The Expression And Function Of Sorting Nexin 17 In Myasthenia Gravis Muscle Cells

BackgroundMyasthenia gravis(MG) is the most common immune-mediated disorder of the neuromuscular junction with a prevalence of 200–300/million population Its and its clinical features include muscle weakness fatigue and muscle weakness intensified after the activity, after rest or sleep. Neuromuscular connection(NMJ) is composed of highly specialized motor neuron presynaptic membrane, synaptic cleft, muscle cell membrane of the postsynaptic membrane. Its effect is responsible for the transmission of signals between nerve and muscle, rapidly translating motor neuron action potentials into muscle contraction.The acetylcholines are released from the nerve terminal activates the acetylcholine receptors(AChRs),leading to channel opening and depolarization of the muscle membrane.The high density and clustering of AChR molecules at the postsynaptic folds ensure the efficiency of the signal transduction.During postnatal development in mammals, the NMJ postsynaptic membrane forms infolded structure and AChRs are concentrated at the crests of the numerous folds.The estimated density of the clustered AChRs is 10,000–20,000/um2,which only 10/um2 compares with in membranes inextrasynaptic regions.The molecular mechanisms that regulate and stabilize AChR clustering remain unclear.In the past three decades, a number of nerve-derived and muscle-derived factors and signaling pathways that drive differentiation of NMJs have been identified. The synaptic factors contain two major signaling proteins, agrin and neuregulin, which are secreted by motor neurons and play essential roles in NMJ formation and maintenance. Other extracellular proteins are also implicated, including laminin, collagen IV/XIII, and perlecan. Inside the muscle cells, many signaling molecules, such as the adaptor proteins Dok7 and rapsyn, directly interact with the cytoplasmic domain of MuSK to form a multiprotein complex required for AChR clustering. The agrin–LRP4–MuSK signaling pathway plays a crucial role in NMJ development. Our understanding of the molecular interactions by which this complex functions has increased. AChR clustering requires the binding of the motor neuron–derived protein agrin on lipoprotein receptor-related protein 4(LRP4) at the postsynaptic membrane. The agrin-LRP4 complex activates the muscle-specific kinase(MuSK) which leads to AChR clustering.The low-density lipoprotein(LDL) receptor(LDLR) family include the LRP4 and LRP1,and the metabolism of LRP1 in cell is affected by cell sorting nexin 17(SNX17).The sorting nexin(SNX) family includes diverse cytoplasmic and membrane associated proteins, which are involved in endocytosis and endosomal trafficking of transmembrane proteins.SNX17 protein induced LRP1 recycling within the cell and avoid LRP1 into the lysosome degradation. LRP1 and LRP4 has the same structure namely NPxY structure domains, therefore LRP4 has the existence and SNX17 combination.SNX17 is distributed in all tissuses.SNX17 in intercostal muscle cells is over expression in myasthenia gravis group.But no study report the content of SNX17 in muscle. SNX17 for neuromuscular junction is influential LRP4 metabolism still needs further research. ObjectiveThe morphology and distribution of AChR and SNX17 molecular are observed by immunofluorescence muscle in this study to compare the content of SNX17 in two groups by fluorescence quantitative PCR and western blot. Methods1. In rats,MG group and the control group intercostals muscles are removed. All the muscle tissues are put into the 4% paraformaldehyde fixed for 10 minutes. The muscles are placed on the microscope slides, we isolated single muscle fiber with a thin needle.Then the fibers are infiltrated in0.5% Triton X-100 for 20 min. The muscle fibers are fixed with 5%BSA buffer for 60 minutes. The muscles are added to a concentration of 1: 50 mouse antihuman Ig G overnight. Then they are added to a concentration of 1: 100 AF-g350 Goat anti Mouse Ig G and α-BTX staining. These antibody are incubated for 1 hour at 37℃water bath. Then the single muscle fibers are washed five minutes for three times by PBS. We observe the muscle acetylcholine receptors form by a laser confocal microscope.2. Fluorescence quantitative PCR method to detect SNX17 m RNA levels. We search the people SNX17 sequence in Gene Bank.The upstream primer sequences5,-CAATGGGTCTTGCCGAACAG-3,Downstreamprimer sequences5,CGCCTACGT GGCCTATAACAT-3,.Beta actin as inside,The upstream primer sequences5,-GGGCCGGACTCGTCATACT-3,Downstreamprimer sequences5,-CCTGGCACCC AGCACAAT-3,. Each group is weighed 20 mg of muscle homogenates. The total RNA is extracted from the muscle homogenates.The c DNA is reversed transcription by reverse transcription kit.The RNA is detected by fluorescence quantitative 20 ul PCR.we observe the PCR amplification. We set the fluorescent degeneration reaction conditions: 95 ℃ for 30 s;95℃ for 5s;55℃ for 2min;72℃ for 30 s with forty cycles. We put10 ul pcr amplification product into the agarose gel electrophoresis, in order to ensure the specificity of the primer design. The ratio of β-actin gene Ct values and SNX17 gene Ct values is a standardized relative quantitative genes.3.Western blot method to detect SNX17 protein content:Each group get intercostal muscle 20mg:We get the homogenate in Liquid nitrogen.Then the RIPA is droped into the homogenate. The BCA method is used to measured cell protein concentration in 5000 r/min by the centrifugal supernatant after 5 min, Beta actin as inside. The SNX17 antibodies that rat anti people and rabbit anti beta actin antibodys are droped on the sampes incubation 4 ℃ for the night. Antibodys HRP labeled that sheep anti mouse and rabbit are droped on the sampes in 37 ℃ for 1 h incubation, and then they are exposured to film, Using image analysis software Quantity One were determined and beta actin gene average color stripe, the ratio of the average strength of each protein expression.Results1. General clinical data:the MG group(10 cases) and control group(14 cases). The differences have no statistical significance in age and agender of the two groups(table 1).2. AChR, SNX17 morphological observation: AChR generally located in the central part of the vertical axis of muscle fiber, red oval shape, contour line is smooth continuous integrity.Control group AChR has cookies samples, internal fine reticular structure of the branch;And MG group cann,t be observed the complete red outer contour, internal network structure.But SNX17 can be observed In the same position, with the similar shape.The are completely overlapping images(Figure 1 and figure 2).3. The quantitative results of SNX17 gene and protein : SNX17 m RNA content is increased obviously in MG group by q-PCR,and the difference has statistically significant(t=5.75,P<0.05). SNX17 protein content is increased obviously according to the results of western blot method, the difference has statistically significant(t=3.03,P<0.05). Conclusion(1)The expression of SNX17 is higher in MG intercostals muscles cells, and it can promote LRP4 recycle in the cell.(2)SNX17 and AChR are located in the neuromuscular junction, promoting the AChR gathered,And it may be an important material to maintain the Neural signal transmission.