Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts.

Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.

IPIP27A cooperates with OCRL to support endocytic traffic in the zebrafish pronephric tubule.

Endocytosis is a fundamentally important process through which material is internalized into cells from the extracellular environment. In the renal proximal tubule, endocytosis of the abundant scavenger receptor megalin and its co-receptor cubilin play a vital role in retrieving low molecular weight proteins from the renal filtrate. Although we know much about megalin and its ligands, the machinery and mechanisms by which the receptor is trafficked through the endosomal system remain poorly defined. In this study, we show that inositol phosphatase interacting protein of 27 kDa (Ipip27A), an interacting partner of the Lowe syndrome protein oculocerebrorenal syndrome of Lowe (OCRL), is required for endocytic traffic of megalin within the proximal renal tubule of zebrafish larvae. Knockout of Ipip27A phenocopies the endocytic phenotype seen upon loss of OCRL, with a deficit in uptake of both fluid-phase and protein cargo, which is accompanied by a reduction in megalin abundance and altered endosome morphology. Rescue and co-depletion experiments indicate that Ipip27A functions together with OCRL to support proximal tubule endocytosis. The results therefore identify Ipip27A as a new player in endocytic traffic in the proximal tubule in vivo and support the view that defective endocytosis underlies the renal tubulopathy in Lowe syndrome and Dent-2 disease.

A missense mutant of ocrl1 promotes apoptosis of tubular epithelial cells and disrupts endocytosis and the cell cycle of podocytes in Dent-2 Disease.

BACKGROUND: This study aimed to identify an orcl1 mutation in a patient with Dent-2 Disease and investigate the underlying mechanisms. METHODS: The ocrl1 mutation was identified through exome sequencing. Knockdown of orcl1 and overexpression of the orcl1 mutant were performed in HK-2 and MPC5 cells to study its function, while flow cytometry measured reactive oxygen species (ROS), phosphatidylserine levels, and cell apoptosis. Scanning electron microscopy observed crystal adhesion, while transmission electron microscopy examined kidney tissue pathology. Laser scanning confocal microscopy was used to examine endocytosis, and immunohistochemical and immunofluorescence assays detected protein expression. Additionally, podocyte-specific orcl1 knockout mice were generated to investigate the role of orcl1 in vivo. RESULTS: We identified a mutation resulting in the replacement of Histidine with Arginine at position 318 (R318H) in ocrl1 in the proband. orcl1 was widely expressed in the kidney. In vitro experiments showed that knockdown of orcl1 and overexpression of ocrl1 mutant increased ROS, phosphatidylserine exocytosis, crystal adhesion, and cell apoptosis in HK-2 cells. Knockdown of orcl1 in podocytes reduced endocytosis and disrupted the cell cycle while increasing cell migration. In vivo studies in mice showed that conditional deletion of orcl1 in podocytes caused glomerular dysfunction, including proteinuria and fibrosis. CONCLUSION: This study identified an R318H mutation in orcl1 in a patient with Dent-2 Disease. This mutation may contribute to renal injury by promoting ROS production and inducing cell apoptosis in tubular cells, while disrupting endocytosis and the cell cycle, and promoting cell migration of podocytes. Video Abstract.

Endocrine and behavioural features of Lowe syndrome and their potential molecular mechanisms.

BACKGROUND: Lowe syndrome (LS) is an X linked disease caused by pathogenic variants in the OCRL gene that impacts approximately 1 in 500 000 children. Classic features include congenital cataract, cognitive/behavioural impairment and renal tubulopathy. METHODS: This study is a retrospective review of clinical features reported by family based survey conducted by Lowe Syndrome Association. Frequency of non-ocular clinical feature(s) of LS and their age of onset was summarised. An LS-specific therapy effectiveness scale was used to assess the response to the administered treatment. Expression of OCRL and relevant neuropeptides was measured in postmortem human brain by qPCR. Gene expression in the mouse brain was determined by reanalysis of publicly available bulk and single cell RNA sequencing. RESULTS: A total of 137 individuals (1 female, 89.1% white, median age 14 years (range 0.8-56)) were included in the study. Short stature (height <3rd percentile) was noted in 81% (n=111) individuals, and 15% (n=20) received growth hormone therapy. Undescended testis was reported in 47% (n=64), and median age of onset of puberty was 15 years. Additional features were dental problems (n=77, 56%), bone fractures (n=63, 46%), hypophosphataemia (n=60, 44%), developmental delay and behavioural issues. OCRL is expressed in human and mouse hypothalami, and in hypothalamic cell clusters expressing Ghrh, Sst, Oxt, Pomc and pituitary cells expressing Gh and Prl. CONCLUSIONS: There is a wide spectrum of the clinical phenotype of LS. Some of the features may be partly driven by the loss of function of OCRL in the hypothalamus and the pituitary.

OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease.

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/- mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/- kidneys. The OcrlY/- mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/- mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/- and Clcn5Y/- mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.

Effects of Proximal Tubule Shortening on Protein Excretion in a Lowe Syndrome Model.

