Method for aggregating acetylcholinergic receptor for stabilizing neuromuscular junction

A technology of acetylcholine receptors and neuromuscular junctions, which is applied in the field of regulating the stability of acetylcholine receptor aggregates of neuromuscular junctions, and can solve problems such as unclear effector molecules

Inactive Publication Date: 2009-01-21
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effector molecules of Rapsyn are still unknown

Method used

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  • Method for aggregating acetylcholinergic receptor for stabilizing neuromuscular junction
  • Method for aggregating acetylcholinergic receptor for stabilizing neuromuscular junction
  • Method for aggregating acetylcholinergic receptor for stabilizing neuromuscular junction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Example 1 Cholinergic agonist CCh can activate Calpain

[0126] The activity of Calpain is regulated by calcium ions, and cholinergic agonists and depolarization of muscle cells can cause an increase in calcium ions. The present inventors examined whether cholinergic agonists could modulate the activity of Calpain in muscle cells.

[0127] Such as figure 1 As shown in A, treatment of muscle cells with CCh, a cholinergic agonist that is not easily hydrolyzed, can not only induce calcium ion flux in muscle cells, but also increase the activity of Calpain in myotubes. Calpain activity peaked 15 min after CCh stimulation and then decayed. Calpain activation by CCh was inhibited when cells were pretreated with Calpeptin, a membrane-permeable Calpain inhibitor. These results suggest that cholinergic agonists can activate Calpain in myocytes.

[0128] Cdk5 in muscle cells can also be activated by CCh (Lin, W et al., Neurotransmitteracetylcholine negatively regulates neurom...

Embodiment 2

[0130] Example 2 Inhibiting the activity of Calpain can improve the stability of AChR aggregates

[0131] Further, the present inventors examined whether Calpain is involved in the process of cholinergic agonist-induced dissipation of AChR aggregates. Myocytes cultured in vitro were first treated with Agrin to induce AChR aggregates, then transferred to Agrin-free medium, and stimulated with CCh with or without Calpeptin treatment. The stimulation of CCh alone can promote the depolymerization of AChR aggregates, making the total length of AChR aggregates per unit length of myotubes shorter and the total number less ( figure 2 A, 2B and 2C). The reduction of AChR aggregates induced by CCh was improved in Calpeptin-treated cells ( figure 2 B and 2C), suggesting that Calpain is involved in the dissipation of AChR aggregates induced by cholinergic agonists.

[0132] In order to further understand the regulatory effect of Calpain on AChR aggregation, the inventors used the str...

Embodiment 3

[0133] Embodiment 3 Rapsyn can combine with Calpain

[0134] When Rapsyn was used as a bait to find its binding protein in the yeast two-hybrid system, the inventors found the large subunit of m-Calpain. M-Calpain consists of two subunits: a unique large subunit with a size of 80KD, and a small subunit with a size of 28KD shared with μ-Calpain. The large subunit itself also contains four domains: a short domain at the nitrogen end (domain I), a domain with enzymatic catalytic activity (domain II), and a domain that regulates enzymatic activity (domain III) and a calcium ion-binding domain (domain IV) comprising the EF chiral structure. The clone initially screened from yeast contained 266 amino acid sequences (equivalent to the 435-700 amino acid sequence of m-Calpain's own amino acid sequence), including domain IV and part of domain III, suggesting that the region combined with Rapsyn may be in the structure In domain III and domain IV ( image 3 B). In order to delineate...

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Abstract

The invention discloses the stability application of Calpain or the excitant or antagonist of Calpain in preparing AchR aggregate that regulates neuromuscular junction. The invention also discloses a method for screening the agent that regulates the stability of the AchR aggregate of neuromuscular junction by using the Calpain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for regulating the stability of acetylcholine receptor aggregates of neuromuscular joints. Background technique [0002] Neuromuscular junctions form between motor neurons and muscle fibers. AChR forms aggregates on the postsynaptic membrane to ensure efficient and precise synaptic transmission. The process of forming AChR aggregates is a dynamic process. The newly formed AChR aggregates innervated by motor nerve endings can be secreted by motor neurons, a kind of glycoprotein-aggrin Agrin, and the protein synthesized by muscle cells that binds to AChR- Stabilized by the coupling protein Rapsyn. At the same time, motor neurons also release negative regulatory factors to dissipate uninnervated AChR aggregates, thereby optimizing the postsynaptic structure. Genetic evidence suggests that ACh may be such a negative regulator. AChR aggregates were found to be larger in mutant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K38/55A61P21/02C12Q1/37
Inventor 罗振革钱磊陈飞
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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