Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability.

Emily Bowie,Ryan Norris,Kathryn V Anderson,Sarah C Goetz,Susan K Dutcher

doi:10.1371/journal.pgen.1007844

Abstract

Spinocerebellar ataxia type 11 (SCA11) is a rare, dominantly inherited human ataxia characterized by atrophy of Purkinje neurons in the cerebellum. SCA11 is caused by mutations in the gene encoding the Serine/Threonine kinase Tau tubulin kinase 2 (TTBK2) that result in premature truncations of the protein. We previously showed that TTBK2 is a key regulator of the assembly of primary cilia in vivo. However, the mechanisms by which the SCA11-associated mutations disrupt TTBK2 function, and whether they interfere with ciliogenesis were unknown. In this work, we present evidence that SCA11-associated mutations are dominant negative alleles and that the resulting truncated protein (TTBK2SCA11) interferes with the function of full length TTBK2 in mediating ciliogenesis. A Ttbk2 allelic series revealed that upon partial reduction of full length TTBK2 function, TTBK2SCA11 can interfere with the activity of the residual wild-type protein to decrease cilia number and interrupt cilia-dependent Sonic hedgehog (SHH) signaling. Our studies have also revealed new functions for TTBK2 after cilia initiation in the control of cilia length, trafficking of a subset of SHH pathway components, including Smoothened (SMO), and cilia stability. These studies provide a molecular foundation to understand the cellular and molecular pathogenesis of human SCA11, and help account for the link between ciliary dysfunction and neurodegenerative diseases.

Highlights

Primary cilia play a critical role in many aspects of embryonic development
Since the shorter cilia observed for each of the Ttbk2 hypomorphic allele combinations relative to Ttbk2 gene trap (Ttbk2gt)/+ cells could be due to defects in the protein machinery that mediates assembly of the ciliary axoneme, the intraflagellar transport (IFT) machinery, we examined the localization of IFT components in Mouse embryonic fibroblasts (MEFs) of each genotype
We show that the human Spinocerebellar ataxia type 11 (SCA11)- associated mutations to Ttbk2 produce truncated proteins that interfere with the function of full-length Tau tubulin kinase 2 (TTBK2) in cilia formation

Summary

Introduction

Primary cilia play a critical role in many aspects of embryonic development. Cilia are important for the development of the brain and central nervous system, which accounts for the structural brain defects, cognitive impairments, and other neurological disorders that are characteristic of many human ciliopathies[1,2,3]. We identified a Serine/Threonine kinase, Tau tubulin kinase 2 (TTBK2), that is essential for initiating the assembly of primary cilia in the embryo[4]. The protein is comprised of a kinase domain (AA 21–284), and a long C-terminus that is important for targeting TTBK2 to the mother centriole[4], as well as mediating its interactions with end-binding proteins at the microtubule +tips[8], and likely for additional regulation of TTBK2 function. TTBK2 was initially shown to phosphorylate the microtubule-associated proteins TAU and MAP2 in addition to β-Tubulin in vitro[6], and more recent evidence suggests that TTBK2 can phosphorylate the centriolar distal appendage protein CEP164[9], as well as the atypical kinesin KIF2A at the microtubule +tips[10]

Methods

Results

Conclusion