Netrin Receptor Research Articles

PTPRD and DCC Are Novel BACE1 Substrates Differentially Expressed in Alzheimer's Disease: A Data Mining and Bioinformatics Study.

The β-site Amyloid precursor protein Cleaving Enzyme 1 (BACE1) is an extensively studied therapeutic target for Alzheimer’s disease (AD), owing to its role in the production of neurotoxic amyloid beta (Aβ) peptides. However, despite numerous BACE1 inhibitors entering clinical trials, none have successfully improved AD pathogenesis, despite effectively lowering Aβ concentrations. This can, in part, be attributed to an incomplete understanding of BACE1, including its physiological functions and substrate specificity. We propose that BACE1 has additional important physiological functions, mediated through substrates still to be identified. Thus, to address this, we computationally analysed a list of 533 BACE1 dependent proteins, identified from the literature, for potential BACE1 substrates, and compared them against proteins differentially expressed in AD. We identified 15 novel BACE1 substrates that were specifically altered in AD. To confirm our analysis, we validated Protein tyrosine phosphatase receptor type D (PTPRD) and Netrin receptor DCC (DCC) using Western blotting. These findings shed light on the BACE1 inhibitor failings and could enable the design of substrate-specific inhibitors to target alternative BACE1 substrates. Furthermore, it gives us a greater understanding of the roles of BACE1 and its dysfunction in AD.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2′-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

The netrin-1 receptor UNC5C contributes to the homeostasis of undifferentiated spermatogonia in adult mice

In adult testis, the cell mobility is essential for spermatogonia differentiation and is suspected to regulate spermatogonial stem cell fate. Netrin-1 controls cell migration and/or survival according to the cellular context. Its involvement in some self-renewing lineages raises the possibility that Netrin-1 could have a role in spermatogenesis.We show that in addition to Sertoli cells, a fraction of murine undifferentiated spermatogonia express the Netrin-1 receptor UNC5c and that UNC5c contributes to spermatogonia differentiation. Receptor loss in Unc5crcm males leads to the concomitant accumulation of transit-amplifying progenitors and short syncytia of spermatogonia. Without altering cell death rates, the consequences of Unc5c loss worsen with age: the increase in quiescent undifferentiated progenitors associated with a higher spermatogonial stem cell enriched subset leads to the spermatocyte I decline. We demonstrate in vitro that Netrin-1 promotes a guidance effect as it repulses both undifferentiated and differentiating spermatogonia.Finally, we propose that UNC5c triggers undifferentiated spermatogonia adhesion/ migration and that the repulsive activity of Netrin-1 receptors could regulate spermatogonia differentiation, and maintain germ cell homeostasis.

The netrin-1 receptor DCC promotes the survival of a subpopulation of midbrain dopaminergic neurons: Relevance for ageing and Parkinson's disease.

Mechanisms that determine the survival of midbrain dopaminergic (mDA) neurons in the adult central nervous system (CNS) are not fully understood. Netrins are a family of secreted proteins that are essential for normal neural development. In the mature CNS, mDA neurons express particularly high levels of netrin‐1 and its receptor Deleted in Colorectal Cancer (DCC). Recent findings indicate that overexpressing netrin‐1 protects mDA neurons in animal models of Parkinson’s disease (PD), with a proposed pro‐apoptotic dependence function for DCC that triggers cell death in the absence of a ligand. Here, we sought to determine if DCC expression influences mDA neuron survival in young adult and ageing mice. To circumvent the perinatal lethality of DCC null mice, we selectively deleted DCC from mDA neurons utilizing DATcre/loxP gene‐targeting and examined neuronal survival in adult and aged animals. Reduced numbers of mDA neurons were detected in the substantia nigra pars compacta (SNc) of young adult DATcre/DCCfl/fl mice, with further reduction in aged DATcre/DCCfl/fl animals. In contrast to young adults, aged mice also exhibited a gene dosage effect, with fewer SNc mDA neurons in DCC heterozygotes (DATcre/DCCfl/wt). Notably, loss of mDA neurons in the SN was not uniform. Neuronal loss in the SN was limited to ventral tier mDA neurons, while mDA neurons in the dorsal tier of the SN, which resist degeneration in PD, were spared from the effect of DCC deletion in both young and aged mice. In the ventral tegmental area (VTA), young adult mice with conditional deletion of DCC had normal mDA neuronal numbers, while significant loss occurred in aged DATcre/DCCfl/fl and DATcre/DCCfl/wt mice compared to age‐matched wild‐type mice. Our results indicate that expression of DCC is required for the survival of subpopulations of mDA neurons and may be relevant to the degenerative processes in PD.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Multiple Functions of Draxin/Netrin-1 Signaling in the Development of Neural Circuits in the Spinal Cord and the Brain.

