10-ferroptosis suppressor protein 1 (CoQ10-FSP1) pathway.

Counteracting Age-Related Netrin-1 Signaling Insufficiency Ameliorates Endothelial Cell Senescence and Angiogenesis Impairment.

Senescent cells that accumulate are regarded as promising therapeutic targets. However, senolytic therapy failed to achieve satisfactory results. We previously discovered that young human plasma improved vascular endothelial cell senescence, and UNC5B might be a novel intervention target. Netrin-1, as a natural ligand of UNC5B, plays roles in multiple age-related vascular disorders, but its involvement in aging is still unclear. Here, we observed a significant decrease in plasma Netrin-1 levels in old healthy subjects compared to the young. In vivo, adeno-associated-virus-mediated delivery of Netrin-1 into aged mice significantly improved functional recovery in a model of hindlimb ischemia, promoted angiogenesis in ischemic tissues, and activated the endothelial nitric oxide synthase. Furthermore, we revealed that low-dose Netrin-1 recombinant protein significantly reduced senescence-associated-ß-galactosidase-positive cells, inhibited the P53 pathway, promoted cell migration, increased tubule formation, and elevated nitric oxide production in senescent endothelial cells. However, UNC5B inhibition blocked the pro-angiogenesis effect of low-dose Netrin-1 on senescent cells or aortic rings. In summary, this study depicts that modulating Netrin-1 signaling can result in improved vascular health and Netrin-1 may have therapeutic potential for age-related ischemic diseases.

Urinary and circulatory netrin-1 as biomarker in diabetes and its related complications: a systematic review and meta-analysis.

BACKGROUND: Novel biomarkers have been suggested for the diagnosis and prognosis of diabetes mellitus. The biomarker utility of netrin-1 in diabetes as an extracellular protein has been investigated. In this systematic review and meta-analysis, we reviewed the role of netrin-1 as a biomarker in prediabetes, diabetes, and complications of diabetes. METHODS: PubMed, Embase, Scopus, and Web of Science were systematically searched for studies that measured circulatory and/or urinary netrin-1 levels in diabetes and compared them with non-diabetic patients or evaluated the prognostic role of this marker. Standardized mean difference (SMD) and 95% confidence interval (CI) were calculated using random-effect meta-analysis to compare netrin-1 levels between groups. The impact of mean age, male sex percentage, sample size, mean body mass index, and publication year on the overall heterogeneity was assessed using meta-regression. RESULTS: Among 413 records from international databases, 19 original studies were included with 2061 cases (1137 diabetics, 196 prediabetics, and 728 healthy controls). Meta-analysis of eight studies measuring netrin-1 in patients with diabetes and comparing it with healthy controls showed no significant difference between the two groups (SMD 0.69, 95% CI -0.78 to 2.16, I2 = 98%, p-value = 0.36). On the other hand, a meta-analysis of netrin-1 levels in patients with prediabetes in comparison with healthy controls revealed that they had lower levels (SMD -0.51, 95% CI -0.81 to -0.21, p-value < 0.01). Diabetic patients with microalbuminuria and macroalbuminuria had significantly higher circulatory netrin-1 levels compared to normoalbuminuric group SMD 1.18, 95% CI 0.83 to 1.53, p-value < 0.01 and SMD 1.67, 95% CI 0.76 to 2.58, p-value < 0.01, respectively). Moreover, no difference in urinary netrin-1 levels was found between micro-, macro-, and normoalbuminuric groups (p-value > 0.05). CONCLUSION: Netrin-1 showed promising results as a biomarker in diabetes prognosis. However, more studies are required to confirm our findings, and higher sample size studies are needed to evaluate the diagnostic utility of this marker.

Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling.

The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Stromal netrin 1 coordinates renal arteriogenesis and mural cell differentiation.

The kidney vasculature has a complex architecture that is essential for renal function. The molecular mechanisms that direct development of kidney blood vessels are poorly characterized. We identified a regionally restricted, stroma-derived signaling molecule, netrin 1 (Ntn1), as a regulator of renal vascular patterning in mice. Stromal progenitor (SP)-specific ablation of Ntn1 (Ntn1SPKO) resulted in smaller kidneys with fewer glomeruli, as well as profound defects of the renal artery and transient blood flow disruption. Notably, Ntn1 ablation resulted in loss of arterial vascular smooth muscle cell (vSMC) coverage and in ectopic SMC deposition at the kidney surface. This was accompanied by dramatic reduction of arterial tree branching that perdured postnatally. Transcriptomic analysis of Ntn1SPKO kidneys revealed dysregulation of vSMC differentiation, including downregulation of Klf4, which we find expressed in a subset of SPs. Stromal Klf4 deletion similarly resulted in decreased smooth muscle coverage and arterial branching without, however, the disruption of renal artery patterning and perfusion seen in Ntn1SPKO. These data suggest a stromal Ntn1-Klf4 axis that regulates stromal differentiation and reinforces stromal-derived smooth muscle as a key regulator of renal blood vessel formation.

Syntaxin-1 is necessary for UNC5A-C/Netrin-1-dependent macropinocytosis and chemorepulsion.

Introduction: Brain connectivity requires correct axonal guidance to drive axons to their appropriate targets. This process is orchestrated by guidance cues that exert attraction or repulsion to developing axons. However, the intricacies of the cellular machinery responsible for the correct response of growth cones are just being unveiled. Netrin-1 is a bifunctional molecule involved in axon pathfinding and cell migration that induces repulsion during postnatal cerebellar development. This process is mediated by UNC5 homolog receptors located on external granule layer (EGL) tracts. Methods: Biochemical, imaging and cell biology techniques, as well as syntaxin-1A/B (Stx1A/B) knock-out mice were used in primary cultures and brain explants. Results and discussion: Here, we demonstrate that this response is characterized by enhanced membrane internalization through macropinocytosis, but not clathrin-mediated endocytosis. We show that UNC5A, UNC5B, and UNC5C receptors form a protein complex with the t-SNARE syntaxin-1. By combining botulinum neurotoxins, an shRNA knock-down strategy and Stx1 knock-out mice, we demonstrate that this SNARE protein is required for Netrin1-induced macropinocytosis and chemorepulsion, suggesting that Stx1 is crucial in regulating Netrin-1-mediated axonal guidance.

Enhanced Neuron Growth and Electrical Activity by a Supramolecular Netrin-1 Mimetic Nanofiber.

Neurotrophic factors are essential not only for guiding the organization of the developing nervous system but also for supporting the survival and growth of neurons after traumatic injury. In the central nervous system (CNS), inhibitory factors and the formation of a glial scar after injury hinder the functional recovery of neurons, requiring exogenous therapies to promote regeneration. Netrin-1, a neurotrophic factor, can initiate axon guidance, outgrowth, and branching, as well as synaptogenesis, through activation of deleted in colorectal cancer (DCC) receptors. We report here the development of a nanofiber-shaped supramolecular mimetic of netrin-1 with monomers that incorporate a cyclic peptide sequence as the bioactive component. The mimetic structure was found to activate the DCC receptor in primary cortical neurons using low molar ratios of the bioactive comonomer. The supramolecular nanofibers enhanced neurite outgrowth and upregulated maturation as well as pre- and postsynaptic markers over time, resulting in differences in electrical activity similar to neurons treated with the recombinant netrin-1 protein. The results suggest the possibility of using the supramolecular structure as a therapeutic to promote regenerative bioactivity in CNS injuries.