Repulsive Guidance Research Articles

Retinal ganglion cell-derived semaphorin 6A segregates starburst amacrine cell dendritic scaffolds to organize the inner retina.

To form functional circuits, neurons must settle in their appropriate cellular locations and then project and elaborate neurites to contact their target synaptic neuropils. Laminar organization within the vertebrate retinal inner plexiform layer (IPL) facilitates pre- and postsynaptic neurite targeting, yet the precise mechanisms underlying establishment of functional IPL subdomains are not well understood. Here we explore mechanisms defining the compartmentalization of OFF and ON neurites generally, and OFF and ON direction-selective neurites specifically, within the developing IPL. We show that semaphorin 6A (Sema6A), a repulsive axon guidance cue, is required for delineation of OFF versus ON circuits within the IPL: in the Sema6a null IPL, the boundary between OFF and ON domains is blurred. Furthermore, Sema6A expressed by retinal ganglion cells (RGCs) directs laminar segregation of OFF and ON starburst amacrine cell (SAC) dendritic scaffolds, which themselves serve as a substrate upon which other retinal neurites elaborate. These results demonstrate that RGCs, the first neuron-type born within the retina, play an active role in functional specialization of the IPL.

Development and validation of an ultra-performance liquid chromatography with tandem mass spectrometry method for determination of soluble repulsive guidance molecule A in human serum and cerebrospinal fluid.

Aim: Repulsive guidance molecule A (RGMa) is upregulated in neurodegenerative diseases. To assess RGMa levels in human serum and cerebrospinal fluid (CSF), a quantification method was developed and validated according to ICH M10 guideline.Methods & results: Sample preparation consisted of immunoprecipitation (IP, only for serum), digestion and purification followed by MS.Conclusion: An UPLC-MS/MS method was established and used to assess normal range of soluble RGMa levels in serum and CSF of healthy controls, and patients with mild cognitive impairment or Alzheimer's disease. The normal range was between 13.0-44.8ng/ml (CSF) and 9.9-20.9ng/ml (serum) in healthy controls. In the CSF of patients with mild cognitive impairment and Alzheimer's disease, total soluble RGMa was twofold lower while unchanged in serum.

RGM Family Involved in the Regulation of Hepcidin Expression in Anemia of Chronic Disease

The persistent production of inflammatory cytokines causes anemia of chronic disease (ACD). Playing a central role in the pathophysiology of ACD is hepcidin, a key regulator of iron metabolism. The regulation of hepcidin expression is a complex process intricately controlled by multiple pathways. These include the BMP/SMAD, the HFE–TFR2, and the IL-6/STAT3 pathway, each playing a significant role in this regulation. We detail the critical role of the repulsive guidance molecule (RGM) family, especially hemojuvelin (HJV/RGMc), in regulating hepcidin expression in ACD. HJV functions as a co-receptor for BMPs and positively regulates hepcidin expression. RGMa and RGMb may also regulate hepcidin expression and inflammatory responses. RGM family proteins play essential roles in the interplay between inflammation, iron metabolism, and the immune system, and elucidating them could lead to a better understanding of the pathophysiology of ACD and the development of new therapeutic strategies.

PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade

Programmed cell death ligand 2 (PD-L2), a ligand for the receptor programmed cell death 1 (PD-1), has an identity of 34% with its twin ligand PD-L1 and exhibits higher binding affinity with PD-1 than PD-L1. However, the role of PD-L2 in non-small cell lung cancer (NSCLC) progression, especially tobacco-induced cancer progression, has not been fully understood. Here, we found that PD-L2 promoted tumor growth in murine models with recruitment of regulatory T cells (Tregs). In patients with NSCLC, PD-L2 expression level in tumor samples was higher than in counterpart normal controls and was positively associated with patients’ response to anti-PD-1 treatment. Mechanismly, PD-L2 bound its receptor Repulsive guidance molecule B (RGMB) on cancer cells and activated extracellular signal-regulated kinase (Erk) and nuclear factor κB (NFκB), leading to increased production of chemokine CCL20, which recruited Tregs and contributed to NSCLC progression. Consistently, knockdown of RGMB or NFκB p65 inhibited PD-L2-induced CCL20 production, and silencing of PD-L2 repressed Treg recruitment by NSCLC cells. Furthermore, cigarette smoke and carcinogen benzo(a)pyrene (BaP) upregulated PD-L2 in lung epithelial cells via aryl hydrocarbon receptor (AhR)-mediated transcription activation, whose deficiency markedly suppressed BaP-induced PD-L2 upregulation. These results suggest that PD-L2 mediates tobacco-induced recruitment of Tregs via the RGMB/NFκB/CCL20 cascade, and targeting this pathway might have therapeutic potentials in NSCLC.

The intracellular domain of Sema6A is essential for development of the zebrafish retina.

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

Protein biomarker signature in patients with spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a slowly progressing disease with limited sensitive biomarkers that support clinical research. We analyzed plasma and serum samples from patients with SBMA and matched healthy controls in multiple cohorts, identifying 40 highly reproducible SBMA-associated proteins out of nearly 3,000 measured. These proteins were robustly enriched in gene sets of skeletal muscle expression and processes related to mitochondria and calcium signaling. Many proteins outperformed currently used clinical laboratory tests (e.g., creatine kinase [CK]) in distinguishing patients from controls and in their correlations with clinical and functional traits in patients. Two of the 40 proteins, Ectodysplasin A2 receptor (EDA2R) and Repulsive guidance molecule A (RGMA), were found to be associated with decreased survival and body weight in a mouse model of SBMA. In summary, we identified what we believe to be a robust and novel set of fluid protein biomarkers in SBMA that are linked with relevant disease features in patients and in a mouse model of disease. Changes in these SBMA-associated proteins could be used as an early predictor of treatment effects in clinical trials.

Hemojuvelin-mediated hepcidin induction requires both bone morphogenetic protein type I receptors ALK2 and ALK3

AbstractHemojuvelin (HJV) is a glycosylphosphatidylinositol-anchored protein of the repulsive guidance molecule family acting as a bone morphogenetic protein (BMP) coreceptor to induce the hepatic iron regulatory protein hepcidin. Hepcidin causes ubiquitination and degradation of the sole known iron exporter ferroportin, thereby limiting iron availability. The detailed signaling mechanism of HJV in vivo has yet to be investigated. In the current manuscript, we used an established model of adeno-associated virus (AAV)-mediated liver-specific overexpression of HJV in murine models of hepatocyte-specific deficiency of the BMP type I receptors Alk2 or Alk3. In control mice, HJV overexpression increased hepatic Hamp messenger RNA (mRNA) levels, soluble HJV (sHJV), splenic iron content (SIC), as well as phosphorylated small mothers against decapentaplegic protein (pSMAD1/5/8) levels. In contrast, in Alk2fl/fl;Alb-Cre and Alk3fl/fl;Alb-Cre mice, which present with moderate and severe iron overload, respectively, the administration of AAV-HJV induced HJV and sHJV. However, it did not rescue the iron overload phenotypes of those mice. Serum iron levels were induced in Alk2fl/fl;Alb-Cre mice after HJV overexpression. In phosphate-buffered saline–injected Alk3fl/fl;Alb-Cre mice, serum iron levels and the expression of duodenal ferroportin remained high, whereas Hamp mRNA levels were decreased to 1% to 5% of the levels detected in controls. This was reduced even further by AAV-HJV overexpression. SIC remained low in mice with hepatocyte-specific Alk2 or Alk3 deficiency, reflecting disturbed iron homeostasis with high serum iron levels and transferrin saturation and an inability to induce hepcidin by HJV overexpression. The data indicate that ALK2 and ALK3 are both required in vivo for the HJV-mediated induction of hepcidin.

Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

Analysis of guidance and levitation forces between HTS magnets and conductive tubes for Hyperloop

High-temperature superconducting (HTS) magnets combined with linear synchronous motors and electrodynamic suspension (EDS) are considered as one of the most suitable technologies for Hyperloop. However, HTS magnets on pods generate strong magnetic fields, inducing eddy currents on conductive tubes when pods undergo movement through conductive tubes. The induced eddy currents affect HTS magnets, leading to electromagnetic (EM) drag, guidance, and levitation forces on the pods, thus reducing the propulsion efficiency and dynamic stability of pods. This study continues the existing research on EM drag forces between HTS magnet and tube for Hyperloop by comprehensively analyzing the EM guidance and levitation forces between HTS magnets and conductive tubes. Importantly, the full-scale 3D finite element analysis (FEA) simulations show that different steel tubes, such as AISI 1010 and high-manganese (Hi-Mn) steels, should be adopted, depending on the operating velocities, v, to avoid attractive forces in the guidance direction decreasing horizontal stiffnesses, For example, Hi-Mn tubes generating repulsive guidance forces are adopted when v is below 300 km/h while AISI 1010 tubes are used when v is over 300 km/h to minimize the construction cost of vacuum tubes to guarantee the increase in kx. For the proposed concept, the effect of the different tube connections on guidance and levitation forces is confirmed by the full-scale 3D FEA simulation. Moreover, levitation forces generated between HTS magnets and conductive tubes are nearly 0.5% of the EDS forces of pods, i.e., 200 kN over v of 150 km/h in the levitation direction. Therefore, the effect on vertical stiffnesses, ky, might be limited. In the end, to validate the proposed tube connections, the effect of the two different tube connections on guidance and levitation forces is confirmed by the full-scale 3D FEA simulation, and it was found that the sudden change in the guidance and levitation forces could be low enough to be neglected in the acceleration (from Hi-Mn to AISI 1010 steel tubes) and deceleration (from AISI 1010 to Hi-Mn steel tubes) regions.

Structural insights into the formation of repulsive netrin guidance complexes.

Netrins dictate attractive and repulsive responses during axon growth and cell migration, where the presence of the receptor Uncoordinated-5 (UNC-5) on target cells results in repulsion. Here, we showed that UNC-5 is a heparin-binding protein, determined its structure bound to a heparin fragment, and could modulate UNC-5-heparin affinity using a directed evolution platform or structure-based rational design. We demonstrated that UNC-5 and UNC-6/netrin form a large, stable, and rigid complex in the presence of heparin, and heparin and UNC-5 exclude the attractive UNC-40/DCC receptor from binding to UNC-6/netrin to a large extent. Caenorhabditis elegans with a heparin-binding-deficient UNC-5 fail to establish proper gonad morphology due to abrogated cell migration, which relies on repulsive UNC-5 signaling in response to UNC-6. Combining UNC-5 mutations targeting heparin and UNC-6/netrin contacts results in complete cell migration and axon guidance defects. Our findings establish repulsive netrin responses to be mediated through a glycosaminoglycan-regulated macromolecular complex.

Abstract WP275: Longitudinal Biomarker Analysis in Patients With Ischemic Stroke: Exploratory Targeted and Omics Assessments From a Phase 2a Clinical Trial

