Regulation of Primary Cilia Disassembly Through HUWE1-Mediated TTBK2 Degradation Plays a Crucial Role in Cerebellar Development and Medulloblastoma Growth

Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB. Overall design: To investigate the therapeutic potential of targeting TTBK2 in SHH-MB, we generated TTBK2 knockout (TTBK2-/-) Daoy cells, a well-established SHH-MB cell line.

小脑发育需要对颗粒神经元祖细胞（granule neuron progenitor, GNP）的增殖进行精准调控。尽管已知初级纤毛（primary cilia）对维持GNP增殖必不可少，但调控GNPs内初级纤毛动态变化的确切分子机制仍未阐明。本研究阐明了中心体激酶TTBK2（Tau tubulin kinase-2）与E3泛素连接酶HUWE1在GNP增殖中的关键作用。我们发现，在音猬因子（Sonic Hedgehog, SHH）信号通路激活的增殖状态GNP中，TTBK2呈高表达，这与活跃的GNP增殖及初级纤毛的存在高度吻合。TTBK2通过抑制初级纤毛解聚来稳定其结构，进而促进SHH信号介导的GNP增殖。机制层面，我们鉴定出HUWE1是一种新型中心体E3泛素连接酶，可通过靶向降解TTBK2促进初级纤毛解聚。初级纤毛解聚是触发GNP分化的关键信号，可促使其从小脑外颗粒层（external granule layer, EGL）迁移至内颗粒层（internal granule layer, IGL）以完成后续成熟过程。此外，我们还建立了TTBK2与SHH型髓母细胞瘤（SHH-type medulloblastoma, SHH-MB）之间的关联，这类肿瘤以GNP增殖失控为核心特征。敲除TTBK2可抑制SHH-MB的增殖，提示TTBK2或可成为该类癌症的潜在治疗靶点。综上，本研究揭示了调控小脑发育的分子机制，并为SHH-MB提供了潜在的抗癌治疗策略。
实验整体设计：为探究靶向TTBK2治疗SHH-MB的潜力，我们构建了TTBK2基因敲除（TTBK2-/-）的Daoy细胞——这是一种经典的SHH-MB细胞系。