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

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 (TTBK2 SCA11 ) 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, TTBK2 SCA11 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, includingSmoothened (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. Author SummaryDefects in primary cilia structure and function are linked to a number of recessive genetic disorders, now collectively referred to as ciliopathies. Most of the characteristics of these disorders arise from disruptions to embryonic development, with the requirements for primary cilia in adult tissues being less well-defined. We previously showed that a kinase associated with an adult-onset neurodegenerative condition is required for cilium assembly and ciliary signaling during development. Here, we show that the human disease-associated

TTBK2 and primary cilia are essential for the connectivity and survival of cerebellar Purkinje neurons

“…Ttbk2 is essential both for the initiation of cilium assembly as well as for the structure and stability of cilia (Bowie et al, 2018;Goetz et al, 2012). Consistent with this, we have found that primary cilia are lost from cells within the cerebellum of Ttbk2 cmut mice ( Figure 5) prior to the onset of the phenotypes we observe in neuronal connectivity and survival.…”

“…In this work, we present evidence that loss or impaired function of TTBK2 within the brain results in degeneration of PCs due largely to the requirement for TTBK2 in mediating the assembly and stability of primary cilia. This points to ciliary dysfunction as being a major mechanism underlying the pathology of SCA11, which is caused by truncating mutations to TTBK2 (Houlden et al, 2007) that act as dominant negatives (Bowie et al, 2018). In addition, our work raises the possibility that cilia play an important, largely unappreciated role in maintaining neuronal connectivity within the brain, and may also be required for the viability of some types of neurons.…”

“…SCA11 is somewhat unusual among spinocerebellar ataxias in part because the reported causal mutations are base pair insertions or deletions (Houlden et al, 2007;Johnson et al, 2008;Lindquist et al, 2017), rather than CAG repeats which is the genetic cause of most SCAs (Hersheson et al, 2012). While mice heterozygous for a knockin of one of the human-disease associated SCA11 mutations do not exhibit degenerative phenotypes (Bouskila et al, 2011;Bowie et al, 2018), we postulated based on our previous studies that a knockout of Ttbk2 from the adult mouse brain might cause neurodegenerative phenotypes that recapitulate SCA11. To test this, we obtained a conditional allele of Ttbk2 (Ttbk2 tm1c(EUCOMM)Hmgu ) from the European Mutant Mouse Cell Repository, (referred to from here as Ttbk2 fl ).…”

“…Our prior work demonstrated that mutations associated with SCA11, which result in the expression of a truncated protein, interfere with the function of full-length TTBK2. In particular, these mutations dominantly interfere with cilia formation in embryos and cultured cells (Bowie et al, 2018). Throughout the adult cerebellum and other regions of the hindbrain, neurons as well as glia possess primary cilia ( Figure 5A-5C).…”

“…SCA11 is characterized by the loss of Purkinje cells (PC) in the cerebellum, causing ataxia and other motor coordination deficits (Houlden et al, 2007;Seidel et al, 2012). Recently, we demonstrated that SCA11-associated alleles of Ttbk2 act as dominant negatives, causing defects in cilium assembly, stability, and function (Bowie et al, 2018).…”