Introduction: Elezanumab (EZB), a monoclonal antibody specific to repulsive guidance molecule A (RGMa), is under investigation to treat acute ischemic stroke. Biomarker evidence in ischemic stroke and for the RGMa pathway is limited. Methods: This blinded, in-study analysis from a 52-week phase 2a, multicenter, randomized (1:1; EZB:placebo) clinical trial (NCT04309474) included patients aged 30-90 years with an acute ischemic stroke (onset ≤ 24 hours before baseline), and a NIHSS score of 7-21. Blinded analyses did not distinguish between treatment arms. Healthy adult controls were procured outside this trial. Blood samples were taken post-stroke across 52 weeks, or 1 time (baseline) for healthy adults. Plasma concentrations of neurodegenerative markers neurofilament light chain protein (NfL) and glial fibrillary acidic protein (GFAP) were measured (validated Simoa Human Neurology 4-Plex A assay; Quanterix). Protein, mRNA, and miRNA were assessed with omics approaches. Results: This preliminary analysis included 34 patients with stroke (mean [SD] age 67.1 [12.3] years, 58.8% male) and 31 healthy adults (61.8 [8.0] years, 71.0% male). The mean (SD) baseline NIHSS total score was 10.9 (4.3). NfL and GFAP concentrations were elevated on day 1 in patients with stroke vs healthy adults (healthy adults are included for reference only). Mean concentrations of NfL were 48.7 pg/mL vs 13.9 pg/mL, respectively; mean concentrations of GFAP were 746.0 pg/mL vs 137.2 pg/mL, respectively. In patients with stroke, agnostic to treatment, peak NfL and GFAP elevations occurred at day 28 and days 2-4, respectively; NfL and GFAP concentrations were lower at week 52 than day 1. Conclusions: Preliminary blinded analysis of plasma markers of neurodegeneration confirm increased NfL and GFAP levels during acute stroke, which diminish over time with different temporal patterns. Final unblinded results will provide a robust natural time course of specific biomarkers of neurodegeneration including GAP43. Forthcoming analyses evaluate broad panels of proteins, mRNA, and miRNA markers. Identifying relevant post stroke biomarkers with directed and exploratory omics may allow development of validated assays to assess efficacy and support decision making in clinical trials.

Inhibition of repulsive guidance molecule-a ameliorates compromised blood–spinal cord barrier integrity associated with neuromyelitis optica in rats

The influx of pathogenic aquaporin-4 antibodies (AQP4-Abs) across the blood–spinal cord barrier (BSCB) is crucial for the development and exacerbation of neuromyelitis optica (NMO). We examined whether prophylactic intravenous administration of anti-repulsive guidance molecule-a antibodies (RGMa-Abs) has disease-modifying effects on BSCB dysfunction using an NMO model elicited by peripheral administration of AQP4-Abs to rats. RGMa-Ab treatment attenuated the acute exacerbation of perivascular astrocytopathy in the spinal cord and clinical symptoms, which were highly correlated with neurofilament light chain levels in both the cerebrospinal fluid (CSF) and serum. Additionally, RGMa-Ab treatment suppressed the expression of proinflammatory cytokines/chemokines and the infiltration of inflammatory cells into the spinal cord. CSF analysis of NMO rats revealed that RGMa-Ab treatment improved the CSF/serum albumin ratio and suppressed AQP4-Abs influx. RGMa inhibition using RGMa-Abs is suggested as a potential therapeutic option for BSCB dysfunction associated with NMO.

Comparability of Elezanumab Safety, Tolerability, and Pharmacokinetics in Healthy Japanese, Chinese, and White Participants.

Elezanumab is a fully human monoclonal antibody, which is directed against repulsive guidance molecule A. The safety, tolerability, pharmacokinetics (PK), and immunogenicity of elezanumab were assessed in 2 Phase 1 clinical studies. The objective of this study was to assess the PK, safety, tolerability, and immunogenicity following intravenous infusion of elezanumab in healthy adult Japanese, Han Chinese, and Caucasian participants as well as Western participants from the single-ascending-dose study. Elezanumab exposures were approximately 20% higher in Japanese and Han Chinese participants compared to White participants without controlling for body weight. After statistically controlling for body weight by including it as a covariate, the PK of elezanumab in White participants were comparable to those in Japanese and Han Chinese participants. The clinical implications of these exposure differences are yet to be determined. All adverse events were assessed by the investigator as having no reasonable possibility of being related to the study drugs and were mild in severity. No positive immunogenicity effect was observed that impacted elezanumab exposure or safety.

Repulsive guidance molecules b (RGMb): molecular mechanism, function and role in diseases.

