Plexin A4 Research Articles

Host genetic polymorphisms involved in long-term symptoms of COVID-19

ABSTRACT Host genetic polymorphisms are recognized as a critical determinant of diversity in clinical symptoms of Coronavirus disease 2019 (COVID-19). Accordingly, this study aimed to determine possible associations between single nucleotide polymorphisms (SNPs) in 37 candidate genetic variants and clinical consequences of COVID-19 – especially long-term symptoms, Long COVID. A total of 260 COVID-19 patients, divided into mild (n = 239) and severe (n = 21) and further categorized based on the presence of Long COVID (no, n = 211; yes, n = 49), were recruited. Genotyping of selected polymorphisms responsible for viral entry, immune response, and inflammation was performed using MassARRAY system. Out of 37 SNPs, 9 including leucine zipper transcription factor like-1 (LZTFL1) rs10490770 C allele, LZTFL1 rs11385942 dupA allele, nicotinamide adenine dinucleotide synthetase-1 (NADSYN1) rs12785878 TT genotype, plexin A-4 (PLXNA4) rs1424597 AA genotype, LZTFL1 rs17713054 A allele, interleukin-10 (IL10) rs1800896 TC genotype and C allele, angiotensin converting enzyme-2 (ACE2) rs2285666 T allele, and plasmanylethanolamine desaturase-1 (PEDS1) rs6020298 GG genotype and G allele were significantly associated with an increased risk of developing Long COVID, whereas interleukin-10 receptor subunit beta (IL10RB) rs8178562 GG genotype was significantly associated with a reduced risk of Long COVID. Kaplan-Meier curve displayed that the above gene polymorphisms were significantly associated with cumulative rate of Long COVID occurrence. Polymorphisms in LZTFL1 rs10490770, LZTFL1 rs11385942, LZTFL1 rs17713054, NADSYN1 rs12785878, PLXNA4 rs1424597, IL10 rs1800896, ACE2 rs2285666, PEDS1 rs6020298, and IL10RB rs8178562 appear to be genetic factors involved in development of Long COVID.

Modulation of DRG neurons response to semaphorin 3A via substrate stiffness.

Semaphorin 3A (Sema3a) is a chemotropic protein that acts as a neuronal guidance cue and plays a major role in dorsal root ganglion (DRG) sensory neurons projection during embryo development. The present study evaluated the impact of stiffness in the repulsive response of DRG neurons to Sema3a when cultured over substrates of variable stiffness. Stiffness modified DRG neurons morphology and regulated their response to Sema3a, reducing the collapse of growth cones when they were cultured on softer substrates. Sema3a receptors expression was also regulated by stiffness, neuropilin-1 was overexpressed and plexin A4 mRNA was downregulated in stiffer substrates. Cytoskeleton distribution was also modified by stiffness. In softer substrates, βIII-tubulin and actin co-localized up to the leading edge of the growth cones, and as the substrate became stiffer, βIII-tubulin was confined to the transition and peripheral domains of the growth cone. Moreover, a decrease in the α-actinin adaptor protein was also observed in softer substrates. Our results show that substrate stiffness plays an important role in regulating the collapse response to Sema3a and that the modulation of cytoskeleton distribution and Sema3a receptors expression are related to the differential collapse responses of the growth cones.

Interactions between semaphorins and plexin–neuropilin receptor complexes in the membranes of live cells

Signaling of semaphorin ligands via their plexin–neuropilin receptors is involved in tissue patterning in the developing embryo. These proteins play roles in cell migration and adhesion but are also important in disease etiology, including in cancer angiogenesis and metastasis. While some structures of the soluble domains of these receptors have been determined, the conformations of the full-length receptor complexes are just beginning to be elucidated, especially within the context of the plasma membrane. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy allows direct insight into the formation of protein–protein interactions in the membranes of live cells. Here, we investigated the homodimerization of neuropilin-1 (Nrp1), plexin A2, plexin A4, and plexin D1 using pulsed-interleaved excitation fluorescence cross-correlation spectroscopy. Consistent with previous studies, we found that Nrp1, plexin A2, and plexin A4 are present as dimers in the absence of exogenous ligand. Plexin D1, on the other hand, was monomeric under similar conditions, which had not been previously reported. We also found that plexin A2 and A4 assemble into a heteromeric complex. Stimulation with semaphorin 3A or semaphorin 3C neither disrupts nor enhances the dimerization of the receptors when expressed alone, suggesting that activation involves a conformational change rather than a shift in the monomer–dimer equilibrium. However, upon stimulation with semaphorin 3C, plexin D1 and Nrp1 form a heteromeric complex. This analysis of interactions provides a complementary approach to the existing structural and biochemical data that will aid in the development of new therapeutic strategies to target these receptors in cancer.

