INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse

Abstract

The primary cilium is an antenna-like structure that protrudes from the cell surface of quiescent/differentiated cells and participates in extracellular signal processing [1][2][3] . Here, we report that mice deficient for the lipid 5-phosphatase Inpp5e develop a multiorgan disorder associated with structural defects of the primary cilium. In ciliated mouse embryonic fibroblasts, Inpp5e is concentrated in the axoneme of the primary cilium. Inpp5e inactivation did not impair ciliary assembly but altered the stability of pre-established cilia after serum addition. Blocking phosphoinositide 3-kinase (PI3K) activity or ciliary platelet-derived growth factor receptor a (PDGFRa) restored ciliary stability. In human INPP5E, we identified a mutation affecting INPP5E ciliary localization and cilium stability in a family with MORM syndrome, a condition related to Bardet-Biedl syndrome. Together, our results show that INPP5E plays an essential role in the primary cilium by controlling ciliary growth factor and PI3K signaling and stability, and highlight the consequences of INPP5E dysfunction.Lipid 5-phosphatases selectively remove the phosphate from position D-5 of the inositol ring of phosphoinositides and inositolphosphates 4,5 . To characterize the functions of the 5-phosphatase Inpp5e 6-8 , we generated Inpp5e D/+ mice ( Supplementary Fig. 1a). We obtained no adult Inpp5e D/D mutant mice from intercrosses between Inpp5e D/+ mice. However, at embryonic day 13.5 (E13.5) and E18.5, 16.9% (11/65) and 14.8% (12/81) of embryos were homozygous for the deletion allele, respectively. The mutant mice died soon after birth, indicating that total inactivation of Inpp5e led to embryonic and postnatal death. Analyses confirmed the absence of Inpp5e protein in mutant cells and tissues (Fig. 1a). Inpp5e D/D mice presented with bilateral anophthalmos (100%, n ¼ 43) and postaxial hexadactyly (62.5%, n ¼ 16; Fig. 1b,c). Histological analyses revealed that eye development ceased at the optic vesicle stage, just before the appearance of the optic cup (Fig. 1d). Analysis of kidneys from the mice revealed the presence of multiple cysts (100%, n ¼ 10; Fig. 1e). Of the cysts, 84% expressed AQP2 and 14% expressed AQP1, indicating an origin in cortical collecting and connecting ducts (when AQP2 + ) as well as proximal tubules and the descending limb of the loop of Henle (when AQP1 + ) ( Supplementary Fig. 2). Only 2% of the renal glomeruli were cystic. Inpp5e D/D embryos had skeletal abnormalities such as a bifid sternum (50%, n ¼ 6), delayed ossification of metacarpals and phalanges (100%, n ¼ 5) and cleft palate (75%, n ¼ 4; Fig. 1f-h). We identified cerebral developmental defects, such as anencephaly and exencephaly, in 30% of Inpp5e D/D embryos at E15.5 (n ¼ 30; Fig. 1i,j). We did not detect liver alterations, laterality defects or respiratory cilium defects in mutant animals. The tissue localization of lesions observed in Inpp5e D/D embryos matched the tissue expression of Inpp5e mRNA during mouse embryogenesis ( Supplementary Fig. 3).Becau...

Phenotypic spectrum and prevalence of INPP5E mutations in Joubert Syndrome and related disorders

“…15 In particular, INPP5E has been shown to promote ciliary stabilization, and some mutations have been previously shown to alter the INPP5E enzymatic activity and promote premature cilia destabilization in response to specific stimuli. [10][11] Our data indicate a mutation prevalence of about 2.7% among JSRD, while no pathogenic changes were detected in 75 MKS fetuses, suggesting that INPP5E is not causative of the MKS phenotype. Lack of allelism between these two conditions has been already described for other ciliary genes such as ARL13B and CEP41, that are mutated only in JSRD.…”

“…Among JSRD, we identified 12 INPP5E mutations of which 10 novel, raising to 16 the number of distinct pathogenic changes so far reported. [10][11] We describe for the first time a homozygous nonsense mutation (p.Y543X), resulting in the production of a truncated protein lacking the final part of the catalytic domain and the C-terminus transmembrane domain. All the remaining mutations are missense changes affecting evolutionarily conserved amino-acid residues clustered within or flanking the enzymatically active phosphatase domain ( Figure 1).…”

Cilia and cell cycle re-entry

“…of certain phospholipids, wherein a disturbance of this balance could lead to destabilization of the primary cilium in response to a mitogenic stimulus that could, in turn, result in faster ciliary resorption and faster cell cycle re-entry. 26,27 Mice lacking INPP5E show numerous developmental defects, ranging from kidney cysts to polydactyly and delayed ossification of metacarpals and phalanges to anencephaly and exencephaly, 26,27 highlighting the important role of ciliary resorption, per se, during embryonic development.…”

“…26,27 INPP5E is a lipid phosphatase localized exclusively at the cilia and removes the 5-phosphate group from PI(3,4,5)P3 and PI(4,5)P2. 26,27 Loss of INPP5E results in an excess of these precursors in the axonemal membrane.…”

Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1

“…Thus, the correct spatial and temporal regulation of these enzymes is of central importance (1,2). As a consequence, not only the lack of these enzymes but also the disruption of their interactions can have profound effects on cell function and result in pathological conditions (2)(3)(4)(5)(6)(7)(8)(9).…”