Abstract
Joubert syndrome (JBTS) is the archetypal ciliopathy caused by mutation of genes encoding ciliary proteins leading to multi-system phenotypes, including a cerebello-retinal-renal syndrome. JBTS is genetically heterogeneous, however mutations in CEP290 are a common underlying cause. The renal manifestation of JBTS is a juvenile-onset cystic kidney disease, known as nephronophthisis, typically progressing to end-stage renal failure within the first two decades of life, thus providing a potential window for therapeutic intervention. In order to increase understanding of JBTS and its associated kidney disease and to explore potential treatments, we conducted a comprehensive analysis of primary renal epithelial cells directly isolated from patient urine (human urine-derived renal epithelial cells, hURECs). We demonstrate that hURECs from a JBTS patient with renal disease have elongated and disorganized primary cilia and that this ciliary phenotype is specifically associated with an absence of CEP290 protein. Treatment with the Sonic hedgehog (Shh) pathway agonist purmorphamine or cyclin-dependent kinase inhibition (using roscovitine and siRNA directed towards cyclin-dependent kinase 5) ameliorated the cilia phenotype. In addition, purmorphamine treatment was shown to reduce cyclin-dependent kinase 5 in patient cells, suggesting a convergence of these signalling pathways. To our knowledge, this is the most extensive analysis of primary renal epithelial cells from JBTS patients to date. It demonstrates the feasibility and power of this approach to directly assess the consequences of patient-specific mutations in a physiologically relevant context and a previously unrecognized convergence of Shh agonism and cyclin-dependent kinase inhibition as potential therapeutic targets.
Highlights
Joubert syndrome (JBTS) is a ciliopathy syndrome which is characterized most often by cerebellar defects and retinal dystrophy
We studied a non-consanguineous English family in whom two children (JBTS II: 1 and JBTS II: 2) were severely affected with JBTS (Fig. 1A), manifesting as early onset nystagmus and Leber congential amaurosis (LCA) resulting in visual loss (Table 1)
Non-transformed renal epithelial cells derived from a JBTS patient with a cerebello-retinal-renal phenotype carrying pathogenic compound heterozygous CEP290 mutations, we observed abnormally long cilia with a mismatch in cilia staining between alpha-acetylated tubulin, an axonemal protein, and ADP-ribosylation factor-like protein 13B (ARL13B), a ciliary membrane associated protein
Summary
Joubert syndrome (JBTS) is a ciliopathy syndrome which is characterized most often by cerebellar defects and retinal dystrophy. CEP290 (alias NPHP6) mutations underlie JBTS type 5 [2,3] and are the commonest genetic lesion associated with a cerebello-oculo-renal phenotype [4]. Clinical phenotypes associated with CEP290 mutations may be diverse and include isolated Leber congential amaurosis (LCA) [5,6], MeckelGruber syndrome [7,8] and Bardet-Biedl syndrome [9]. Elegant work in Chlamydomonas has revealed that CEP290 is an integral part of the transition zone, the specialized region at the most proximal part of the cilium, which together with transition fibers forms the ciliary gate. In murine photoreceptor cells Cep290 localizes to the connecting cilium [2], a structure which represents a specialized transition zone that extends between the inner and outer segments of the photoreceptor cell [10]
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