Downregulation of Endothelial Plexin A4 Under Inflammatory Conditions Impairs Vascular Integrity.

Objective: Besides hyperlipidemia, inflammation is an important determinant in the initiation and the progression of atherosclerosis. As Neuroimmune Guidance Cues (NGCs) are emerging as regulators of atherosclerosis, we set out to investigate the expression and function of inflammation-regulated NGCs.Methods and results: NGC expression in human monocytes and endothelial cells was assessed using a publicly available RNA dataset. Next, the mRNA levels of expressed NGCs were analyzed in primary human monocytes and endothelial cells after stimulation with IL1β or TNFα. Upon stimulation a total of 14 and 19 NGCs in monocytes and endothelial cells, respectively, were differentially expressed. Since plexin A4 (PLXNA4) was strongly downregulated in endothelial cells under inflammatory conditions, the role of PLXNA4 in endothelial function was investigated. Knockdown of PLXNA4 in endothelial cells markedly impaired the integrity of the monolayer leading to more elongated cells with an inflammatory phenotype. In addition, these cells showed an increase in actin stress fibers and decreased cell-cell junctions. Functional assays revealed decreased barrier function and capillary network formation of the endothelial cells, while vascular leakage and trans-endothelial migration of monocytes was increased.Conclusion: The current study demonstrates that pro-inflammatory conditions result in differential expression of NGCs in endothelial cells and monocytes, both culprit cell types in atherosclerosis. Specifically, endothelial PLXNA4 is reduced upon inflammation, while PLXNA4 maintains endothelial barrier function thereby preventing vascular leakage of fluids as well as cells. Taken together, PLXNA4 may well have a causal role in atherogenesis that deserves further investigation.

Activity-induced secretion of semaphorin 3A mediates learning.

The semaphorin family is a well‐characterized family of secreted or membrane‐bound proteins that are involved in activity‐independent neurodevelopmental processes, such as axon guidance, cell migration, and immune functions. Although semaphorins have recently been demonstrated to regulate activity‐dependent synaptic scaling, their roles in Hebbian synaptic plasticity as well as learning and memory remain poorly understood. Here, using a rodent model, we found that an inhibitory avoidance task, a hippocampus‐dependent contextual learning paradigm, increased secretion of semaphorin 3A in the hippocampus. Furthermore, the secreted semaphorin 3A in the hippocampus mediated contextual memory formation likely by driving AMPA receptors into hippocampal synapses via the neuropilin1–plexin A4–semaphorin receptor complex. This signaling process involves alteration of the phosphorylation status of collapsin response mediator protein 2, which has been characterized as a downstream molecule in semaphorin signaling. These findings implicate semaphorin family as a regulator of Hebbian synaptic plasticity and learning.

MicroRNA‑564 inhibits the progression of non‑small cell lung cancer via targeting plexin A4

Νon-small cell lung cancer (NSCLC) is the most frequently diagnosed type of cancer, and the most prevalent cause of cancer-associated mortality. The present study aimed to investigate whether microRNA (miR)-564 influences NSCLC progression by regulating NSCLC cell growth and migration, via targeting plexin A4. Therefore, the expression levels of miR-564 and plexin A4 were evaluated in NSCLC specimens or cells using reverse transcription-quantitative PCR. Furthermore, colony formation and Cell Counting Kit-8 assays were performed to determine the proliferative ability of NSCLC cells. The cell migration capacity was assessed using a Transwell assay. In addition, to examine the binding ability of miR-564 on the plexin A4 3'-untranslated region (3'UTR), a dual-luciferase reporter assay was performed. A mouse xenograft model was established to evaluate the effect of miR-564 knockdown on tumor growth in vivo, whereas the expression of plexin A4 and Ki67 in NSCLC tissues was detected using immunohistochemistry. Notably, miR-564 was downregulated in both NSCLC cell lines and tissues, while its overexpression, following transfection with miR-564 mimics, attenuated the proliferation and proliferation, migration and invasion of NSCLC cells. By contrast, silencing of miR-564 using a miR-564 inhibitor promoted NSCLC cell proliferation, migration and invasion. The luciferase assay revealed that miR-564 directly targeted the plexin A4 3'UTR in A549 and H460 cells. Additionally, the overexpression of plexin A4 rescued the effect of miR-564 on NSCLC cell proliferation, migration and invasion abilities. Further in vivo studies demonstrated that miR-564 knockdown promoted NSCLC growth, while miR-564 overexpression resulted in the opposite effect in nude mice. Overall, the results of the present study revealed that miR-564 promotes the proliferation and migration of NSCLC cells, both in vitro and in vivo, via targeting plexin A4. Therefore, miR-564 may be considered as a possible therapeutic target for NSCLC.

