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An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

ABSTRACT: Mutations in human CILK1 (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human CILK1 A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate from the kinase domain. Mouse embryo fibroblasts (MEFs) with either heterozygous or homozygous A612T mutant alleles exhibited a higher ciliation rate, shorter individual cilia, and upregulation of ciliary Hedgehog signaling. Thus, a single A612T mutant allele was sufficient to impair primary cilia and ciliary signaling in MEFs. Gene expression profiles of wild-type versus mutant MEFs revealed profound changes in cilia-related molecular functions and biological processes. The CILK1 A615T mutant protein was not increased to the same level as the wild-type protein when co-expressed with scaffold protein KATNIP (katanin-interacting protein). Our data show that KATNIP regulation of a JME-associated single-residue variant of CILK1 is compromised, and this impairs the maintenance of primary cilia and Hedgehog signaling.

SUBMITTER: Limerick A

PROVIDER: S-EPMC11311665 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

Limerick Ana A McCabe Ellie A EA Turner Jacob S JS Kuang Kevin W KW Brautigan David L DL Hao Yi Y Chu Cheuk Ying CY Fu Sean H SH Ahmadi Sean S Xu Wenhao W Fu Zheng Z

Cells 20240726 15

Mutations in human <i>CILK1</i> (ciliogenesis associated kinase 1) are linked to ciliopathies and epilepsy. Homozygous point and nonsense mutations that extinguish kinase activity impair primary cilia function, whereas mutations outside the kinase domain are not well understood. Here, we produced a knock-in mouse equivalent to the human <i>CILK1</i> A615T variant identified in juvenile myoclonic epilepsy (JME). This residue is in the intrinsically disordered C-terminal region of CILK1 separate f ...[more]

PMID: 39120290

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Project description:BackgroundPrimary cilia are small non-motile microtubule and cell membrane protrusions expressed on most vertebrate cells, including cortical and hippocampal neurons. These small organelles serve as sensory structures sampling the extracellular environment and reprogramming the transcriptional machinery in response to environmental change. Primary cilia are decorated with a variety of receptor proteins and are necessary for specific signaling cascades such as the Sonic hedgehog (Shh) pathway. Disrupting cilia structure or function results in a spectrum of diseases collectively referred to as ciliopathies. Common to human ciliopathies is cognitive impairment, a symptom also observed in Alzheimer's disease (AD). One hallmark of AD is accumulation of senile plaques composed of neurotoxic Amyloid-β (Aβ) peptide. The Aβ peptide is generated by the proteolytic cleavage of the amyloid precursor protein (APP). We set out to determine if Aβ affects primary cilia structure and the Shh signaling cascade.MethodsWe utilized in vitro cell-based assays in combination with fluorescent confocal microscopy to address our study goals. Shh signaling and cilia structure was studied using two different cell lines, mouse NIH3T3 and human HeLa cells. To investigate how Aβ levels affect Shh signaling and cilia structure in these cells, we utilized naturally secreted Aβ as well as synthetic Aβ. Effects on Shh signaling were assessed by luciferase activity while cilia structure was analyzed by fluorescent microscopy.ResultsHere, we report that APP localizes to primary cilia and Aβ treatment results in distorted primary cilia structure. In addition, we demonstrate that Aβ treatment interrupts canonical Shh signal transduction.ConclusionsOverall, our study illustrates that Aβ can alter primary cilia structure suggesting that elevated Aβ levels, like those observed in AD patients, could have similar effects on neuronal primary cilia in the brain. Additionally, our study suggests that Aβ impairs the Shh signaling pathway. Together our findings shed light on two novel targets for future AD therapeutics.

Dataset Information

An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

Publications

An Epilepsy-Associated CILK1 Variant Compromises KATNIP Regulation and Impairs Primary Cilia and Hedgehog Signaling.

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