Receptor UNC5C Research Articles

Evidence of positive selection of genetic variants associated with PCOS.

Was polycystic ovary syndrome (PCOS), which impairs fertility and adheres to the evolutionary paradox, subject to evolutionary selection during ancestral times and did rapidly diminish in prevalence? This study strengthened the hypothesis that positive selection of genetic variants occurred and may account for the high prevalence of PCOS observed today. PCOS is a complex endocrine disorder characterized by both reproductive and metabolic disturbances. As a heritable disease that impairs fertility, PCOS should diminish rapidly in prevalence; however, it is the most common cause of female subfertility globally. Few scientific genetic studies have attempted to provide evidence for the positive selection of gene variants underlying PCOS. We performed an evolutionary analysis of 2,504 individuals from 14 populations of the 1000 Genomes Project. We tested the signature of positive selection for 37 single-nucleotide polymorphisms (SNPs) associated with PCOS in previous genome-wide association studies using six parameters of positive selection. Analyzing the evolutionary indices together, there was obvious positive selection at the PCOS-related SNPs loci, especially within the original evolution window of humans, demonstrated by significant Tajima's D values. Compared to the genome background, six of the 37 SNPs in or close to five genes (DENN domain-containing protein 1A: DENND1A, chromosome 9 open reading frame 3: AOPEP, aminopeptidase O: THADA, diacylglycerol kinase iota: DGKI, and netrin receptor UNC5C: UNC5C) showed significant evidence of positive selection, among which DENND1A, AOPEP, and THADA represent the set of most established susceptibility genes for PCOS. First, only well-documented SNPs were selected from well-designed experiments. Second, it is difficult to determine which hypothesis of PCOS evolution is at play. After considering the most significant functions of these genes, we found that they had a wide variety of functions with no obvious association between them. Our findings provide additional evidence for the positive evolution of PCOS. Our analyses require confirmation in a larger study with more evolutionary indicators and larger data range. Further research to identify the roles of the DENND1A, AOPEP, THADA, DGKI, and UNC5C genes is also necessary. This study was supported by the National Key Research and Development Program of China (2021YFC2700400 and 2021YFC2700701), Basic Science Center Program of NSFC (31988101), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), National Natural Science Foundation of China (82192874, 31871509, and 82071606), Shandong Provincial Key Research and Development Program (2020ZLYS02), Taishan Scholars Program of Shandong Province (ts20190988), and Fundamental Research Funds of Shandong University. The authors have no conflicts of interest to disclose. N/A.

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.

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.

Disease-associated mutations in human TUBB3 disturb netrin repulsive signaling.

Missense mutations in the human TUBB3 gene cause a variety of neurological disorders associated with defects in axon guidance and neuronal migration, but the underlying molecular mechanisms are not well understood. Recent studies have shown that direct coupling of dynamic TUBB3 in microtubules with netrin receptors is required for netrin-1-mediated axon guidance, and the interaction of netrin-1 repulsive receptor UNC5C with TUBB3 is involved in netrin-1 mediated axonal repulsion. Here, we report that TUBB3 mutations perturb netrin-1/UNC5C repulsive signaling in the developing nervous system. Among twelve mutants reported in previous studies, five of them show significantly reduced interaction with UNC5C in comparison to the wild-type TUBB3. TUBB3 mutants R262C and R62Q exhibit decreased subcellular colocalization with UNC5C in the peripheral area of the growth cone of primary mouse neurons. Netrin-1 reduces the colocalization of UNC5C with wild-type TUBB3, but not TUBB3 mutants R262C or R62Q, in the growth cone. Results from the in vitro cosedimentation assay indicate that netrin-1 inhibits cosedimentation of UNC5C with polymerized microtubules in primary mouse neurons expressing the wild-type TUBB3, but not R262C or R62Q. Expression of either R262C or R62Q not only blocks netrin-1-induced growth cone collapse and axonal repulsion of primary EGL cells in vitro, but also results in axon projections defects of chicken dorsal root ganglion neurons in ovo. Our study reveals that human TUBB3 mutations specifically perturb netrin-1/UNC5C-mediated repulsion.

A Retino-retinal Projection Guided by Unc5c Emerged in Species with Retinal Waves

SummaryThe existence of axons extending from one retina to the other has been reported during perinatal development in different vertebrates. However, it has been thought that these axons are either a labeling artifact or misprojections. Here, we show unequivocally that a small subset of retinal ganglion cells (RGCs) project to the opposite retina and that the guidance receptor Unc5c, expressed in the retinal region where the retinal-retinal (R-R) RGCs are located, is necessary and sufficient to guide axons to the opposite retina. In addition, Netrin1, an Unc5c ligand, is expressed in the ventral diencephalon in a pattern that is consistent with impeding the growth of Unc5c-positive retinal axons into the brain. We also have generated a mathematical model to explore the formation of retinotopic maps in the presence and absence of a functional connection between both eyes. This model predicts that an R-R connection is required for the bilateral coordination of axonal refinement in species where refinement depends upon spontaneous retinal waves. Consistent with this idea, the retinal expression of Unc5c correlates with the existence and size of an R-R projection in different species and with the extent of axonal refinement in visual targets. These findings demonstrate that active guidance drives the formation of the R-R projection and suggest an important role for these projections in visual mapping to ensure congruent bilateral refinement.

