JDB, Vol. 10, Pages 51: The Role of Primary Cilia-Associated Phosphoinositide Signaling in Development

4.1. INPP5EINPP5E dephosphorylates the 5-position on the inositol ring of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and converts them to PtdIns4P and PtdIns(3,4)P2, respectively [1]. In non-ciliated cells, INPP5E resides at distal appendages of the mother centriole and maintains a specific centrosomal PtdIns4P pool [26]. PtdIns4P binds to both tau-tubulin kinase-2 (TTBK2) and the distal appendage protein CEP164, which compromises the TTBK2-CEP164 interaction and inhibits the recruitment of TTBK2 [26]. In quiescent cells, INPP5E translocates to cilia with a decreased PtdIns4P level at centrosome, leading to the recruitment of TTBK2 and the initiation of ciliogenesis [26]. During ciliogenesis, INPP5E translocates to the ciliary membrane, where it maintains the stability and signaling function of primary cilia by preserving a PtdIns4P-dominant environment (Figure 3) [23,26]. INPP5E contains an N-terminal proline-rich domain, an inositol polyphosphate 5-phosphatase domain, and a C-terminal CAAX prenylation motif which is necessary for its ciliary localization [1,23]. As reported, phosphodiesterase PDE6D, the transition fiber protein CEP164, and the small GTPase ARL13B coordinate to recruit INPP5E into the ciliary membrane [77,78,79].Current knowledge from human patients and animal models supports that INPP5E is essential for development. Two human ciliopathy syndromes are associated with INPP5E mutations: Joubert syndrome (JBTS) and MORM syndrome (Table 1 and Table 2) [80,81,82,83]. JBTS is an autosomal recessive neurodevelopmental disorder characterized by the appearance of a “molar tooth sign” on axial MRI, which results from the abnormal development of the cerebellar vermis and the brainstem [84]. The most common clinical features of JBTS include ataxia, hyperpnea, sleep apnea, ocular motor apraxia, hypotonia, and cystic dysplastic kidney (Figure 4 and Table 2) [85]. JBTS-associated INPP5E mutations are mainly missense and cluster in the catalytic domain (Table 1). In vitro experiments also confirmed that these muted INPP5E exhibit decreased phosphatase activity and cause impaired PI distribution in cilia [81,83,85,86,87]. On the other hand, MORM syndrome is resulted from C-terminal deletions of INPP5E (Table 1), which leads to loss of the CAAX motif and exclusion of INPP5E from the ciliary membrane without affecting the catalytic activity [23,88]. Correspondingly, in addition to similar symptoms as JBTS including intellectual disability and retinal dystrophy, MORM syndrome shows phenotypes unseen in JBTS, including obesity and micropenis, as observed in other ciliopathies such as Bardet–Biedl syndrome and Cohen syndrome (Figure 4 and Table 2) [23,88,89]. Compared with JBTS-associated INPP5E mutants which lead to a loss of INPP5E activity in the whole cell and whole body, MORM-associated INPP5E mutants only damage the INPP5E activity in cilia but may increase the INPP5E activity in other cellular locales due to mislocalization. In this context, the phenotypic differences between JBTS and MORM may be caused by the abnormal non-ciliary activity of INPP5E, which could be seen when other ciliary abnormalities disturb the ciliary targeting of INPP5E.Results from animal models reinforce the conclusion in human patients that INPP5E function in primary cilia is required for the development of multiple organs [23,24,90]. Inpp5eD/D mice (deletion of exons 7 and 8) are embryonic and postnatal lethal with ciliopathy features, including bilateral anophthalmos, postaxial hexadactyly, renal cyst, skeletal abnormalities, as well as cerebral developmental defects, suggesting that INPP5E is essential for primary cilium-mediated functions [23]. Interestingly, the inactivation of Inpp5e in mice on the postnatal day-28 did not affect the survival of adult mice; however, still caused ciliopathy phenotypes such as higher body weight, retinal dystrophy, and cystic glomeruli [23]. This result supports the involvement of INPP5E in the cilium-dependent homeostasis of mature tissues/organs, whereas also indicates that the INPP5E-dependent ciliary function is more important for the embryonic development. Further studies using the Inpp5eD/D MEFs suggest that the Inpp5e deletion increases the ciliary level of the Hedgehog suppressor GPR161 via PtdIns(4,5)P2-dependent recruitment of TULP3, which promotes GLI3R formation and reduces Hedgehog signaling [28]. Consistent with the Inpp5eD/D mice, another mouse model of Inpp5e-/- (deletion of exons 2 to 6) also recapitulates JBTS features, including polycystic