Abstract

Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.

Highlights

  • Missense mutations in Abelson-helper integration site 1 (AHI1) result in the neurodevelopmental ciliopathy called Joubert syndrome

  • The Joubert syndrome (JBTS)-causing Mutation, V443D, in AHI1 Alters Binding to HAP1—To further explore the binding of AHI1 and HAP1, which occurs in a region of AHI1 that appears not to contain any known protein motif, we examined the known JBTS-causing mutation in AHI1, V443D, that is located in this region

  • We show that the function of AHI1 is affected by one of the known JBTS-associated missense mutations in AHI1 with the resulting amino acid substitution, V443D, causing an inability for AHI1-V443D to localize to the basal body and cellcell junctions

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Summary

Background

Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. Ahi1-deficient mice display retinal degeneration resulting from defective ciliary protein trafficking [12, 13] These studies suggest a crucial role for Ahi in cilium formation and function. We have shown that expression of AHI1-V443D and AHI1-R351L in IMCD3 cells resulted in an inability of mutant AHI1 to localize at cellcell junctions and at the basal body of primary cilia, with a corresponding reduction in cilium formation in AHI1-V443Dand AHI1-R351L-transfected cells. These results suggest that the V443D and R351L mutations affect the distribution of AHI1, possibly due to these disrupted protein interactions. Because Hap knock-out mice were only reported to have cerebellar vermal hypoplasia and superior cerebellar peduncle defects without other JBTS-associated phenotypes (i.e. photoreceptor degeneration and cystic kidney disease) [14], this suggests that the JBTS-like phenotype in Hap knock-out mice may be through a defect in an unknown non-ciliary mediated mechanism or that Hap loss results in cerebellar developmental defects through a non-JBTS pathway

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