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Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete netrin-1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, dorsal root ganglion neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for netrin-1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g., netrin-1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.

Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Na v 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain. Spinal pain is a major clinical problem. Here the authors show that osteoclasts create porous area of endplates of the vertebrae and sensory innervation of porous endplates by Netrin-1 release from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

Blood-brain barrier (BBB) integrity is critical for proper function of the central nervous system (CNS). Here, we show that the endothelial Unc5B receptor controls BBB integrity by maintaining Wnt/β-catenin signaling. Inducible endothelial-specific deletion of Unc5B in adult mice leads to BBB leak from brain capillaries that convert to a barrier-incompetent state with reduced Claudin-5 and increased PLVAP expression. Loss of Unc5B decreases BBB Wnt/β-catenin signaling, and β-catenin overexpression rescues Unc5B mutant BBB defects. Mechanistically, the Unc5B ligand Netrin-1 enhances Unc5B interaction with the Wnt co-receptor LRP6, induces its phosphorylation and activates Wnt/β-catenin downstream signaling. Intravenous delivery of antibodies blocking Netrin-1 binding to Unc5B causes a transient BBB breakdown and disruption of Wnt signaling, followed by neurovascular barrier resealing. These data identify Netrin-1-Unc5B signaling as a ligand-receptor pathway that regulates BBB integrity, with implications for CNS diseases. The authors show that Netrin-1-Unc5B signaling controls blood-brain barrier integrity by maintaining Wnt/b-catenin signaling and that delivery of antibodies blocking Netrin-1 binding to Unc5B causes transient and size-selective BBB breakdown.

Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix (ECM) fragmentation and inflammation. However, the mechanisms by which these events are coupled thereby fueling focal vascular damage are undefined. Here we report through single-cell RNA-sequencing of diseased aorta that the neuronal guidance cue netrin-1 can act at the interface of macrophage-driven injury and ECM degradation. Netrin-1 expression peaks in human and murine aneurysmal macrophages. Targeted deletion of netrin-1 in macrophages protects mice from developing AAA. Through its receptor neogenin-1, netrin-1 induces a robust intracellular calcium flux necessary for the transcriptional regulation and persistent catalytic activation of matrix metalloproteinase-3 (MMP3) by vascular smooth muscle cells. Deficiency in MMP3 reduces ECM damage and the susceptibility of mice to develop AAA. Here, we establish netrin-1 as a major signal that mediates the dynamic crosstalk between inflammation and chronic erosion of the ECM in AAA. Abdominal aortic aneurysms (AAA) are characterized by extensive extracellular matrix degradation. Here Hadi et al. identify a netrin-1/neogenin-based crosstalk between macrophages and vascular smooth muscle cells (VSMCs), leading to the secretion of the matrix metalloproteinase MMP-3 by VSMCs and subsequent matrix degradation in AAA lesions.

Axon guidance molecules direct growing axons toward their targets, assembling the intricate wiring of the nervous system. One of these molecules, Netrin-1, and its receptor, DCC (deleted in colorectal cancer), has profound effects, in laboratory animals, on the adolescent expansion of mesocorticolimbic pathways, particularly dopamine. Now, a rapidly growing literature suggests that (1) these same alterations could occur in humans, and (2) genetic variants in Netrin-1 and DCC are associated with depression, schizophrenia, and substance use. Together, these findings provide compelling evidence that Netrin-1 and DCC influence mesocorticolimbic-related psychopathological states that emerge during adolescence.

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

Mechanical overload of the vascular wall is a pathological hallmark of life-threatening abdominal aortic aneurysms (AAA). However, how this mechanical stress resonates at the unicellular level of vascular smooth muscle cells (VSMC) is undefined. Here we show defective mechano-phenotype signatures of VSMC in AAA measured with ultrasound tweezers-based micromechanical system and single-cell RNA sequencing technique. Theoretical modelling predicts that cytoskeleton alterations fuel cell membrane tension of VSMC, thereby modulating their mechanoallostatic responses which are validated by live micromechanical measurements. Mechanistically, VSMC gradually adopt a mechanically solid-like state by upregulating cytoskeleton crosslinker, α-actinin2, in the presence of AAA-promoting signal, Netrin-1, thereby directly powering the activity of mechanosensory ion channel Piezo1. Inhibition of Piezo1 prevents mice from developing AAA by alleviating pathological vascular remodeling. Our findings demonstrate that deviations of mechanosensation behaviors of VSMC is detrimental for AAA and identifies Piezo1 as a novel culprit of mechanically fatigued aorta in AAA. Mechanobiological signals have been reported to contribute to abdominal aortic aneurysm (AAA) development. Here the authors report that the microskeletal stiffness and a Piezo1-mediated mechanism influence vascular smooth muscle cell mechanosensation and AAA disease development.