A Retino-Retinal Connection Guided by Unc5c Emerged in Species with Retinal Waves

The existence of axons extending from one retina to the other has been reported during perinatal development in different vertebrates. However, it has been thought that these axons are either a labelling artefact or misprojections. Here, we unequivocally show that a small subset of retinal ganglion cells (RGCs) project to the opposite retina and that the guidance receptor Unc5c, expressed in the retinal region where the R-R neurons are located, is necessary and sufficient to guide axons to the opposite retina. In addition, Netrin1, an Unc5c ligand, is expressed in the ventral diencephalon in a pattern that is consistent with impeding the growth of Unc5c retinal axons into the brain. We have also generated a mathematical model to explore the formation of retinotopic maps in the presence and absence of a functional connection between both eyes. This model predicts that an R-R connection is required for the bilateral coordination of axonal refinement in species where refinement depends upon spontaneous retinal waves. Consistent with this idea, the retinal expression of Unc5c correlates with the existence and size of an R-R projection in different species and with the extent of axonal refinement in visual targets. These findings demonstrate that active guidance drives the formation of the R-R projection and suggest an important role for these projections in visual mapping to ensure congruent bilateral refinement.

Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion.

Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the in vitro cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of DRG toward the spinal cord in vivo Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion.SIGNIFICANCE STATEMENT Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic β-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.

Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons.

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin-ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

Expression and significance of netrin-1 and its receptor UNC5C in precocious puberty female rat hypothalamus.

To study expressions of netrin-1 and its receptor UNC5C in female precocious puberty rat hypothalamus, and explore its effect on precocious puberty process. Forty female one-week-old SD rats were randomly divided into four groups: experimental group A (precocious puberty early youth), experimental group B (precocious puberty medium youth), group A (normal pre-puberty), group B (normal early youth) with 10 rats in each group. Precocious puberty experimental rats were induced with Danazol and rats in control group were injected with saline. Uterus and ovaries were removed, specimens were weighed, uterus index and ovarian index were calculated, and amount of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were detected from the blood by ELISA. Real-time PCR was used to detect netrin-1 and its receptor UNC5C, as well as hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression in hypothalamus tissues; and then, a co-immunoprecipitation study of interactions between netrin-1 and its receptor UNC5C was carried out. Relative target gene expression levels of control group A, control group B, experimental group A, and experimental group B (with β -actin as an internal control for normalization) were as follows: Netrin-1: 3.5±0.9, 5.4±0.7, 4.9±1.0, 5.3±0.3; UNC5C: 0.8± 0.04, 1.7±0.2, 1.82±0.23, 1.58±0.4; GnRH: 1.2±0.3, 2.7±0.3, 2.4±0.7, 3.2±0.4. LH and FSH concentrations, netrin-1 and its receptor expression are increased in precocious puberty animal models.

Abstract 3430: Recurrent somatic mutations of nitric oxide synthase NOS3, netrin receptor UNC5C and DNA repair genes in muscle-invasive bladder carcinoma

