Pathogenic variants of TUBB8 cause oocyte spindle defects by disrupting with EB1-CAKP5 interactions and potential treatment targeting microtubule acetylation through HDAC6 inhibition.

Journal:Clinical and Translational Medicine

Place of Publication:UK

Key Words:D417N variant,female infertility,meiotic arrest,oocyte

Abstract:Background: Numerous pathogenic variants causing human oocyte maturation  arrest have been reported on the primate-specific TUBB8 gene. The main etiology  is the dramatic reduction of tubulin α/β dimer, but still large numbers of variants  remain unexplained.Methods: Using microinjection mRNA and genome engineering to reintroduce  the conserved pathogenic missense variants into oocytes or in generating TUBB8  variant knock-in mouse models, we investigated that the human deleterious variants alter microtubule nucleation and spindle assembly during meiosis. Live-cell  imaging and immunofluorescence were utilised to track the dynamic expression of microtubule plus end-tracking proteins in vivo and analysed microtubule  nucleation or spindle assembly in vitro, respectively. Immunoprecipitation-mass  spectrometry and ultramicro-quantitative proteomics were performed to identify  the differential abundance proteins and affected interactome of TUBB8 protein.Results: First, we observed a significant depletion of the EB1 signal upon  microinjection of mutated TUBB8 mRNA (including R262Q, M300I, and D417N  missense variants), indicating disruption of microtubule nucleation caused by  these introduced TUBB8 missense variants. Mechanically, we demonstrated  that the in vivo TUBB8-D417N missense variant diminished the affinity of  EB1 and microtubules. It also harmed the interaction between microtubules  and CKAP5/TACC3, which are crucial for initiating microtubule nucleation.  Attenuated Ran-GTP pathway was also found in TUBB8-D417N oocytes, leading to disrupted spindle assembly. Stable microtubule was largely abolished on  the spindle of TUBB8-D417N oocytes, reflected by reduced tubulin acetylation  and accumulated HDAC6. More importantly, selective inhibition of HDAC6 by  culturing TUBB8-D417N oocytes with Tubacin or Tubastatin A showed morphologically normal spindle and drastically recovered polar-body extrusion rate.  These rescue results shed light on the strategy to treat meiotic defects in a certain  group of TUBB8 mutated patients.  Conclusion: Our study provides a comprehensive mechanism elucidating  how TUBB8 missense variants cause oocyte maturation arrest and offers new  therapeutic avenues for treating female infertility in the clinic.

All the Authors:Jianhua Chen,Cao Li,Tian Wu,Siyue Yin,Guangping Yang,Yipin Wang,Zhihan Guo,Saifei Hu,Yanni He,Yingnan Wang,Yao Chen,Youqiang Su,Congxiu Miao,Yun Qian

First Author:Hui Luo

Indexed by:Research Article

Correspondence Author:Ruizhi Feng

Document Code:10.1002/ctm2.70193

Volume:15

Issue:1

Page Number:e70193

ISSN No.:2001-1326

Translation or Not:no

Date of Publication:2025-01-20

Date of Publication:2025-01-20