kidneys, cleft palate, polydactyly, edema, and ossification delays [23,24,90]. Inpp5e-/- MEFs exhibited the defective Hedgehog signaling with reduced ciliary accumulation of SMO and GLI2 when treated with the SMO agonist [24]. Expression of a constitutively active SMO mutant (SMOM2) in Inpp5e-/- mice partially restored the associated embryonic development defects, emphasizing the involvement of Hedgehog signaling in INPP5E regulation in embryonic development [24]. However, whether the expression of SMOM2 can rescue the defects in Inpp5e-/- adult mice is not determined in this study.Although most INPP5E-associated JBTS patients mainly show neurologic symptoms with rare kidney or hepatic features, both Inpp5eD/D and Inpp5e-/- mice showed polycystic kidneys with a 100% penetrance [23,24]. Moreover, the renal-specific deletion of Inpp5e exons 2-6 in mice resulted in severe polycystic kidneys and renal failure, likely caused by the hyperactivation of PI3K/Akt and mTORC1 pathway [90]. Despite the possibility that INPP5E may carry slightly diverse functions in different species, that the PKD phenotype in Inpp5e-inactivated mice is absent in INPP5E-mutated human patients more likely reflects the dosage difference of functional INPP5E in both models. Mouse models suffer from a complete loss of INPP5E protein during the embryonic and/or postnatal development of kidneys [23,90], whereas in patients, INPP5E mutations are often hypomorphic with reduced protein level, weakened phosphatase activity, or defective ciliary localization [23,83], which may be sufficient to support the development and homeostasis of kidneys. In other animal model such as zebrafish, Inpp5e knockdown in morphants also impairs cilia formation and function in the Kupffer’s vesicle and pronephric ducts, thus leads to ciliopathy-like phenotypes including body axis asymmetry, microphthalmia, pericardial edema, kinked tail, and pronephric cyst formation [91]. Moreover, expression of human INPP5E rescues the defects in the Inpp5e knockdown morphants [91], suggesting that the functionality of INPP5E is mostly conserved in vertebrate development.Especially, INPP5E patient mutations exhibit reduced cilia stability compared with the wild-type protein [23]. Taken together, INPP5E is the most well-studied PI enzyme, which plays a critical role in cilia assembly, stability, and signaling pathways [6,29,92,93], highlighting the importance of INPP5E in development. 4.2. OCRL and INPP5BOCRL and INPP5B are both members of the inositol polyphosphate 5-phosphatase family as INPP5E, but mainly hydrolyze PtdIns(4,5)P2 to generate PtdIns4P [1]. OCRL localizes to the cilium and basal body, as well as the endocytic network, and functions in the assembly and maintenance of primary cilia (Figure 3) [94,95,96,97,98]. OCRL is a multi-domain protein including PH, 5-phosphatase, ASH (ASPM-SPD2-Hydin), and catalytically inactive RhoGAP (Rho GTPase-activating protein) domains [1]. At the early stage of ciliogenesis, OCRL is recruited by the small GTPase RAB8 to cilia through direct binding [95,99], which is essential for primary cilia assembly [94]. Mutations of OCRL in the 5-phosphatase domain interrupts its ciliary localization, whereas deletion of the RhoGAP domain eliminates OCRL in the ciliary proper and restrains it near the ciliary base [95]. This is consistent with the discovery that patients’ fibroblasts with OCRL mutations exhibit defective ciliogenesis and shortened cilia [95,96], indicating the diverse disorders present in OCRL-mutated patients may be due to the dysregulation of primary cilia. As a paralog of OCRL, INPP5B shares similar structural domains [1] but contains a C-terminal CAAX prenylation domain, which is essential for the ciliary localization of INPP5B [100]. Knockdown of INPP5B results in a significant decrease of ciliogenesis and ciliary length in both cultured mammalian cells and zebrafish Kupffer’s vesicle, but the detailed mechanism remains unclear [100].OCRL mutations are identified as causative of two human diseases: oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, and Dent-2 disease (Table 1 and Table 2) [101,102,103,104,105]. Both Lowe syndrome and Dent-2 disease are rare X-linked genetic disorders. Lowe syndrome patients exhibit defective cilium assembly and ciliopathy symptoms such as intellectual disability, congenital cataracts, and renal dysfunction (Figure 4) [105]. Dent-2 is often described as a milder form of Lowe syndrome, as most patients only develop renal symptoms and a few have mild intellectual disability, hypotonia, cataracts, and rickets (Figure 4) [101]. Genetic analyses showed that Lowe syndrome-associated