Axon guidance proteins play key roles in the formation of neural circuits during development. We previously identified an axon guidance cue, named draxin, that has no homology with other axon guidance proteins. Draxin is essential for the development of various neural circuits including the spinal cord commissure, corpus callosum, and thalamocortical projections. Draxin has been shown to not only control axon guidance through netrin-1 receptors, deleted in colorectal cancer (Dcc), and neogenin (Neo1) but also modulate netrin-1-mediated axon guidance and fasciculation. In this review, we summarize the multifaceted functions of draxin and netrin-1 signaling in neural circuit formation in the central nervous system. Furthermore, because recent studies suggest that the distributions and functions of axon guidance cues are highly regulated by glycoproteins such as Dystroglycan and Heparan sulfate proteoglycans, we discuss a possible function of glycoproteins in draxin/netrin-1-mediated axon guidance.

UNC5B-AS1 promotes the proliferation, migration and EMT of hepatocellular carcinoma cells via regulating miR-4306/KDM2A axis

ABSTRACT Being one of the most prevalent malignancies, hepatocellular carcinoma (HCC) threatens the health of population all over the world. Numerous researches have confirmed that long noncoding RNAs (lncRNAs) play an important role in tumor progression. Nonetheless, the mechanisms of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) in HCC remain obscure. Thus, this study aims to investigate the regulatory role and mechanism of UNC5B-AS1 in HCC cells. In our research, UNC5B-AS1 was subjected to gene expression analysis by RT-qPCR. Biological functions of UNC5B-AS1 in HCC cells were measured by MTT, colony formation, EdU and transwell assays. The combination between UNC5B-AS1, lysine demethylase 2A (KDM2A) and miR-4306 was validated by mechanism assays. Result showed UNC5B-AS1 was upregulated in HCC tissues and cells, contributing to the development of cancer staging and survival rate of HCC patients. Moreover, UNC5B-AS1 deficiency inhibited the proliferation, migration and epithelial–mesenchymal transition (EMT) of HCC cells. Furthermore, UNC5B-AS1 could interact with miR-4306 in HCC cells. Similarly, KDM2A was proved as the target gene of miR-4306. Finally, miR-4306 downregulation or KDM2A overexpression reversed the prohibitive role of UNC5B-AS1 knockdown in HCC progression. In short, UNC5B-AS1 accelerates the proliferation, migration and EMT of HCC cells via the regulation of miR-4306/KDM2A axis.

Netrin-1 as a Multitarget Barrier Stabilizer in the Peripheral Nerve after Injury

The blood–nerve barrier and myelin barrier normally shield peripheral nerves from potentially harmful insults. They are broken down during nerve injury, which contributes to neuronal damage. Netrin-1 is a neuronal guidance protein with various established functions in the peripheral and central nervous systems; however, its role in regulating barrier integrity and pain processing after nerve injury is poorly understood. Here, we show that chronic constriction injury (CCI) in Wistar rats reduced netrin-1 protein and the netrin-1 receptor neogenin-1 (Neo1) in the sciatic nerve. Replacement of netrin-1 via systemic or local administration of the recombinant protein rescued injury-induced nociceptive hypersensitivity. This was prevented by siRNA-mediated knockdown of Neo1 in the sciatic nerve. Mechanistically, netrin-1 restored endothelial and myelin, but not perineural, barrier function as measured by fluorescent dye or fibrinogen penetration. Netrin-1 also reversed the decline in the tight junction proteins claudin-5 and claudin-19 in the sciatic nerve caused by CCI. Our findings emphasize the role of the endothelial and myelin barriers in pain processing after nerve damage and reveal that exogenous netrin-1 restores their function to mitigate CCI-induced hypersensitivity via Neo1. The netrin-1-neogenin-1 signaling pathway may thus represent a multi-target barrier protector for the treatment of neuropathic pain.

IL-20 is involved in obesity by modulation of adipogenesis and macrophage dysregulation.

IL-20 is a proinflammatory cytokine of the IL-10 family and involved in several diseases. However, the regulatory role of IL-20 in obesity is not well understood. We explored the function of IL-20 in the pathogenesis of obesity-induced insulin resistance by ELISA, Western blotting and flow cytometry. The therapeutic potential of IL-20monoclonal antibody 7E for ameliorating diet-induced obesity was analysed in murine models. Higher serum IL-20levels were detected in obese patients. It was upregulated in leptin-deficient (ob/ob), leptin-resistant (db/db) and high-fat diet (HFD)-induced murine obesity models. In vitro, IL-20 regulated the adipocyte differentiation and the polarization of bone marrow-derived macrophages into proinflammatory M1 type. It also caused inflammation and macrophage retention in adipose tissues by upregulating TNF-α, monocyte chemotactic protein 1 (MCP-1), netrin 1 and unc5b (netrin receptor) expression in macrophages and netrin 1, leptin and MCP-1 in adipocytes. IL-20 promoted insulin resistance by inhibiting glucose uptake in mature adipocytes through the SOCS-3 pathway. In HFD-induced obesity in mice, 7E treatment reduced body weight and improved glucose tolerance and insulin sensitivity; it also reduced local inflammation and the number of M1-like macrophages in adipose tissues. We have identified a critical role of IL-20 in obesity-induced inflammation and insulin resistance, and we conclude that IL-20may be a novel target for treating obesity and insulin resistance in patients with metabolic disorders.

