Functional Characterization and Rescue of a Deep Intronic Mutation in OCRL Gene Responsible for Lowe Syndrome.

Dent-2 disease and Lowe syndrome are two pathologies caused by mutations in inositol polyphosphate 5-phosphatase OCRL gene. Both conditions share proximal tubulopathy evolving to chronic kidney failure. Lowe syndrome is in addition defined by a bilateral congenital cataract, intellectual disability, and hypotonia. The pathology evolves in two decades to a severe condition with renal complications and a fatal issue. We describe here a proof of principle for a targeted gene therapy on a mutation of the OCRL gene that is associated with Lowe syndrome. The affected patient bears a deep intronic mutation inducing a pseudo-exon inclusion in the mRNA, leading to a OCRL-1 protein loss. An exon-skipping strategy was designed to correct the effect of the mutation in cultured cells. We show that a recombinant U7-modified small RNA efficiently triggered the restoration of normal OCRL expression at mRNA and protein levels in patient's fibroblasts. Moreover, the PI(4,5)P2 accumulation and cellular alterations that are hallmark of OCRL-1 dysfunction were also rescued. Altogether, we provide evidence that the restoration of OCRL-1 protein, even at a reduced level, through RNA-based therapy represents a potential therapeutic approach for patients with OCRL splice mutations.

OCRL-mutated fibroblasts from patients with Dent-2 disease exhibit INPP5B-independent phenotypic variability relatively to Lowe syndrome cells.

OCRL mutations are associated with both Lowe syndrome and Dent-2 disease, two rare X-linked conditions. Lowe syndrome is an oculo-cerebro-renal disorder, whereas Dent-2 patients mainly present renal proximal tubulopathy. Loss of OCRL-1, a phosphoinositide-5-phosphatase, leads in Lowe patients' fibroblasts to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, with defects in F-actin network, α-actinin distribution and ciliogenesis, whereas fibroblasts of Dent-2 patients are still uncharacterized. To search for mechanisms linked to clinical variability observed between these two OCRL mutation-associated pathologies, we compared dermal fibroblasts from independent patients, four affected by Dent-2 disease and six with Lowe syndrome. For the first time, we describe that Dent-2 fibroblasts with OCRL loss-of-function (LOF) mutations exhibit decrease in actin stress fibers, appearance of punctate α-actinin signals and alteration in primary cilia formation. Interestingly, we quantified these phenotypes as clearly intermediate between Lowe and control fibroblasts, thus suggesting that levels of these defects correlate with clinical variations observed between patients with OCRL mutations. In addition, we show that Lowe and Dent-2 fibroblasts display similar PI(4,5)P2 accumulation levels. Finally, we analyzed INPP5B, a paralogous gene already reported to exhibit functional redundancy with OCRL, and report neither differences in its expression at RNA or protein levels, nor specific allelic variations between fibroblasts of patients. Altogether, we describe here differential phenotypes between fibroblasts from Lowe and Dent-2 patients, both associated with OCRL LOF mutations, we exclude direct roles of PI(4,5)P2 and INPP5B in this phenotypic variability and we underline potential key alterations leading to ocular and neurological clinical features in Lowe syndrome.

Novel IL1RAPL1 mutations associated with intellectual disability impair synaptogenesis.

Mutations in interleukin-1 receptor accessory protein like 1 (IL1RAPL1) gene have been associated with non-syndromic intellectual disability (ID) and autism spectrum disorder. This protein interacts with synaptic partners like PSD-95 and PTPδ, regulating the formation and function of excitatory synapses. The aim of this work was to characterize the synaptic consequences of three IL1RAPL1 mutations, two novel causing the deletion of exon 6 (Δex6) and one point mutation (C31R), identified in patients with ID. Using immunofluorescence and electrophysiological recordings, we examined the effects of IL1RAPL1 mutant over-expression on synapse formation and function in cultured rodent hippocampal neurons. Δex6 but not C31R mutation leads to IL1RAPL1 protein instability and mislocalization within dendrites. Analysis of different markers of excitatory synapses and sEPSC recording revealed that both mutants fail to induce pre- and post-synaptic differentiation, contrary to WT IL1RAPL1 protein. Cell aggregation and immunoprecipitation assays in HEK293 cells showed a reduction of the interaction between IL1RAPL1 mutants and PTPδ that could explain the observed synaptogenic defect in neurons. However, these mutants do not affect all cellular signaling because their over-expression still activates JNK pathway. We conclude that both mutations described in this study lead to a partial loss of function of the IL1RAPL1 protein through different mechanisms. Our work highlights the important function of the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis and as such in ID in the patients.

From Lowe syndrome to Dent disease: correlations between mutations of the OCRL1 gene and clinical and biochemical phenotypes.

Mutations of OCRL1 are associated with both the Lowe oculocerebrorenal syndrome, a multisystemic and Dent-2 disease, a renal tubulopathy. We have identified a mutation in 130 Lowe syndrome families and 6 affected by Dent-2 disease with 51 of these mutations being novel. No founding effect was evidenced for recurrent mutations. Two mutations initially reported as causing Dent-2 disease were identified in patients, including two brothers, presenting with Lowe syndrome thus extending the clinical variability of OCRL1 mutations. mRNA levels, protein content, and PiP(2) -ase activities were analyzed in patient's fibroblasts. Although mRNA levels were normal in cells harboring a missense mutation, the OCRL1 content was markedly lowered, suggesting that enzymatic deficiency resulted mainly from protein degradation rather than from a catalytic inactivation. Analysis of a splicing mutation that led to the elimination of the initiation codon evidenced the presence of shortened forms of OCRL1 that might result from the use of alternative initiation codons. The specific mapping of the frameshift and nonsense mutations, exclusively identified in exons 1-7 and exons 8-23, respectively, for Dent disease and Lowe syndrome together with the possible use of alternative initiation codons might be related to their clinical expression, that is, Lowe syndrome or Dent-2 disease.

