白内障             1258                                      PO0025

    Fiber cell denucleation and gap junction communication is inhibited by γD-crystallin (V76D) mutation during lens development

    Kaijun Wang, Haiquan Liu, Lin Li, Chunhong Xia, Ke Yao, Xiaohua Gong

    浙江大学医学院附属二院眼科中心 310009

    Purpose: To identify new cataract mutation from ENU-mutagenized mice and to characterize the mechanism of congenital cataract caused by this mutation.

    Methods: Mutagenized mice were generated by a forward genetic approach using alkylating agent N-ethyl-N-nitrosourea (ENU) to induce random mutations in mouse genome. The phenotype of mouse lens was evaluated by slit lamp examination. Chromosome location of the mutation was determined by genome-wide linkage analysis. Mutational analysis of candidate genes was performed by direct sequencing. The cellular and biochemical changes were characterized by standard histology, dapi staining, Immunohistochemical staining and biochemical methods.
    Results: From a screen of ENU-mutagenized mice, we have identified a dominant cataract caused by the γD-V76D mutation. Heterozygous mice developed normal size eyeballs and lenses with nuclear cataracts while homozygous mice showed posteriorly ruptured lenses at the age of 3 weeks. Mutant lens histopathology showed large aggregates in fiber cell nuclei. Incompletion of fiber cell denucleation was evidenced by the presence of DAPI-stained nuclei and sustained chromosomal DNA. Immunohistochemical staining data confirmed γ-crystallin protein aggregates in fiber cell nuclei and a reduction of gap junctions consisting of α3 and α8 connexins in those fiber cells. Unlike wild-type γD proteins, mutant γD-V76D proteins also aggregated in the nuclei of transfected culture cells.
    Conclusion: These data suggest that γD-V76D mutant proteins interrupt fiber cell denucleation by forming nuclear aggregates and decrease gap junction communication to cause cataracts. Thus, γD-crystallin proteins probably play a role in the regulation of cell denucleation and intercellular gap junction communication in lens fiber cells during lens development.

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