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

Abstract

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. In C . elegans , mutations in ccpp-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 in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4 , ttll-5 , or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-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 . elegans NEKL-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 identifying C . elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.

CCP1, a Tubulin Deglutamylase, Increases Survival of Rodent Spinal Cord Neurons following Glutamate-Induced Excitotoxicity

“…Furthermore, the loss of primary cilia causes neurodegeneration in mammals ( Bowie and Goetz, 2020 ). Because CCP1 and glutamylation are implicated in ciliary degeneration and dysfunction, as well as neurodegeneration in rodents and humans ( Campbell et al, 2002 ; Fernandez-Gonzalez et al, 2002 ; Chakrabarti et al, 2008 ; Kim et al, 2010 ; O’Hagan et al, 2011 ; Kubo et al, 2015 ; Branham et al, 2016 ; Power et al, 2020 ), we hypothesized that the neuroprotective effects of CCP1 arise from ciliation of spinal cord neurons. However, cilia of spinal cord neurons have not been well-documented.…”

“…M14D carboxypeptidases, such as CCP1 ( Rodriguez de la Vega et al, 2007 ), remove or reduce the length of glutamate side-chains ( Rogowski et al, 2010 ). When deglutamylase function is lost, hyperglutamylation affects ciliary motor transport and causes degeneration of some types of neuronal sensory cilia in Caenorhabditis elegans ( O’Hagan et al, 2011 ; Power et al, 2020 ) . In mice, loss of CCP1 leads to the degeneration of retinal photoreceptors and sperm defects ( Fernandez-Gonzalez et al, 2002 ).…”

The Emerging Roles of Axonemal Glutamylation in Regulation of Cilia Architecture and Functions

“…In the CEM (Cephalic male) cilia of C. elegans , the cooperation between TTLL-11 and CCPP-1 remodels the axonemal doublets into a special formation of 18 singlets and then maintains them in this conformation ( O’Hagan et al, 2017 ). Meanwhile, in the amphid neurons, TTLL4/5/11-dependent glutamylation of the axoneme counteracts with CCPP-1-mediated deglutamylation ( Power et al, 2020 ). Hyperglutamylation of the axonemal tubulins due to TTLL4 overexpression or CCPP-1 deficiency may induce the spastin-dependent MT severing of the B tubules, which eventually leads to progressive defects in the ciliary structures and progressive degeneration as consequence ( O’Hagan et al, 2011 ; O’Hagan and Barr, 2012 ).…”

The tubulin code specializes neuronal cilia for extracellular vesicle release

“…Tubulin tyrosine ligase like enzymes (TTLLs) add glutamate side chains to the C‐terminal tail of α‐ and β‐tubulin to promote ciliary MT glutamylation. The C. elegans genome encodes five TTLLs ‐4, 5, 9, 11, and 15 with TTLL‐4 and TTLL‐11 being the major ciliary MT polyglutamylases (Chawla et al., 2016; Kimura et al., 2010; O'Hagan et al., 2011, 2017; Power et al., 2020) (Figure 4c). The ttll‐11 gene encodes two isoforms—A and B.…”

“…Microtubule glutamylation levels alter motor localizations, velocities, and changes in IFT in response to environmental stimuli (Kimura et al., 2018; Power et al., 2020). In CEM cilia, microtubule glutamylation regulates the velocities of OSM‐3 and KLP‐6 (O'Hagan et al., 2011, 2017) (see section 4.3 and Figure 5c).…”