Common Variants in PLXNA4 and Correlation to CSF-related Phenotypes in Alzheimer's Disease

The Plexin-A 4 (PLXNA4) gene, has recently been identified in genome wide association studies (GWAS), as a novel genetic player associated with Alzheimer's disease (AD). Additionally, PLXNA4 genetic variations were also found to increase AD risk by tau pathology in vitro. However, the potential roles of PLXNA4 variants in the amyloid-β (Aβ) pathology, were not evaluated. Five targeted loci capturing the top common variations in PLXNA4, were extracted using tagger methods. Multiple linear regression models were used to explore whether these variations can affect the cerebrospinal fluid (CSF) (Aβ1−42, T-tau, and P-tau) phenotypes in the Alzheimer's disease Neuroimaging Initiative (ADNI) dataset. We detected that two loci (rs6467431, rs67468325) were significantly associated with CSF Aβ1−42 levels in the hybrid population (rs6467431: P = 0.01376, rs67468325: P = 0.006536) and the significance remained after false discovery rate (FDR) correction (rs6467431: Pc = 0.03441, rs67468325: Pc = 0.03268). In the subgroup analysis, we further confirmed the association of rs6467431 in the cognitively normal (CN) subgroup (P = 0.01904, Pc = 0.04761). Furthermore, rs6467431-A carriers and rs67468325-G carriers showed higher CSF Aβ1−42 levels than non-carriers. Nevertheless, we did not detect any significant relationships between the levels of T-tau, P-tau and these PLXNA4 loci. Our findings provided preliminary evidence that PLXNA4 variants can confer AD risk through modulating the Aβ deposition.

B7-H4 Modulates Regulatory CD4+ T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex.

The potent immune regulatory function of an agonistic B7-H4-Ig fusion protein (B7-H4Ig) has been demonstrated in multiple experimental autoimmune models; however, the identity of a functional B7-H4 receptor remained unknown. The biological activity of B7-H4 is associated with decreased inflammatory CD4+ T cell responses as supported by a correlation between B7-H4-expressing tumor-associated macrophages and Foxp3+ T cells within the tumor microenvironment. Recent data indicate that members of the semaphorin (Sema)/plexin/neuropilin (Nrp) family of proteins both positively and negatively modulate immune cell function. In this study, we show that B7-H4 binds the soluble Sema family member Sema3a. Additionally, B7-H4Ig-induced inhibition of inflammatory CD4+ T cell responses is lost in both Sema3a functional mutant mice and mice lacking Nrp-1 expression in Foxp3+ T cells. These findings indicate that B7-H4Ig binds to Sema3a, which acts as a functional bridge to stimulate an Nrp-1/Plexin A4 heterodimer to form a functional immunoregulatory receptor complex resulting in increased levels of phosphorylated PTEN and enhanced regulatory CD4+ T cell number and function.

720 - Semaphorin 3A and Plexin A4 Signaling as a Novel Regulator of Intestinal Neuroimmune Interaction

720 - Semaphorin 3A and Plexin A4 Signaling as a Novel Regulator of Intestinal Neuroimmune Interaction

Dual leucine zipper kinase regulates expression of axon guidance genes in mouse neuronal cells.

BackgroundRecent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells.ResultsOur analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules.ConclusionsCollectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13064-016-0068-8) contains supplementary material, which is available to authorized users.

Identification of plexin A4 as a novel clusterin receptor links two Alzheimer’s disease risk genes

