SNX17 can increase LRP4 membrane expression, promote acetylcholine receptor aggregation, and improve EAMG

Keywords: myasthenia gravis; LRP4； Therapeutic targets

Myasthenia gravis ( MG) is most prominently characterized by impaired aggregation of the neuromuscular junction (NMJ), postsynaptic membrane (endplate membrane), acetylcholine receptor (AChR) due to autoimmune damage.

and as a low-density lipoprotein receptor ( LDLRs) family of low-density lipoprotein receptor-associated protein 4 (LRP4), due to the Agrin-LRP4-MuSK signaling cascade, plays an important role
in the AChR aggregation of the endplate membrane.

Sorting microtubule junction protein 17 (SNX17) is an intracellular adaptor protein that can regulate the degradation and recycling of a variety of internalized membrane proteins, and its unique PX and FERM domains can promote the circulation of internalized membrane proteins by specifically recognizing the "-NPxY-" motif in the cytosolic domain of LDLR family members.
In turn, the receptor function
is re-exerted.
However, it is unclear
whether this mechanism has a similar effect in MG.

In October 2022, the Gao team of the Department of Neuroimmunology, Henan Institute of Medical Sciences, Zhengzhou University, published a report entitled "Sorting nexin 17 increases low-density lipoprotein receptor-related protein 4 membrane expression: A novel mechanism of" at Front Immunol acetylcholine receptor aggregation in myasthenia gravis", using EAMG mouse model as the research object, explored the relationship between SNX17 and LRP4, and studied the change of aggregation status and improvement of muscle weakness of AChR
。

This paper is also one of the most watched papers in myasthenia gravis and related papers published in October (see [November 22] last month summary of important papers on myasthenia gravis research), the study design is rigorous, the conclusions are objective and reliable, and it has high scientific significance
.

Therefore, we invited He Xiaoxiao, the first author of the paper, to compile and interpret, and the corresponding author Professor Gao Gao did the review
.

Study design

1.
C2C12 myotube cells were selected to induce the internalization of LRP4 protein on the cell surface by applying LRP4 antibody stimulation, and the intracellular interaction relationship
between SNX17 and LRP4 was explored by co-immunoprecipitation technology.
By knocking down and overexpressing intracellular SNX17, the effects
of SNX17 on membrane expression of LRP4, MuSK phosphorylation and AChR aggregation were explored at the cellular level.

2.
Establish an EAMG mouse model, and explore the regulatory effect of SNX17 on the Agrin-LRP4-MuSK signaling cascade pathway from the animal level by knocking down and overexpressing SNX17 in the gastrocnemius muscle of mice, so as to evaluate the effect of SNX17 on EAMG as a whole Effects
of action in mouse models.

Figure 1.
Animal experimental procedures

Key findings

1.
SNX17 overexpression increased the expression of LRP4 on the cell surface, phosphorylation of MuSK and aggregation of AChR

By imposing LRP4 antibody stimulation induces cell surface LRP4 protein internalization, and the interaction between SNX17 and LRP4 is demonstrated by co-immunoprecipitation (Figure 2A).

When SNX17 in myotubular cells was overexpressed, the expression of LRP4 on the cell surface (Figure 2B), phosphorylation of MuSK (Figure 2C), and the number of AChR aggregations on the cell surface (Figure 2D) increased
significantly.
The effect of SNX17 in promoting MuSK phosphorylation and AChR aggregation by increasing the amount of circulating return to internalized LRP4 was demonstrated.

Figure 2.
SNX17 knockout reduces cell surface LRP4 expression, MuSK phosphorylation, and AChR aggregation
.

2.
The expression of SNX17 in the gastrocnemius muscle of EAMG mice was reduced, and the AChR at NMJ was fragmented

Research utilization The AChR-α subunit (Rα 97~116) peptide was used as an immunogen to establish the EAMG model by active immunization, and the expression of SNX17 in the gastrocnemius muscle of EAMG model mice was found to be reduced by Western blot detection (Figure 3A ）
。 Also, LRP4 expression and MuSK phosphorylation levels were lower than normal mice (Figure 3B).

In addition, the AChR morphology at NMJ in the normal group and the solvent control group was normal and showed a complete pretzel structure, but the area of AChR on the muscle filament of EAMG mice was significantly reduced and significantly fragmented (Figure 3C), suggesting that SNX17 may be affected AChR
at NMJ.

Figure 3.
SNX17 expression was reduced in the gastrocnemius muscle of EAMG mice, and AChR was fragmented at NMJ

3.
SNX17 overexpression increased the expression of LRP4 and MuSK phosphorylation in the gastrocnemius muscle of EAMG mice, and improved AChR morphology and leg weakness at NMJ

When After overexpression of SNX17 in the gastrocnemius muscle of EAMG mice, the expression of LRP4 and the phosphorylation level of MuSK (Figure 3A) and the area of AChR at NMJ were increased, improving the fragmentation morphology of AChR (Figure 3B).
）
。 In addition, no obvious damage phenomena such as inflammatory cell infiltration and myofiber atrophy were observed in the gastrocnemius myofibers of SNX17-overexpressed mice (Figure 3C
).
Compared with the attenuation rate of the fifth amplitude of 27.
12% ± 1.
63% in the model control group, the fifth amplitude attenuation rate in the SNX17 overexpression group was reduced to 13.
07% ±0.
89% (Figure 3D), and the leg strength was still different from that of normal mice.
But it also got some recovery (Figure 3E).

Figure 3.
SNX17 overexpression increased the expression of LRP4 and MuSK phosphorylation in the gastrocnemius muscle of EAMG mice, and improved AChR morphology and leg weakness at NMJ

Conclusion

This study sheds light on SNX17 increases the expression of cell surface LRP4 by interacting with internalized LRP4, thereby promoting MuSK phosphorylation and AChR aggregation, ultimately improving the mechanism of
action of muscle weakness in EAMG mice.

The process may be: SNX17 recognizes and binds LRP4 to early endosomes due to internalization, and mediates the transformation of LRP4 into circulating endosomes and back to myocyte membranes
.
The LRP4 of the return membrane induces AChR aggregation through the Agrin-LRP4-MuSK signaling pathway, thereby receiving ACh released by the presynaptic membrane, further restoring neuromuscular excitation transmission (Figure 4).

This new mechanism is not only SNX17 is involved in the beneficial support and complementation of the conceptual connotation of receptor recycling, and reveals that SNX17 may be a new target for future MG therapy
.

Figure 4.
The underlying mechanism
by which SNX17 increases cell surface LRP4 expression to promote AChR aggregation and recovery of neuromuscular excitatory transmission.