Inhibition of the long non-coding RNA UNC5B-AS1/miR-4455/RSPO4 axis reduces cervical cancer growth in vitro and in vivo.

Long non-coding RNAs (lncRNAs) are significant regulatory factors for the initiation and development of numerous malignant tumors, including cervical cancer (CC). The expression of lncRNA unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1, also known as UASR1) is up-regulated in tissues of cervical squamous cell carcinoma and endocervical adenocarcinoma compared to in normal tissues based on the GEPIA database. In the present study, we explored the functions of UNC5B-AS1 and its underlying mechanism with respect to CC development. A real-time quantitative polymerase chain reaction was applied for the detection of UNC5B-AS1 expression in CC cells. Cell counting kit-8, colony formation and transwell assays, as well as western blot and flow cytometry analyses, were employed to detect the biological effects of UNC5B-AS1 knockdown on malignant phenotypes of CC cells in vitro. In addition, the combination between microRNA-4455 (miR-4455) and UNC5B-AS1 or R-spondin 4 (RSPO4) was explored by RNA immunoprecipitation, luciferase reporter and RNA pulldown assays. A tumor xenograft nude mice model was established to explore the effect of UNC5B-AS1 depletion or miR-4455 overexpression on tumor growth. UNC5B-AS1 is up-regulated in CC tissues and cells. The knockdown of UNC5B-AS1 inhibits CC cell proliferation, migration and invasion and promotes CC cell apoptosis. Mechanistically, UNC5B-AS1 binds with miR-4455 to up-regulate RSPO4 expression. RSPO4 is targeted by miR-4455 and its expression is negatively regulated by miR-4455 expression. In vivo assays revealed that UNC5B-AS1 depletion or miR-4455 overexpression inhibits tumor growth by regulating RSPO4 expression. Inhibition of UNC5B-AS1/miR-4455/RSPO4 reduces CC growth both in vitro and in vivo, furnishing new insights into molecular studies on UNC5B-AS1 with respect to CC development.

A ligand-insensitive UNC5B splicing isoform regulates angiogenesis by promoting apoptosis

The Netrin-1 receptor UNC5B is an axon guidance regulator that is also expressed in endothelial cells (ECs), where it finely controls developmental and tumor angiogenesis. In the absence of Netrin-1, UNC5B induces apoptosis that is blocked upon Netrin-1 binding. Here, we identify an UNC5B splicing isoform (called UNC5B-Δ8) expressed exclusively by ECs and generated through exon skipping by NOVA2, an alternative splicing factor regulating vascular development. We show that UNC5B-Δ8 is a constitutively pro-apoptotic splicing isoform insensitive to Netrin-1 and required for specific blood vessel development in an apoptosis-dependent manner. Like NOVA2, UNC5B-Δ8 is aberrantly expressed in colon cancer vasculature where its expression correlates with tumor angiogenesis and poor patient outcome. Collectively, our data identify a mechanism controlling UNC5B’s necessary apoptotic function in ECs and suggest that the NOVA2/UNC5B circuit represents a post-transcriptional pathway regulating angiogenesis.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

Locomotor recovery after spinal cord injury: intimate dependence between axonal regeneration and re-connection.

The HSPG syndecan is a core organizer of cholinergic synapses.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.

Regulation of UNC-40/DCC and UNC-6/Netrin by DAF-16 promotes functional rewiring of the injured axon.

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.

Anti-Inflammatory Role of Netrin-4 in Diabetic Retinopathy.

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.

Netrin-1 functions as a suppressor of bone morphogenetic protein (BMP) signaling

Netrin-1 is a secreted protein that is well known for its involvement in axonal guidance during embryonic development and as an enhancer of cancer cell metastasis. Despite extensive efforts, the molecular mechanisms behind many of the physiological functions of netrin-1 have remained elusive. Here, we show that netrin-1 functions as a suppressor of bone morphogenetic protein (BMP) signaling in various cellular systems, including a mutually inhibitory interaction with the BMP-promoting function of leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins. The BMP inhibitory function of netrin-1 in mouse embryonic fibroblasts was dependent on the netrin receptor neogenin, with the expression level regulated by both netrin-1 and LRIG proteins. Our results reveal a previously unrecognized function of netrin-1 that may help to explain several of the developmental, physiological, and cancer-promoting functions of netrins at the signal transduction level.