Although abundant genetic and biochemical evidence strongly links Clusterin (CLU) to Alzheimer disease (AD) pathogenesis, the receptor for CLU within the adult brain is currently unknown. Using unbiased approaches, we identified Plexin A4 (PLXNA4) as a novel, high-affinity receptor for CLU in the adult brain. PLXNA4 protein expression was high in brain with much lower levels in peripheral organs. CLU protein levels were significantly elevated in the cerebrospinal fluid (CSF) of Plxna4-/- mice and, in humans, CSF levels of CLU were also associated with PLXNA4 genotype. Human AD brains had significantly increased the levels of CLU protein but decreased levels of PLXNA4 by ∼50%. To determine whether PLXNA4 levels influenced cognition, we analyzed the behaviour of Plxna4+/+, Plxna4+/-, and Plxna4-/- mice. In comparison to WT controls, both Plxna4+/- and Plxna4-/- mice were hyperactive in the open field assay while Plxna4-/- mice displayed a hyper-exploratory (low-anxiety phenotype) in the elevated plus maze. Importantly, both Plxna4+/- and Plxna4-/- mice displayed prominent deficits in learning and memory in the contextual fear-conditioning paradigm. Thus, even a 50% reduction in the level of PLXNA4 is sufficient to cause memory impairments, raising the possibility that memory problems seen in AD patients could be due to reductions in the level of PLXNA4. Both CLU and PLXNA4 have been genetically associated with AD risk and our data thus provide a direct relationship between two AD risk genes. Our data suggest that increasing the levels of PLXNA4 or targeting CLU-PLXNA4 interactions may have therapeutic value in AD.

Association study of the PLXNA4 gene with the risk of Alzheimer's disease.

The Plexin-A 4 (PLXNA4) gene has recently been recognized as a functional candidate gene of late-onset Alzheimer's disease (LOAD). The single nucleotide polymorphism (SNP) rs13232207 of PLXNA4 gene has been reported to be associated with Alzheimer's disease (AD) in Japanese cohorts. We sought to clarify whether this novel locus gains the same effect in northern Han Chinese. To investigate the relationship between SNP rs13232207 and AD sufferers, a case-control study of unrelated individuals was conducted with a total sample size of 2,318 subjects (978 cases and 1,340 age and gender matched healthy controls) in a Northern Han Chinese population. SPSS 22.0 was applied for the statistical process. No significant difference in polymorphic distribution of rs13232207 was observed on LOAD risk independently under dominant (P=0.057), additive (P=0.233) or recessive model (P=0.392). In terms of interaction with apolipoprotein E (APOE), there is also no positive interaction in dominant (P=0.438), additive (P=0.055) or recessive model (P=0.095). Replication of association between the PLXNA4 rs13232207 and AD in a Han ethnic group indicates that this link is not the result of chance.

TrkA mediates retrograde semaphorin 3A signaling through plexin A4 to regulate dendritic branching

Semaphorin 3A (Sema3A), a secretory semaphorin, exerts various biological actions through a complex between neuropilin-1 and plexin-As (PlexAs). Sema3A induces retrograde signaling, which is involved in regulating dendritic localization of GluA2 (also known as GRIA2), an AMPA receptor subunit. Here, we investigated a possible interaction between retrograde signaling pathways for Sema3A and nerve growth factor (NGF). Sema3A induces colocalization of PlexA4 (also known as PLXNA4) signals with those of tropomyosin-related kinase A (TrkA, also known as NTRK1) in growth cones, and these colocalized signals were then observed along the axons. The time-lapse imaging of PlexA4 and several TrkA mutants showed that the kinase and dynein-binding activity of TrkA were required for Sema3A-induced retrograde transport of the PlexA4-TrkA complex along the axons. The inhibition of the phosphoinositide 3-kinase (PI3K)-Akt signal, a downstream signaling pathway of TrkA, in the distal axon suppressed Sema3A-induced dendritic localization of GluA2. The knockdown of TrkA suppressed Sema3A-induced dendritic localization of GluA2 and that suppressed Sema3A-regulated dendritic branching both in vitro and in vivo These findings suggest that by interacting with PlexA4, TrkA plays a crucial role in redirecting local Sema3A signaling to retrograde axonal transport, thereby regulating dendritic GluA2 localization and patterning.

Axon guidance: Setting up for seeing in slow motion.

Interactions among semaphorin 6A, plexin A2 and plexin A4, and between contactin 4 and amyloid precursor protein, are crucial for the normal development of the circuits underlying vertical and horizontal optokinetic reflexes, respectively.

Plexin a4 expression in adult rat cranial nerves

PlexinsA1–A4 participate in class 3 semaphorin signaling as co-receptors to neuropilin 1 and 2. PlexinA4 is the latest member of the PlexinA subfamily to be identified. In previous studies, we described the expression of PlexinA4 in the brain and spinal cord of the adult rat. Here, antibodies to PlexinA4 were used to reveal immunolabeling in most of the cranial nerve surveyed. Labeling was found in the olfactory, optic, oculomotor, trochlear, trigeminal, abducens, facial, vestibulocochlear, glossopharyngeal, vagus, and hypoglossal nerves. This is the first detailed description of the cellular and subcellular distribution of PlexinA4 in the adult cranial nerves. The findings will set the basis for future studies on the potential role of PlexinA4 in regeneration and repair of the adult central and peripheral nervous system.