Abstract Bladder cancer is the fourth most common cancer type in males and approximately 72,500 individuals were diagnosed in the United States in 2012. Patients with superficial (low-grade papillary) tumors generally have a good prognosis with a 5-year survival rate exceeding 90%, but those with muscle-invasive or locally advanced disease at surgical resection have a significant risk of recurrence with 5-year survival rates of 30-60%. The systemic chemotherapy options for bladder cancer are very limited, and no new drugs have been approved in the United States for metastatic bladder cancer in over 20 years. Although a number of newer, molecularly targeting drugs have been developed and approved for multiple cancer types in the last two decades, no such drug has been developed for the treatment of bladder cancer. This is likely due, in part, to the relatively limited molecular understanding of invasive bladder cancers and lack of knowledge about potential genetic drivers of invasive forms of the disease. Hence, it is of priority to discover novel genetic pathways involved in the carcinogenesis of muscle-invasive bladder cancer for the assessment of risk stratification and for the development of novel drugs. We analyzed exomes of 43 muscle-invasive bladder carcinomas and identified two novel significantly mutated genes, NOS3 (18.6%) and UNC5C (16.3%), in addition to previously reported frequently mutated genes like TP53 (37.2%) and KDM6A (18.6%). Both NOS3 and UNC5C are known to be involved in the apoptotic pathways, with NOS3 encoding for endothelial nitric oxide synthase (eNOS), and UNC5C for netrin receptor, a member of the dependence receptor family. When we correlated the mutational profiles with clinicopathological outcomes by stratifying patients according to disease recurrence status and observing for specific clustering of genetic mutations, we found that somatic mutations in six DNA repair genes (ATM, ERCC2, FANCD2, RAD51AP1, PALB2, BRCA2) were significantly associated with enhanced recurrence-free survival (hazard ratio of 0.091 with 95% CI of 0.018 to 0.45; P=0.0034) even after adjustment for pathologic TN staging, suggestive of their mutations as favorable prognostic events in bladder cancer. Citation Format: Kai Lee Yap, Kazuma Kiyotani, Kenji Tamura, Miran Jang, Magdeline Montoya, Cory Ganshert, Tomoaki Fujioka, Gary Steinberg, Peter O'Donnell, Yusuke Nakamura. Recurrent somatic mutations of nitric oxide synthase NOS3, netrin receptor UNC5C and DNA repair genes in muscle-invasive bladder carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3430. doi:10.1158/1538-7445.AM2014-3430

Abstract 28: The Netrin-1 receptor UNC5C is a tumor suppressor in colorectal malignancies

Abstract The Netrin-1 receptors UNC5A, B and C belong to the functional family of dependence receptors that share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer a tumor suppressor activity to these receptors. Expression of one of these dependence receptors at the surface of a tumor cell is thus speculated to render this cell dependent on ligand for its survival, hence inhibiting cell proliferation or migration outside the ligand availability zone. Here we show that the expression of the UNC5C gene is down-regulated in colorectal cancers, mainly because of tumor-associated specific promoter methylation. In addition, inactivation of UNC5C in mice is associated with increased intestinal tumor progression, demonstrating its tumor suppressor role. Moreover, mutations in UNC5C that turn down the pro-apoptotic activity of the receptor are found in patients with familial form of colorectal cancer. This germline coding mutation in UNC5C has been detected in five different families from France and USA and co-segregates with the disease. We further, determine that this anti-apoptotic mutation arose from a common founder. In summary, our study strongly indicates that the genetic inactivation of the tumor suppressor UNC5C may be a major promoter of familial predisposition to colorectal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 28. doi:1538-7445.AM2012-28

Covering the Cover

Covering the Cover

Variants in the Netrin-1 Receptor UNC5C Prevent Apoptosis and Increase Risk of Familial Colorectal Cancer

Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk of colorectal cancer (CRC). We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, United States, and Finland). Almost all cases from France and the United States had familial CRC; of the Finnish cases, 92 of 984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis to determine the origin of this variant. Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio, 8.8; Wald's 95% confidence interval, 1.47-52.93; P = .03) and in 2 families in the US cohort (odds ratio, 1.9; P = .6) but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109-kilobase identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in white subjects (14 generations; 95% confidence interval, 6-36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared with wild-type UNC5C, measured in an assay of active caspase-3. Inherited mutations in UNC5C prevent apoptosis and increase risk of CRC.

Regulation and Function of the Rhox5 Homeobox Gene

Recently, a large cluster of homeobox genes was discovered on the X chromosome that is expressed in reproductive tissues after birth. It is postulated that these reproductive homeobox genes on the X chromosome (Rhox) encode transcription factors that regulate gametogenesis. In support of this, male mice lacking the founding member of this gene cluster, Rhox5, are subfertile, exhibiting increased germ-cell apoptosis and a defect in sperm motility. To identify RHOX5 targets, microarray analyses were used to identify genes differently expressed in postnatal testes from Rhox5-null and control littermates. Highly overrepresented were genes that encode proteins involved in cellular metabolism. Several lines of evidence indicated that one of these, insulin II, is a direct target of RHOX5. Microarray analysis was also used to identify genes differentially expressed in response to physiological levels of Rhox5 in a Sertoli-cell line. Among the few genes identified, the netrin-1 receptor UNC5c, a proapoptotic molecule that is inhibited by RHOX5, was also regulated in vivo, and is thus a candidate to be downstream of RHOX5 in a prosurvival germ-cell pathway. To understand the means by which Rhox5 expression is restricted to Sertoli nurse cells in the testis, a variety of molecular approaches were used in both Sertoli-cell lines and mice. This analysis revealed that both positive and negative cis elements collaborate to confer Sertoli cell-specific gene expression. Acting on the positive cis elements are androgen receptor and GATA transcription factors. Collectively, the results of this study provide an initial glimpse into the regulatory networks that control spermatogenesis.