Phosphoregulation of MgcRacGAP in mitosis involves Aurora B and Cdk1 protein kinases and the PP2A phosphatase.

MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56epsilon, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.

Iodide transporter NIS regulates cancer cell motility and invasiveness by interacting with the Rho guanine nucleotide exchange factor LARG.

A number of solute carrier (SLC) proteins are subject to changes in expression and activity during carcinogenesis. Whether these changes play a role in carcinogenesis is unclear, except for some nutrients and ion carriers whose deregulation ensures the necessary reprogramming of energy metabolism in cancer cells. In this study, we investigated the functional role in tumor progression of the sodium/iodide symporter (NIS; aka SLC5A5), which is upregulated and mislocalized in many human carcinomas. Notably, we found that NIS enhanced cell migration and invasion without ion transport being involved. These functions were mediated by NIS binding to leukemia-associated RhoA guanine exchange factor, a Rho guanine exchange factor that activates the small GTPase RhoA. Sequestering NIS in intracellular organelles or impairing its targeting to the cell surface (as observed in many cancers) led to a further increase in cell motility and invasiveness. In sum, our results established NIS as a carrier protein that interacts with a major cell signaling hub to facilitate tumor cell locomotion and invasion.

A circadian clock in hippocampus is regulated by interaction between oligophrenin-1 and Rev-erbα.

Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.

Lowe syndrome protein Ocrl1 is translocated to membrane ruffles upon Rac GTPase activation: a new perspective on Lowe syndrome pathophysiology.

Oculocerebrorenal Lowe syndrome is a rare X-linked disorder characterized by bilateral cataract, mental retardation and renal Fanconi syndrome. The Lowe syndrome protein Ocrl1 is a PIP2 5-phosphatase, primarily localized to the trans-Golgi network (TGN), which 'loss of function' mutations result in PIP2 accumulation in patient's cells. Although PIP2 is involved in many cell functions including signalling, vesicle trafficking and actin polymerization, it has been difficult so far to decipher molecular/cellular mechanisms responsible for Lowe syndrome phenotype. We have recently shown that, through its C-terminal RhoGAP domain, Ocrl1 forms a stable complex with Rac GTPase within the cell. In line with this finding, we report here that upon epidermal growth factor induced Rac activation in COS-7 cells, a fraction of Ocrl1 translocates from TGN to plasma membrane and concentrates in membrane ruffles. In order to investigate the functionality of Ocrl1 in plasma membrane, we have analysed PIP2 distribution in human dermal fibroblasts (HDFs) from Lowe patients versus control HDFs. As revealed by both immunodetection and green fluorescent protein-PH binding, PIP2 was found strikingly to accumulate in PDGF induced ruffles in Lowe HDFs when compared with control. This suggests that Ocrl1 is active as a PIP2 5-phosphatase in Rac induced membrane ruffles. Cellular properties such as cell migration and establishment of cell-cell contacts, which depend on ruffling and lamellipodia formation, should be further investigated to understand the pathophysiology of Lowe syndrome.

Lowe syndrome protein OCRL1 interacts with Rac GTPase in the trans-Golgi network.

The oculocerebrorenal syndrome of Lowe (OCRL) is a rare X-linked disorder characterized by severe mental retardation, congenital cataracts and renal Fanconi syndrome. OCRL1 protein is a phosphatidylinositol 4,5-bisphosphate 5-phosphatase with a C-terminal RhoGAP domain. Considering the pleiotropic cellular functions of Rho GTPases (Rho, Rac and Cdc42) and their dysregulation in several forms of mental retardation, we have investigated the so far unexplored function of the RhoGAP domain of OCRL1. Activated Rac GTPase was found to stably associate with the OCRL1 RhoGAP domain in vitro and to co-immunoprecipitate with endogenous OCRL1. Contrasting with other GAPs, OCRL1 RhoGAP exhibited a significant interaction with GDP bound Rac in vitro. As compared to Rac, other Rho GTPases tested showed reduced (Cdc42) or no binding (RhoA, RhoG) to OCRL1 RhoGAP. Immunofluorescence studies in HEK and COS7 cells and Golgi perturbation assays with Brefeldin A demonstrated that a fraction of endogenous Rac co-localizes with OCRL1 and gamma-adaptin in the trans-Golgi network. The OCRL1 RhoGAP domain showed low Rac GAP activity in vitro, and when expressed in Swiss 3T3 cells induced specific inhibition of RacGTP dependent ruffles, consistent with OCRL1 being an active RacGAP. OCRL1 appears to be a bifunctional protein which, in addition to its PIP2 5-phosphatase activity, binds to Rac GTPase. This novel property may play a role in localizing OCRL1 to the trans-Golgi network. Moreover, loss of OCRL1 RhoGAP and the resulting alteration in Rho pathways may contribute to mental retardation in Lowe syndrome, as illustrated in other forms of X-linked mental retardation.

Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells.

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP -null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2-MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.