Identification of beryllium-dependent peptides recognized by CD4+ T cells in chronic beryllium disease

Chronic beryllium disease (CBD) is a granulomatous disorder characterized by an influx of beryllium (Be)-specific CD4⁺ T cells into the lung. The vast majority of these T cells recognize Be in an HLA-DP–restricted manner, and peptide is required for T cell recognition. However, the peptides that stimulate Be-specific T cells are unknown. Using positional scanning libraries and fibroblasts expressing HLA-DP2, the most prevalent HLA-DP molecule linked to disease, we identified mimotopes and endogenous self-peptides that bind to MHCII and Be, forming a complex recognized by pathogenic CD4⁺ T cells in CBD. These peptides possess aspartic and glutamic acid residues at p4 and p7, respectively, that surround the putative Be-binding site and cooperate with HLA-DP2 in Be coordination. Endogenous plexin A peptides and proteins, which share the core motif and are expressed in lung, also stimulate these TCRs. Be-loaded HLA-DP2–mimotope and HLA-DP2–plexin A4 tetramers detected high frequencies of CD4⁺ T cells specific for these ligands in all HLADP2+ CBD patients tested. Thus, our findings identify the first ligand for a CD4⁺ T cell involved in metal-induced hypersensitivity and suggest a unique role of these peptides in metal ion coordination and the generation of a common antigen specificity in CBD.

The p75 neurotrophin receptor, semaphorins, and sympathetic traffic in the heart

the sympathetic and parasympathetic branches of the autonomic nervous system control cardiac function through the coordinated activity generated by neurons within the intracardiac autonomic ganglia and central nervous system (3). Activation of the sympathetic efferent projections to the heart increases heart rate, modifies the pattern and conduction velocity of the impulse through the heart, and augments both atrial and ventricular contractility while hastening myocardial relaxation. In the vasculature, the sympathetic nervous system (SNS) reduces venous capacitance and constricts resistance vessels, increasing blood pressure. Conversely, the parasympathetic nervous system (PSNS) decreases heart rate and cardiac impulse conduction. More recent evidence has suggested that the PSNS may also affect ventricular function, indirectly by countering β-adrenergic action or directly via inhibition of L-type Ca2+ channels (34). A transient imbalance between these two branches can lead to increased heart rate and blood pressure when cardiac output is increased. When subnormal cardiac PSNS activity concomitant with sympathetic hyperactivation persists, such as after myocardial infarction, this disequilibrium may increase the risk for cardiac adverse events, including arrhythmias, hypertension, cardiac hypertrophy, and, ultimately, heart failure. This condition is called “autonomic neural remodeling” and can greatly contribute to the induction of ventricular fibrillation, thus increasing the patient's susceptibility to sudden cardiac death (41, 47). It is plausible that neurotrophic factors, target organ-secreted proteins essential for the development and differentiation of neurons, are involved in the remarkable neuroplasticity exhibited by the SNS in the adult life, including autonomic remodeling after infarction and/or in the onset and progression of congestive heart failure (17, 30, 32).

Plexin A3 and plexin A4 convey semaphorin signals during facial nerve development

In vertebrates, class 3 semaphorins (SEMA3) control axon behaviour by binding to neuronal cell surface receptors composed of a ligand binding subunit termed neuropilin (NRP) and a signal transduction subunit of the A-type plexin family (PLXNA). We have determined the requirement for SEMA3/NRP/PLXN signalling in the development of the facial nerve, which contains axons from two motor neuron populations, branchiomotor and visceromotor neurons. Loss of either SEMA3A/NRP1 or SEMA3F/NRP2 caused defasciculation and ectopic projection of facial branchiomotor axons. In contrast, facial visceromotor axons selectively required SEMA3A/NRP1. Thus, the greater superficial petrosal nerve was defasciculated, formed ectopic projections and failed to branch in its target area when either SEMA3A or NRP1 were lost. To examine which A-type plexin conveyed SEMA3/neuropilin signals during facial nerve development, we combined an expression analysis with loss of function studies. Even though all four A-type plexins were expressed in embryonic motor neurons, PLXNA1 and PLXNA2 were not essential for facial nerve development. In contrast, loss of PLXNA4 phenocopied the defects of SEMA3A and NRP1 mutants, and loss of PLXNA3 phenocopied the defects of SEMA3F and NRP2 mutants. The combined loss of PLXNA3 and PLXNA4 impaired facial branchiomotor axon guidance more severely than loss of either plexin alone, suggesting that SEMA3A and SEMA3F signals, even though both essential, are partially redundant.