Repulsive guidance molecule b (RGMb), a glycosylphosphatidylinositol-anchored member of the RGM family, is initially identified as a co-receptor of bone morphogenetic protein (BMP) in the nervous system. The expression of RGMb is transcriptionally regulated by dorsal root ganglion 11 (DRG11), which is a transcription factor expressed in embryonic DRG and dorsal horn neurons and plays an important role in the development of sensory circuits. RGMb is involved in important physiological processes such as embryonic development, immune response, intercellular adhesion and tumorigenesis. Furthermore, RGMb is mainly involved in the regulation of RGMb-neogenin-Rho and BMP signalling pathways. The recent discovery of programmed death-ligand 2 (PD-L2)-RGMb binding reveals that the cell signalling network and functional regulation centred on RGMb are extremely complex. The latest report suggests that down-regulation of the PD-L2-RGMb pathway in the gut microbiota promotes an anti-tumour immune response, which defines a potentially effective immune strategy. However, the biological function of RGMb in a variety of human diseases has not been fully determined, and will remain an active research field. This article reviews the properties and functions of RGMb, focusing on its role under various physiological and pathological conditions.

Repulsive guidance molecule A (RGMA) is widely expressed in the CNS of the sea lamprey Petromyzon marinus during early development.

RGM interactions with its receptor Neogenin play an important role in the regulation of axonal guidance or cell death in the developing central nervous system. The sea lamprey RGMA transcript has been recently identified. However, its expression has been only studied in the spinal cord of mature (premetamorphic) larval sea lampreys. Here, we report the expression of the sea lamprey RGMA transcript in developing embryos and prolarvae by means of in situ hybridization. Our data show that the RGMA transcript is broadly expressed in the central nervous system of embryos and prolarvae and with a rostro-caudal gradient of expression.

Proteomic insights into the associations between obesity, lifestyle factors, and coronary artery disease

BackgroundWe aimed to investigate the protein pathways linking obesity and lifestyle factors to coronary artery disease (CAD).MethodsSummary-level genome-wide association statistics of CAD were obtained from the CARDIoGRAMplusC4D consortium (60,801 cases and 123,504 controls) and the FinnGen study (R8, 39,036 cases and 303,463 controls). Proteome-wide Mendelian randomization (MR) analysis was conducted to identify CAD-associated blood proteins, supplemented by colocalization analysis to minimize potential bias caused by linkage disequilibrium. Two-sample MR analyses were performed to assess the associations of genetically predicted four obesity measures and 13 lifestyle factors with CAD risk and CAD-associated proteins’ levels. A two-step network MR analysis was conducted to explore the mediating effects of proteins in the associations between these modifiable factors and CAD.ResultsGenetically predicted levels of 41 circulating proteins were associated with CAD, and 17 of them were supported by medium to high colocalization evidence. PTK7 (protein tyrosine kinase-7), RGMB (repulsive guidance molecule BMP co-receptor B), TAGLN2 (transgelin-2), TIMP3 (tissue inhibitor of metalloproteinases 3), and VIM (vimentin) were identified as promising therapeutic targets. Several proteins were found to mediate the associations between some modifiable factors and CAD, with PCSK9, C1S, AGER (advanced glycosylation end product-specific receptor), and MST1 (mammalian Ste20-like kinase 1) exhibiting highest frequency among the mediating networks.ConclusionsThis study suggests pathways explaining the associations of obesity and lifestyle factors with CAD from alterations in blood protein levels. These insights may be used to prioritize therapeutic intervention for further study.

