Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.     

Serum amyloid A facilitates expansion of CD4+ T cell and CD19+ B cell subsets implicated in the severity of myasthenia gravis patients

Serum amyloid A (SAA) is a clinically useful inflammatory marker involved in the pathogenesis of autoimmune diseases. This study aimed to explore the SAA levels in a cohort of patients with myasthenia gravis (MG) in relation to disease-related clinical parameters and myasthenic crisis (MC) and elucidate the effects of SAA on immune response. A total of 82 MG patients including 50 new-onset MG patients and 32 MC patients were enrolled in this study. Baseline data and laboratory parameters of all enrolled MG patients were routinely recorded through electronic medical systems. SAA levels were measured by enzyme-linked immunosorbent assay (ELISA) kit. CD4⁺ T and CD19⁺ B cell subsets were analyzed by flow cytometry. In vitro, human recombinant SAA (Apo-SAA) was applied to stimulate peripheral blood mononuclear cells (PBMCs) from MG patients to observe the effect on T and B cell differentiation. Our results indicated that SAA levels in new-onset MG patients were higher than those in controls and were positively correlated with QMG score, MGFA classification, plasmablast cells, IL-6, and IL-17 levels. Subgroup analysis revealed that SAA levels were increased in generalized MG (GMG) patients than in ocular MG (OMG), as well as elevated in late-onset MG (LOMG) than in early-onset MG (EOMG) and higher in MGFA III/IV compared with MGFA I/II. The ROC curve demonstrated that SAA showed good diagnostic value for MC, especially when combined with NLR. In vitro, Apo-SAA promoted the Th1 cells, Th17 cells, plasmablast cells, and plasma cells differentiation in MG PBMCs. The present findings suggested that SAA was increased in MG patients and promoted expansion of CD4⁺ T cell and CD19⁺ B cell subsets, which implicated in the severity of MG patients.

     

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.     

IRF5调控AChR特异性T细胞参与EAMG疾病发生、发展的实验研究

目的:体内外实验探讨干扰素调节因子5(Interferon regulatory factor 5,IRF5)在乙酰胆碱受体(Acetylcholine Receptor,ACh R)特异性T、B细胞中的表达情况以及与实验性自身免疫性重症肌无力(Experimental Autoimmune Myasthenia Gravis,EAMG)疾病的发生存在的可能关系。方法:ELISA法检测不同发病时相大鼠血清中IRF5的含量;流式细胞仪法检测早期晚期发病时相CD4+T细胞以及CD45R+B细胞中IRF5的表达情况;QPCR法及Western blotting法分别检测体外ACh R刺激不同时间点的T、B细胞中IRF5的m RNA和蛋白水平的表达差异。结果:血清学检测结果显示,IRF5高表达于EAMG大鼠的晚期发病时相的血清中,并随着疾病进程的不断加重呈现逐渐增高的表达,与CFA对照组大鼠相比较差异显著,有统计学意义(***,P0.001);流式结果表明,来自EAMG大鼠早、晚期发病时相的无论是淋巴结还是脾脏中的ACh R特异性T细胞均高表达IRF5,且与CFA对照组相比较,有统计学差异,而B细胞中IRF5的表达即便是在晚期发病时相也与CFA对照组无明显区别;QPCR结果发现,EAMG晚期发病时相的ACh R特异性T细胞在经过ACh R体外刺激0 h,72 h后,均可见IRF5 m RNA水平的高表达,而B细胞中IRF5的m RNA水平则在两组中无统计学差异;Western blotting结果进一步证实,IRF5蛋白水平在ACh R特异性T细胞中呈现高表达。结论:IRF5是通过对ACh R特异性T淋巴细胞的调控进而参与EAMG疾病的发生和发展过程的。     

Generation and characterization of a high affinity anti-human FcRn antibody,rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).     

Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effect of modulators

1. Loss of response after prolonged or repeated application of stimulus is generally termed desensitization. A wide variety of phenomena occurring in living organisms falls under this general definition of desensitization. There are two main types of desensitization processes: specific and non-specific. 2. Desensitization of the nicotinic acetylcholine receptor is triggered by prolonged or repeated exposure to agonists and results in inactivation of its ion channel. It is a case of specific desensitization and is an intrinsic molecular property of the receptor. 3. Desensitization of the nicotinic acetylcholine receptor at the neuromuscular junction was first reported by Katz and Thesleff in 1957. Desensitization of the receptor has been demonstrated by rapid kinetic techniques and also by the characteristic "burst kinetics" obtained from single-channel recordings of receptor activity in native as well as in reconstituted membranes. In spite of a number of studies, the detailed molecular mechanism of the nicotinic acetylcholine receptor desensitization is not known with certainty. The progress of desensitization is accompanied by an increase in affinity of the receptor for its agonist. This change in affinity is attributed to a conformational change of the receptor, as detected by spectroscopic and kinetic studies. A four-state general model is consistent with the major experimental observations. 4. Desensitization of the nicotinic acetylcholine receptor can be potentially modulated by exogenous and endogenous substances and by covalent modifications of the receptor structure. Modulators include the noncompetitive blockers, calcium, the thymic hormone peptides (thymopoietin and thymopentin), substance P, the calcitonin gene-related peptide, and receptor phosphorylation. Phosphorylation is an important posttranslational covalent modification that is correlated with the regulation and desensitization of the receptor through various protein kinases. 5. Although the physiological significance of desensitization of the nicotinic receptor is not yet fully understood, desensitization of receptors probably plays a significant role in the operation of the neuronal networks associated in memory and learning processes. Desensitization of the nicotinic receptor could also possibly be related to the neuromuscular disease, myasthenia gravis.     