mutations are mostly in exons 8–23 (5-phosphatase, ASH, and RhoGAP domains), whereas Dent-2-associated mutations are typically in exons 1-7 (PH domain) (Table 1) [101,104], suggesting that the distinct symptoms in these two disorders result from different mutations. Interestingly, one study using different Lowe and Den-2 fibroblasts displays similar reduced protein and accumulated PtdIns(4,5)P2 levels but milder ciliogenesis defects in Den-2 [96], indicating the ciliation defect may be related to the disease severity. Moreover, the epigenetic differences among individual patients and/or secondary genetic mutations should be considered because the disease severity varies widely between patients who carry the same OCRL mutation [105,106]. However, INPP5B has not been associated with any human diseases. Thus, although INPP5B’s function may be partially redundant with OCRL [107], OCRL is the dominant enzyme accountable for corresponding developmental needs in human.Results from animal studies suggest that mice respond differently from humans to the loss of OCRL [107,108]. Ocrl knockout mice are fertile with normal kidneys, eyes, and brains, failing to recapitulate the phenotypes of Lowe syndrome [107]. However, Ocrl;Inpp5b double knockout mice are embryonic lethal [107] and the kidney-tubule-specific deletion of Inpp5b in Ocrl−/− mice phenocopy the tubulopathy disorder of Lowe Syndrome/Dent-2 [109]. These results indicate that Ocrl and Inpp5b carry redundant functions in mice, and normal mouse development can be conducted if the combined enzyme activity of Ocrl and Inpp5b is above certain threshold. Once levels of Ocrl and Inpp5b drop below this threshold, the severity of developmental defects negatively correlates with the remaining dosage of functional Ocrl and Inpp5b. Interestingly, knock-in of human INPP5B in Ocrl;Inpp5b mice corrected the lethality, but animals still showed phenotypes such as Lowe syndrome/Dent-2 disease including reduced postnatal growth, low molecular weight proteinuria, and aminoaciduria [108]. This is consistent with the previous discovery that although INPP5B and Inpp5b are highly conserved in most exons, the significant differences in exons 7 and 8 lead to different gene transcription, mRNA splicing, and primary protein sequence of between human and mouse [110]. Moreover, one study showed that Inpp5b expression level was dramatically higher in mouse trabecular meshwork cells than the same human cells [100]. This distinct expression may partly explain why Inpp5b and INPP5B compensate the loss of Ocrl in mice differently.Although several studies have confirmed the ciliary localization of OCRL and INPP5B [94,95,100], the investigation of OCRL and INPP5B in cilia has just begun. OCRL can be recruited to cilia by RAB8, which is essential for ciliary assembly, and regulate ciliary protein trafficking in an Rab8- and endosome-dependent manner [94,95]. Knockdown of Ocrl or Inpp5b in zebrafish resulted in defective cilium formation in Kupffer’s vesicle and ciliopathy-like phenotypes including microphthalmia, body-axis asymmetry, microlens, distorted retinas, and hydrocephalus [95,100]. Double knockdown of both Ocrl and Inpp5b in zebrafish showed synergistic effects, suggesting that these two ciliary PI phosphatases may play some non-redundant function in zebrafish development [100]. Consistent with the observation in zebrafish, the lack of OCRL in human retinal pigmented epithelial cells and patients’ fibroblasts results in defective ciliogenesis and shortened cilia [94,95]. However, one study using MDCK cells demonstrated an increased ciliary length when knocking down OCRL [98], indicating a potential cell-specific function of OCRL. Further investigation on the molecular mechanism underlying these differences may help the understanding of the tissue-specific manifestations in Lowe syndrome/Dent-2 disease. As PI phosphatases, how OCRL and INPP5B regulate the disruption of ciliary PIs is also unclear. One study using Lowe syndrome patients’ fibroblasts and Ocrl-null MEFs shows increased PtdIns(4,5)P2 and decreased PtdIns4P in cilia, which is similar to the observation in Inpp5e-null MEFs [97]. Intriguingly, OCRL is also necessary for the activation of Hedgehog signaling, but through a different mechanism from INPP5E. Unlike INPP5E that suppresses the ciliary entry of GPR161 but not SMO [27,28], OCRL deficiency has no effect on GPR161, but disrupts the ciliary translocalization of SMO upon SAG treatment [97]. The underlying molecular mechanism and involvement of corresponding PI species is highly interesting and should be determined in future studies.

留言 (0)

沒有登入
gif