Netrin-1 receptor UNC5C cleavage by active δ-secretase enhances neurodegeneration, promoting Alzheimer's disease pathologies.

Netrin-1, a family member of laminin-related secreted proteins, mediates axon guidance and cell migration during neural development. T835M mutation in netrin receptor UNC5C predisposes to the late-onset Alzheimer's disease (AD) and increases neuronal cell death. However, it remains unclear how this receptor is molecularly regulated in AD. Here, we show that δ-secretase selectively cleaves UNC5C and escalates its proapoptotic activity, facilitating neurodegeneration in AD. Netrin deficiency activates δ-secretase that specifically cuts UNC5C at N467 and N547 residues and enhances subsequent caspase-3 activation, additively augmenting neuronal cell death. Blockade of δ-secretase cleavage of UNC5C diminishes T835M mutant's proapoptotic activity. Viral expression of δ-secretase-truncated UNC5C fragments into APP/PS1 mice strongly accelerates AD pathologies, impairing learning and memory. Conversely, deletion of UNC5C from netrin-1-depleted mice attenuates AD pathologies and rescues cognitive disorders. Hence, δ-secretase truncates UNC5C and elevates its neurotoxicity, contributing to AD pathogenesis.

Linking cortical astrocytic neogenin deficiency to the development of Moyamoya disease–like vasculopathy

Moyamoya-like vasculopathy, the “puff of smoke”-like small vessels in the brain, is initially identified in patients with Moyamoya disease (MMD), a rare cerebrovascular disease, and later found in patients with various types of neurological conditions, including Down syndrome, Stroke, and vascular dementia. It is thus of interest to understand how this vasculopathy is developed. Here, we provided evidence for cortical astrocytic neogenin (NEO1) deficiency to be a risk factor for its development. NEO1, a member of deleted in colorectal cancer (DCC) family netrin receptors, was reduced in brain samples of patients with MMD. Astrocytic Neo1-loss resulted in an increase of small blood vessels (BVs) selectively in the cortex. These BVs were dysfunctional, with leaky blood-brain barrier (BBB), thin arteries, and accelerated hyperplasia in veins and capillaries, resembled to the features of moyamoya-like vasculopathy. Additionally, we found that both MMD patient and Neo1 mutant mice exhibited altered gene expression in their cortex in proteins critical for not only angiogenesis [e.g., an increase in vascular endothelial growth factor (VEGFa)], but also axon guidance (e.g., netrin family proteins) and inflammation. In aggregates, these results suggest a critical role of astrocytic NEO1-loss in the development of Moyamoya-like vasculopathy, providing a mouse model for investigating mechanisms of Moyamoya-like vasculopathy.

Targeting netrin‐3 in small cell lung cancer and neuroblastoma

The navigation cue netrin‐1 is well‐documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin‐1. Interestingly, the epitope recognized by NP137 in netrin‐1 shares 90% homology with its counterpart in netrin‐3, the closest member to netrin‐1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin‐3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin‐3 and netrin‐1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL‐1 or NeuroD1 transcription factors in SCLC. Netrin‐3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin‐3 for netrin‐1 receptors and we demonstrated that netrin‐3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin‐3 in NB and SCLC.

A global integrated analysis of UNC5C down-regulation in cancers: insights from mechanism and combined treatment strategy

As the transmembrane receptor of Netrin-1, the tumor suppressor gene Unc-5 Netrin Receptor C (UNC5C) can trigger apoptosis. Although its tumor suppressor effects have been demonstrated in solid tumors such as colon cancer, there is still a lack of systematic research on its expression regulation mechanism. To address this need, we analyzed datasets from The Cancer Genome Atlas (TCGA) database, including multi-omics data for 32 types of cancers and 10,967 cases. Analysis of these data revealed a trend of significantly decreased UNC5C expression in 16 types of solid tumors. Additionally, low UNC5C expression is related to poor prognosis of five types of tumors and restoring the expression of UNC5C can effectively inhibit the proliferation potential of renal cancer cells. Promoter DNA methylation, chromatin remodeling-mediated epigenetic regulation, transcriptional inhibition, RNA-binding protein and miRNA-mediated post-transcriptional inhibition, genetic changes caused by deep deletion and truncated mutations, and ubiquitinating enzyme-mediated protein degradation can synergistically cause the down-regulation of UNC5C expression in solid tumors. This study is the first to analyze the comprehensive molecular mechanism of down-regulation of the tumor suppressor gene UNC5C from multiple dimensions using pan-cancer data. Our results suggest that analyses of gene expression regulation relying on computational biological methods may help guide the targeted therapy of tumor suppressor gene reactivation.