Netrin-1 (NTN1) is a laminin-related secreted protein involved in axon guidance and cell migration. Previous research has established a significant connection between NTN1 and nervous system development. In recent years, mounting evidence indicates that NTN1 also plays a crucial role in tumorigenesis and tumor progression. For instance, inhibiting Netrin-1 has been shown to suppress tumor growth and epithelial-mesenchymal transition (EMT) characteristics in endometrial cancer. To further elucidate the influence of genes on tumors, we utilized a variety of machine learning techniques and found that NTN1 is strongly linked to multiple cancer types, suggesting it as a potential therapeutic target. This study aimed to elucidate the role of NTN1 in pan-cancer using multi-omics data and explore its potential as a prognostic biomarker in SKCM. Analysis of the TCGA, GTEx, and UALCAN databases revealed significant differences in NTN1 expression at both the mRNA and protein levels. Prognostic value was evaluated through univariate Cox regression and Kaplan–Meier methods. Mutation and methylation analyses were conducted using the cBioPortal and SMART databases. We identified genes interacting with and correlated to NTN1 through STRING and GEPIA2, respectively. Subsequently, we performed GO and KEGG enrichment analyses. The results suggested that NTN1 might be involved in crucial biological processes and pathways related to cancer development and progression, including cell adhesion, axon guidance, immune response, and various signaling pathways. We then explored the correlation between NTN1 and immune infiltration as well as immunotherapy using the ESTIMATE package, TIMER2.0, TISIDB, TIDE, TIMSO, and TCIA. The relationship between NTN1 and tumor heterogeneity, stemness, DNA methyltransferases, and MMR genes was also examined. Lastly, we constructed a nomogram based on NTN1 in SKCM and investigated its association with drug sensitivity. NTN1 expression was significantly associated with tumor immune infiltration, molecular subtypes, and clinicopathological features in various cancers. Genetic analysis revealed that Deep deletions were the most common type of NTN1 alteration. Additionally, a positive correlation was observed between NTN1 CNAs and its expression levels. In most cancers, NTN1 showed positive correlations with immune and stromal scores, as well as with specific immune cell populations. Its predictive value for immunotherapy response was comparable to that of tumor mutational burden. Furthermore, NTN1 exhibited positive correlations with tumor heterogeneity, stemness, DNA methyltransferase genes, and MMR genes. In SKCM, NTN1 was identified as an independent risk factor and demonstrated potential associations with multiple drugs. NTN1 exhibits substantial clinical utility as a prognostic marker and indicator of immune response across various tumor types. This comprehensive analysis provides insights into its potential implications in pan-cancer research.

Netrin-1 signaling is an essential prototypical neuronal guidance mechanism during embryonic development that also regulates tumor cell survival in a variety of adult cancer entities. In line with these data, a monoclonal netrin-1 blocking antibody (anti-netrin-1 mAb/NP137) has been preclinically developed and netrin-1 blockade has recently been investigated in phase 1 and 2 clinical trials in several adult cancers. Here, we investigate the role of netrin-1 in the most common malignant pediatric brain cancer, Medulloblastoma. Interestingly, we find that netrin-1 is upregulated in medulloblastoma subgroups associated with developmental dysregulation, in particular in medulloblastoma with Sonic Hedgehog (SHH) activation. First, we demonstrate that genetic deletion of netrin-1 or systemic treatment with the clinical-stage anti-netrin-1 blocking antibody significantly reduces tumor growth in vivo in various orthotopic models of SHH medulloblastomas. Second, in vitro and in vivo, we unexpectedly uncover that SHH medulloblastomas treated with an SHH-inhibitor targeting Smoothened (SMO) increase netrin-1 expression, paving the way for combinatorial therapy. In line with that, we next show that netrin-1 blockade potentiates the efficacy of SMO inhibitor therapy in vivo. Together, our data indicate that, netrin-1 blockade, used as monotherapy or in combination with SMO inhibitors, is a promising therapeutic strategy in SHH medulloblastomas. Despite being an important driver of a subset of medulloblastomas, efforts to therapeutically target Sonic Hedgehog (SHH) signaling, such as with the use of Smoothened (SMO) inhibitors, have had limited success. Here, the authors find that SHH medulloblastomas are sensitive to netrin-1 inhibition and investigate netrin-1 as a mechanism of resistance to SMO inhibition.

The Hippo (MST1/2) pathway plays a critical role in restricting tissue growth in adults and modulating cell proliferation, differentiation, and migration in developing organs. Netrin1, a secreted laminin-related protein, is essential for nervous system development. However, the mechanisms underlying MST1 regulation by the extrinsic signals remain unclear. Here, we demonstrate that Netrin1 reduction in Parkinson’s disease (PD) activates MST1, which selectively binds and phosphorylates netrin receptor UNC5B on T428 residue, promoting its apoptotic activation and dopaminergic neuronal loss. Netrin1 deprivation stimulates MST1 activation and interaction with UNC5B, diminishing YAP levels and escalating cell deaths. Knockout of UNC5B abolishes netrin depletion-induced dopaminergic loss, whereas blockade of MST1 phosphorylating UNC5B suppresses neuronal apoptosis. Remarkably, Netrin1 is reduced in PD patient brains, associated with MST1 activation and UNC5B T428 phosphorylation, which is accompanied by YAP reduction and apoptotic activation. Hence, Netrin1 regulates Hippo (MST1) pathway in dopaminergic neuronal loss in PD via UNC5B receptor.