Mutation of NEKL-4/NEK10 and TTLL genes suppress neuronal ciliary degeneration caused by loss of CCPP-1 deglutamylase function

Author summary Cilia are microtubule-based organelles that play essential roles in human development and health. Ciliopathies are caused by abnormalities in the structure or function of primary cilia, with polycystic kidney disease (PKD) being a common clinical phenotype. As cilia are found on most non-dividing cells in the human body, ciliopathies often display extrarenal manifestations including neurological disorders and retinal degeneration. The Tubulin Code-combinatorial use of tubulin isotypes and post-translational modifications-dictates ciliary structure, motor-based transport, and function. Mutation in the tubulin deglutamylaseccpp-1(cytosolic carboxypeptidase) results in ciliary hyperglutamylation and degeneration.C.elegans ccpp-1ciliary degeneration is suppressed by a mutation in any of three TTLL (tubulin tyrosine ligase-like) glutamylase genes, indicating that regulated glutamylation is critically important for ciliary homeostasis. Pathological hyperglutamylation caused by CCP deglutamylase mutations are associated with human retinal degeneration and murine progressive neurodegeneration and sperm immotility.ccpp-1ciliary degeneration is also suppressed by a mutation in the kinase NEKL-4/NEK10. NEK kinases are implicated in polycystic kidney disease and other ciliopathies and NEKL-4/NEK10 is important for ciliary stability inC.elegans. By identifyingC.elegansgenes important for neuronal and ciliary stability, the worm may inform research into human ciliopathies and neurodegenerative diseases. Ciliary microtubules are subject to post-translational modifications that act as a Tubulin Code to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. InC.elegans, mutations inccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration inccpp-1mutants. We isolated thettll-5(my38)suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation inttll-4,ttll-5, orttll-11gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization inccpp-1cilia. We also identified thenekl-4(my31)suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia.C.elegansNEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifyingC.elegansgenes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.