Selective Inhibition of Repulsive Guidance Molecule C/Hemojuvelin (RGMc/HJV) with SRK-256 to Mobilize Iron in Functional Iron Deficiency

Functional iron deficiency is a serious condition marked by serum iron restriction in the presence of adequate iron stores that can lead to anemia, worsening quality of life, and leading to poor patient outcomes. Functional iron deficiency is characterized by abnormally high levels of hepcidin given serum iron status; it is frequently observed in diseases with chronic inflammation, such as anemia of chronic disease, chronic kidney disease, and myelofibrosis. Current treatments to address this patient population involve non-specifically targeting hepcidin and come with risks of off-target side effects. The secretion of hepcidin from the liver hepatocytes is linked to circulating serum iron levels and is controlled by the Bone Morphogenic Protein (BMP) 2/6 signaling pathway. Repulsive Guidance Molecule C or Hemojuvelin (RGMc/HJV) is a BMP 2/6 co-receptor in liver hepatocytes. Human genetic mutations in this gene result in juvenile hemochromatosis and genetic mouse studies have demonstrated that RGMc/HJV has a specific role in maintaining systemic iron homeostasis through modulation of hepcidin expression. We have generated a highly selective, fully human antibody (SRK-256) against RGMc/HJV which binds to the target with 39 pM affinity. SRK-256 has no detectable binding to RGMa or RGMb, thus avoiding potential side effects of targeting these gene family members with key physiological roles outside of iron metabolism. A high-resolution crystal structure (2.2 Å) elucidates not only the mechanism of action of SRK-256 directly competing for BMP binding to RGMc via contacts within the BMP binding domain, but also identifies binding to regions within this domain highly unique to RGMc over RGMa and RGMb, thus supporting the selectivity of the molecule. This mechanism of action is corroborated by in vitro assay data demonstrating that SRK-256 blocks signaling by BMP 2, 4, and 6. Pharmacodynamic and pharmacokinetic data supportive of a monthly subcutaneous dosing regimen in human patients has been generated in cynomolgus macaques, where SRK-256 demonstrates a dose-proportional duration of effect on suppression of hepcidin expression and elevation of transferrin saturation and achieves ~95% bioavailability via subcutaneous dosing. Furthermore, in the rat peptidoglycan-polysaccharide (PGPS) model of anemia of chronic disease, we have shown that in contrast to treatment with erythropoiesis stimulating agent alone (EPO, serum iron 47.7 ± 9.8 µg/dL), SRK-256 can restore serum iron to normal levels (Healthy control, serum iron 231.9 ± 12.0 µg/dL; SRK-256, serum iron 248.7 ± 60.9 µg/dL; and SRK-256+EPO, serum iron 277.6 ± 92.9 µg/dL) in the context of inflammation-driven, iron-restricted anemia (Figure 1). SRK-256 represents a potent and selective potential therapy for the treatment of patients with iron-restricted anemias across diseases of chronic inflammation.

Regulatory role of RGMb in lung injury promoted by the combination of carbon ion irradiation and anti-PD-1 antibody through Erk1/2 and p38 MAPK pathways

