Hedgehog signaling in Joubert syndrome

Abstract

Joubert syndrome (JS) is a mostly recessive, neurodevelopmental condition diagnosed by the molar tooth sign (MTS) on axial brain imaging. Currently, >40 genes are associated with JS, all of which encode proteins that function in and around the primary cilium, a microtubule-based signaling organelle. The association of >40 cilium-related JS genes with the JS-specific brain malformation suggests a unifying cilium-related disease mechanism across genetic causes; however, the mechanistic details remain unknown. Aberrant Hedgehog (Hh) signaling has been implicated in model systems representing more than half of the JS genes, but specific alterations in the Hh signaling cascade have not been characterized across genetic causes of JS in a single model system. To determine whether a consistent disruption in Hh signaling underlies JS, we evaluated Hh signaling using standard assays for two key steps in a single model system. Using the CRISPR-Cas9 system in hTERT RPE-1 cells, I engineered biallelic frameshift variants in 9 representative JS-associated genes encoding ciliary proteins that localize to each of the major sub-compartments of the cilium: tip, cilium-proper, transition zone, and basal body. Transition zone mutants displayed fewer cilia, and multiple mutants exhibited abnormal cilium length; however, these defects were not uniformly present across mutants. Similarly, relocalization of the key Hh pathway component SMO in response to pathway stimulation was blunted in several mutants across compartments, most severely in the one transition zone mutant that made enough cilia to be tested. Finally, induction of the Hh target gene GLI1 was blunted in cilium-proper and transition zone mutants, but not in tip and basal body mutants. My work indicates that blunted Hh signaling is a key mechanistic feature associated with JS gene dysfunction, potentially identifying a target for pharmacologic treatments. Future work will focus on determining whether JS gene dysfunction impacts the dynamic range, timing, and other aspects of Hh signal transduction, or if we need to look beyond Hh to identify a unifying mechanism underlying JS.