| Backgroud and ObjectiveLRP4(low-density lipoprotein receptor-related protein 4)is a type I single transmembrane protein of the LDLR family with a large extracellular domain,which expresses widely in many organs of mice,such as muscle,brain,bone,mammary and other skin appendage placodes,and kidney.In muscle,LRP4 characteristically expresses in neuromuscular junction(NMJ),interacts with other key proteins(agrin,Mu SK)to regulate the ACh R aggregation in NMJ,which is critical for the formation and maintenance of the NMJ.In brain,LRP4 regionally expresses in hippocampal astrocytes,and astrocytic LRP4 maintains glutamatergic transmission by controlling the release of ATP from astrocytes,which is critical for the maintain of cognitive function.Furthermore,more and more studies have found that LRP4 also plays a very important role in myasthenia gravis,epilepsy,Alzheimer disease,etc.Myasthenia gravis(MG)is a neuromuscular junction(NMJ)organ-specific autoimmune disease,which is frequently caused by autoantibodies against acetylcholine receptor(ACh R)(≈80%)and Muscle-Specific Kinase(≈10%).But about 10% MG patients are double-seronegative for anti-ACh R and anti-Mu SK antibodies,which is defined as DNMG(double-seronegative MG).Autoantibodies to LRP4 exist in the sera of 10% DSMG patients.It has been proved by active EAMG(active experimental autoimmunology myasthenia gravis),mice are immunized with the extracellular domain of LRP4,that anti-LRP4 antibodies are pathogenic and induce MG-like deficits.Up to now,there is no report about LRP4 passive EAMG,and it is still vague whether patient anti-LRP4 antibodies are the causal for MG.The objective of the first chapter of this project is to determine whether human anti-LRP4 antibodies are pathogenic in mice and to investigate underpinning pathogenic mechanisms.Epilepsy is characterized by the typical symptom of seizure,and anti-seizure medications are the main therapeutic method in clinical,but the effects of these therapy have not been satisfactory.To find a better treatment for controlling seizures,it makes sense to further explore the regulatory mechanisms of seizures at genetic level.LRP4 regionally expresses in mice hippocampus where is key to limbic epileptogenesis.It is well known that neurons release a high level of glutamate during seizures,and it has been reported that the reduction of LRP4 in astrocytes downregulates glutamate released from neurons by regulating astrocytic ATP releasing.Whole brain knockout LRP4 can increase the threshold of seizures in mice.However,it is still unclear whether LRP4 expression level can be regulated by seizures,and what roles does LRP4 play in seizures.The objective of the second chapter of this project is to determine the role of LRP4 in seizures and to investigate its specific regulatory mechanism.Methods1.Anti-LRP4 IgG from a MG patient causes muscle weakness and impairs the neuromuscular junction in miceIgG was purified from a MG patient with anti-LRP4 antibodies and transferred to mice.Mice were characterized for body weight,muscle strength,twitch and tetanic force,NMJ functions including CMAP(compound muscle action potential)and endplate potentials,and NMJ structure.Effects of the antibodies on agrin-elicited Mu SK activation and ACh R clustering were studied and the epitopes of these antibodies were identified.2.LRP4 in hippocampal astrocytes serves as a negative feedback factor in seizuresDetect the changes of LRP4 and mi R-351-5P expression levels in the hippocampus of pilocarpine injected mice.Detect the changes of LRP4 and mi R-351-5P expression levels in primary cultured hippocampal astrocytes treated with glutamate only or with glutamate and AP5(an NMDA receptor antagonist).mi R-351-5p mimics and mi R-351-5p inhibitors were transfected into the hippocampal astrocytes to verify whether the changes of LRP4 expression in astrocytes induced by glutamate stimulation were caused by the changes of mi R-351-5p expression.Finally,after stereotactic injected by sh Lrp4 lentivirus in the hippocampus to reduce regional LRP4 expression level,the threshold changes of seizures were dectected in pilocarpine and pentaerythritol(PTZ)injected mice.Results1.Anti-LRP4 IgG from a MG patient causes muscle weakness and impairs the neuromuscular junction in miceAnti-LRP4 IgG-injected mice suffered MG symptoms,including weight lost and muscle weakness.Decreased CMAPs,reduced twitch and tetanus force,compromised neuromuscular transmission,and NMJ fragmentation and distortion were detected in anti-LRP4 Ig G-injected mice.Anti-LRP4 Ig G inhibited agrin-elicited Mu SK activation and ACh R clustering.The patient Ig G recognized the β3 domain of LRP4 and the C-terminus of agrin and reduced agrin-enhanced LRP4-Mu SK interaction.2.LRP4 in hippocampal astrocytes serves as a negative feedback factor in seizuresSeizures induced by pilocarpine decreased LRP4 expression level and increased mi R-351-5p expression level in mice hippocampal astrocytes.Glutamate reduced LRP4 expression and enhanced mi R-351-5p expression in cultured hippocampal astrocytes,and these effects can be partially attenuated by AP5.Furthermore,mi R-351-5p mimics decreaed LRP4 expression in cultured hippocampal astrocytes,mi R-351-5p inhibitors lessened the reduction of LRP4 expression in glutamate treated hippocampal astrocytes.Local reduction of LRP4 in hippocampus by sh Lrp4 lentivirus injection in hippocampus increased the threshold of seizures in pilocarpine or pentylenetetrazol(PTZ)injected mice.Conclusions1.Anti-LRP4 IgG from a MG patient causes muscle weakness and impairs the neuromuscular junction in miceAnti-LRP4 IgG in MG patient is pathogenic.It impairs the NMJ by interrupting agrin-dependent LRP4-Mu SK interaction.2.LRP4 in hippocampal astrocytes serves as a negative feedback factor in seizures.High released glutamate induced by seizures down-regulated astrocytic LRP4 through increasing mi R-351-5p in hippocampal astrocytes via activating astrocytic NMDA receptor,and locally reduction of LRP4 in hippocampus increased the threshold of seizures. |
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