The combination of carbon ion radiotherapy and anti-PD-1 antibody represents a new approach to treating thoracic tumors. However, the lung damage caused by this combination therapy may limit its use, and the potential mechanisms for this are worthy of investigation. The objective of this research was to examine the potential involvement of repulsive guidance molecule b (RGMb) in lung damage promoted by the utilization of carbon ion irradiation combined with an anti-PD-1 antibody. The C57BL/6 mice have been randomly separated into four distinct groups: control, anti-PD-1, whole thorax carbon ion irradiation, and irradiation in combination with anti-PD-1 treatment groups (combination group). Detection of pathological changes in lung tissue using HE staining. Detection of pulmonary fibrosis by Masson staining and the hydroxyproline assay. ELISA to detect TNF-α, TGF-β, IL-6, and IL-1β expression levels within lung homogenates. The expression of RGMb, p38 MAPK, and Erk1/2 pathways was detected using a fully automated digital Western blotting system WES (ProteinSimple, USA). Flow cytometry was employed to analyze tissue-resident memory T cells (TRM) within the lung. Subsequently, the siRNA gene was employed to induce the downregulation of RGMb in mice in order to validate the involvement of RGMb in radiation-immune lung injury. The present study observed a significant increase in both inflammatory and fibrotic indicators within the mice group's lung tissue that received the combination treatment. The combination group exhibited elevated levels of TGF-β, TNF-α, IL-6, and IL-1β in lung homogenates. Anti-PD-1 antibody and carbon ion irradiation, upregulated RGMb, phospho-p38 MAPK and phospho-Erk1/2. The results obtained from the flow cytometry analysis indicated that the combination group was significantly higher in the number of clonal expansion TRMs, which were predominantly characterized by the expression of CD8+CD103+CD69-TRMs. The downregulate of RGMb via siRNA in mice resulted in a decrease in phospho-p38 MAPK and phospho-Erk1/2. The combination group exhibited a reduction in TNF-α, TGF-β, IL-6, and IL-1β in their lung tissues, and the number of CD8+CD103+CD69-TRM was significantly reduced. The combination group exhibited a significant improvement in inflammatory and fibrotic indicators within the lung tissues. Anti-PD-1 antibody and carbon ion irradiation synergistically regulate RGMb, leading to strong clonal expansion of lung TRM through the p38 MAPK and Erk1/2 pathways. The present study offers valuable insights into the treatment of lung injury due to the combined administration of carbon ion radiotherapy and anti-PD-1 antibody therapy.

RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS.

Repulsive guidance molecule A (RGMa) was originally identified as a neuronal growth cone-collapsing factor. Previous reports have demonstrated the multifunctional roles of RGMa mediated by neogenin1. However, the pathogenic involvement of RGMa in amyotrophic lateral sclerosis (ALS) remains unclear. Here, we demonstrated that RGMa concentration was elevated in the cerebrospinal fluid of both patients with ALS and transgenic mice overexpressing the mutant human superoxide dismutase1 (mSOD1 mice). Treatment with humanized anti-RGMa monoclonal antibody ameliorated the clinical symptoms in mSOD1 mice. Histochemical analysis revealed that the anti-RGMa antibody significantly decreased mutant SOD1 protein accumulation in the motor neurons of mSOD1 mice via inhibition of actin depolymerization. In vitro analysis revealed that the anti-RGMa antibody inhibited the cellular uptake of the mutant SOD1 protein, presumably by reinforcing the neuronal actin barrier. Collectively, these data suggest that RGMa leads to the collapse of the neuronal actin barrier and promotes aberrant protein deposition, resulting in exacerbation of the ALS pathology.

RGMa and Neogenin control dendritic spine morphogenesis via WAVE Regulatory Complex-mediated actin remodeling.

Structural plasticity, the ability of dendritic spines to change their volume in response to synaptic stimulation, is an essential determinant of synaptic strength and long-term potentiation (LTP), the proposed cellular substrate for learning and memory. Branched actin polymerization is a major force driving spine enlargement and sustains structural plasticity. The WAVE Regulatory Complex (WRC), a pivotal branched actin regulator, controls spine morphology and therefore structural plasticity. However, the molecular mechanisms that govern WRC activation during spine enlargement are largely unknown. Here we identify a critical role for Neogenin and its ligand RGMa (Repulsive Guidance Molecule a) in promoting spine enlargement through the activation of WRC-mediated branched actin remodeling. We demonstrate that Neogenin regulates WRC activity by binding to the highly conserved Cyfip/Abi binding pocket within the WRC. We find that after Neogenin or RGMa depletion, the proportions of filopodia and immature thin spines are dramatically increased, and the number of mature mushroom spines concomitantly decreased. Wildtype Neogenin, but not Neogenin bearing mutations in the Cyfip/Abi binding motif, is able to rescue the spine enlargement defect. Furthermore, Neogenin depletion inhibits actin polymerization in the spine head, an effect that is not restored by the mutant. We conclude that RGMa and Neogenin are critical modulators of WRC-mediated branched actin polymerization promoting spine enlargement. This study also provides mechanistic insight into Neogenin's emerging role in LTP induction.