血清胆红素与尿酸水平对重症肌无力的临床意义

目的：探讨血清胆红素以及尿酸的水平对重症肌无力患者的临床价值。方法：选取我院确诊的重症肌无力患者作为实验组, 另选择同期体检的健康志愿者作为对照组。检测并比较两组血清直接胆红素(DBIL)、间接胆红素(IBIL)、总胆红素(TBIL)、尿酸 (UA)及肌酐(Cr)等指标水平。结果：实验组血清DBIL、IBIL、TBIL、UA 水平均显著低于对照组,差异有统计学意义(P＜0.05),但实 验组不同类型的MG 患者血清DBIL、IBIL、TBIL、UA水平比较差异均无统计学意义(P>0.05)。同组男性DBIL、IBIL、TBIL、UA值 均明显高于女性,差异具有统计学意义(P<0.05);两组间相同性别比较,实验组DBIL、IBIL、TBIL、UA 均低于对照组,差异有统计 学意义(P＜0.05)。结论：检测血清BIL 和UA水平对诊断重症肌无力具有一定的参考价值。     

The limitation of IL-10-exposed dendritic cells in the treatment of experimental autoimmune myasthenia gravis and myasthenia gravis

Dendritic cells (DC) are highly specialized antigen presenting cells that play critical roles as instigators and regulators of immune responses including B cell function, antibody synthesis and isotype switch. In this study, we compared immunotherapeutic effect of IL-10-treated DC (IL-10-DC) via both intraperitoneal (i.p.) and subcutaneous (s.c.) delivery in rats with incipient experimental autoimmune myasthenia gravis (EAMG). Spleen DC were isolated from onset of EAMG on day 39 post-immunization, exposed in vitro to IL-10, and then injected into incipient EAMG at dose of 1 x 10(6) cells/rat on day 5 after immunization. Intraperitoneal administration of IL-10-DC suppressed clinical scores, anti-acetylcholine receptors (AChR) antibody secreting cells, antigen-specific IL-10/IFN-gamma production and T cell proliferation compared to control EAMG rats. Importantly, IL-10-DC, if given by s.c. route, failed to ameliorate clinical sign of EAMG. Simultaneously, T cell proliferation, anti-AChR antibody secreting cells and IL-10/IFN-gamma production had no alteration, as compared to control EAMG rats. Both in vitro and in vivo experiments showed that treatment of IL-10 inhibited the migration of DC toward MIP-3beta and lymph node, indicating that in vitro manipulation of DC with IL-10 alters the migration of DC that influences the therapeutic effect in the treatment of autoimmune diseases. In MG patients, neither the improvement of clinical symptom nor the alteration of immunological parameter was observed through s.c. delivery of IL-10-DC, suggesting the limitation of IL-10-DC in the treatment of MG patients.     

眼肌型和全身型重症肌无力患者外周血Tfh细胞及相关分子表达研究

目的:阐述Tfh细胞及相关分子在重症肌无力发病中的作用.通过分析Tfh细胞及相关分子在眼肌型和全身型重症肌无力患者外周血的表达差异,阐明眼肌型和全身型重症肌无力患者外周发生了不同的体液免疫反应;在描述重症肌无力患者症状严重程度方面,比较绝对评分和QMG评分的优劣性.方法:运用抗体标记Tfh细胞相关分子CXCR5、ICOS、Bcl-6及CXCL13,应用流式细胞术检测、分析这些分子在非重症肌无力、眼肌型和全身型重症肌无力患者外周血的表达;对病情做QMG评分和绝对评分,分析其与CXCL13表达的相关性.结果:在眼肌型、全身型重症肌无力患者外周血中,CXCR5、ICOS和Bcl-6分子在CD4+T细胞的表达增高,其中CXCR5、Bcl-6分子表达在3组之间的差异存在统计学意义;随MG病情发展,外周血CXCL13的表达上调,CXCL13与QMG评分和绝对评分相关系数分别为0.669和0.797.结论:Tfh细胞及相关分子CXCR5、Bcl-6、CXCL13的表达参与了MG的发生、发展过程;Tfh细胞及相关分子在全身型重症肌无力患者外周血的表达高于眼肌型重症肌无力,这是全身型重症肌无力患者临床症状更重和症状评分更高的原因,也是全身型重症肌无力患者外周血抗体阳性率高于眼肌型重症肌无力的原因;在重症肌无力患者病情评测中,绝对评分较QMG评分更加准确,与临床症状的相关性更高.