巨噬细胞自噬溶酶体融合障碍导致LCD变性TGFBIp贮积的分子机制研究

格子状角膜营养不良(LCD)是TGFBI基因突变导致视力丧失的一种遗传性疾病，其主要病理特征是变性蛋白TGFBIp贮积于角膜，发病机理仍然不清，除了角膜移植，目前没有其它更好的治疗方法。我们前期在一个25人家系中确诊了14人患有LCD，利用该家系资源研究发现，角膜巨噬细胞自噬降解变性TGFBIp功能失常，自噬体和溶酶体不能融合成为自噬溶酶体从而造成变性TGFBIp贮积，但也没有更进一步深入研究其分子机理。此外，我们预实验发现：在内吞正常TGFBIp的巨噬细胞中，Atg14介导形成STX7-SNAP9-VAMP8复合物并促使自噬体和溶酶体融合为自噬溶酶体，在这个过程中自噬体“货物”mtDNA能够激活TLR9信号通路进而促使巨噬细胞表达CD36和CD68，但内吞变性TGFBIp的巨噬细胞没有这种现象，这可能是LCD发病的一种分子机制。本项目拟继续利用该LCD家系资源进一步深入研究这种分子机制。

Lattice corneal dystrophy (LCD) is related to the denaturation of transforming growth factor-β-induced protein (TGFBIp) due to mutation of TGFBI gene. Autophagic degradation of the denatured proteins TGFBIp by macrophages is one of the pathways to remove the denatured proteins. However，there are few related studies to address this mechanism and the detail mechanisms still keep unknown. Thus, in our previous study, we investigated the role of autophagy in the degradation of mutant TGFBIp (MU TGFBIp) in macrophages. We found fourteen members from a family of 25 were identified as LCD sufferers. Significant TGFBIp aggregates and macrophage infiltration were found only in the corneas of LCD sufferers. Marker accumulation of TGFBIp was found in macrophages exposed to MU TGFBIp even at 5 h after the MU TGFBIp was withdrawn. High expressions of CD68 and CD36 were found in macrophages exposed to WT TGFBIp but not to MU TGFBIp. Impaired autophagic flux due to defective autophagosome fusion to lysosomes was found in macrophages exposed to MU TGFBIp. Blockage of the autophagic process suppressed the expression of CD68 and CD36 in macrophages exposed to WT TGFBIp to levels similar to those found in macrophages exposed to MU TGFBIp. These results have published in the world famous ophthalmologic journal “Investigative Ophthalmology & Visual Science”. In addition, we also found that Atg 14 can promote membrane tethering and fusion of autophagosomes to lysosomes by promoting formation of STX7-SNAP9-VAMP8 complex. Atg14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary SNARE complex on autophagosomes. Moreover, we still found a lysosomal response to the arrival of autophagosomal mtDNA and TLR9 as the cargo and the receptor that triggers and mediates TLR9 signaling pathway. We consider our founding may be a potential molecular mechanism that uncovers the autophagic degradation of MU TGFBIp in LCD. Therefore, this study will further use the clinical resource of LCD family found in our hospital, and continue to discover the more detail mechanisms related to the impaired autophagic degradation of transforming growth factor-β-induced protein by macrophages in LCD.