BACKGROUND: Lowe syndrome (LS) is an X-linked recessive disorder caused by mutations in OCRL, which encodes the enzyme OCRL. Symptoms of LS include proximal tubule (PT) dysfunction typically characterized by low molecular weight proteinuria, renal tubular acidosis (RTA), aminoaciduria, and hypercalciuria. How mutant OCRL causes these symptoms isn't clear. METHODS: We examined the effect of deleting OCRL on endocytic traffic and cell division in newly created human PT CRISPR/Cas9 OCRL knockout cells, multiple PT cell lines treated with OCRL-targeting siRNA, and in orcl-mutant zebrafish. RESULTS: OCRL-depleted human cells proliferated more slowly and about 10% of them were multinucleated compared with fewer than 2% of matched control cells. Heterologous expression of wild-type, but not phosphatase-deficient, OCRL prevented the accumulation of multinucleated cells after acute knockdown of OCRL but could not rescue the phenotype in stably edited knockout cell lines. Mathematic modeling confirmed that reduced PT length can account for the urinary excretion profile in LS. Both ocrl mutant zebrafish and zebrafish injected with ocrl morpholino showed truncated expression of megalin along the pronephric kidney, consistent with a shortened S1 segment. CONCLUSIONS: Our data suggest a unifying model to explain how loss of OCRL results in tubular proteinuria as well as the other commonly observed renal manifestations of LS. We hypothesize that defective cell division during kidney development and/or repair compromises PT length and impairs kidney function in LS patients.

A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy.

Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial-mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

Megalin mediates plasma membrane to mitochondria cross-talk and regulates mitochondrial metabolism.

Mitochondrial intracrines are extracellular signaling proteins, targeted to the mitochondria. The pathway for mitochondrial targeting of mitochondrial intracrines and actions in the mitochondria remains unknown. Megalin/LRP2 mediates the uptake of vitamins and proteins, and is critical for clearance of amyloid-ß protein from the brain. Megalin mutations underlie the pathogenesis of Donnai-Barrow and Lowe syndromes, characterized by brain defects and kidney dysfunction; megalin was not previously known to reside in the mitochondria. Here, we show megalin is present in the mitochondria and associates with mitochondrial anti-oxidant proteins SIRT3 and stanniocalcin-1 (STC1). Megalin shuttles extracellularly-applied STC1, angiotensin II and TGF-ß to the mitochondria through the retrograde early endosome-to-Golgi transport pathway and Rab32. Megalin knockout in cultured cells impairs glycolytic and respiratory capacities. Thus, megalin is critical for mitochondrial biology; mitochondrial intracrine signaling is a continuum of the retrograde early endosome-to-Golgi-Rab32 pathway and defects in this pathway may underlie disease processes in many systems.

Identification and functional analysis of a novel oculocerebrorenal syndrome of Lowe (OCRL) gene variant in two pedigrees with varying phenotypes including isolated congenital cataract.

Purpose: To identify the genetic variation in two unrelated probands with congenital cataract and to perform functional analysis of the detected variants. Methods: Clinical examination and phenotyping, segregation, and functional analysis were performed for the two studied pedigrees. Results: A novel OCRL gene variant (c.1964A>T, p. (Asp655Val)) was identified. This variant causes defects in OCRL protein folding and mislocalization to the cytoplasm. In addition, the variant's location close to the Rab binding site is likely to be associated with membrane targeting abnormalities. Conclusions: The results highlight the importance of early genetic diagnosis in infants with congenital cataract and show that mutations in the OCRL gene can present as apparently isolated congenital cataract.

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.

Primary cilia signaling mediates intraocular pressure sensation.

Lowe syndrome is a rare X-linked congenital disease that presents with congenital cataracts and glaucoma, as well as renal and cerebral dysfunction. OCRL, an inositol polyphosphate 5-phosphatase, is mutated in Lowe syndrome. We previously showed that OCRL is involved in vesicular trafficking to the primary cilium. Primary cilia are sensory organelles on the surface of eukaryotic cells that mediate mechanotransduction in the kidney, brain, and bone. However, their potential role in the trabecular meshwork (TM) in the eye, which regulates intraocular pressure, is unknown. Here, we show that TM cells, which are defective in glaucoma, have primary cilia that are critical for response to pressure changes. Primary cilia in TM cells shorten in response to fluid flow and elevated hydrostatic pressure, and promote increased transcription of TNF-α, TGF-ß, and GLI1 genes. Furthermore, OCRL is found to be required for primary cilia to respond to pressure stimulation. The interaction of OCRL with transient receptor potential vanilloid 4 (TRPV4), a ciliary mechanosensory channel, suggests that OCRL may act through regulation of this channel. A novel disease-causing OCRL allele prevents TRPV4-mediated calcium signaling. In addition, TRPV4 agonist GSK 1016790A treatment reduced intraocular pressure in mice; TRPV4 knockout animals exhibited elevated intraocular pressure and shortened cilia. Thus, mechanotransduction by primary cilia in TM cells is implicated in how the eye senses pressure changes and highlights OCRL and TRPV4 as attractive therapeutic targets for the treatment of glaucoma. Implications of OCRL and TRPV4 in primary cilia function may also shed light on mechanosensation in other organ systems.

Inositol 5-phosphatases: insights from the Lowe syndrome protein OCRL.

The precise regulation of phosphoinositide lipids in cellular membranes is crucial for cellular survival and function. Inositol 5-phosphatases have been implicated in a variety of disorders, including various cancers, obesity, type 2 diabetes, neurodegenerative diseases and rare genetic conditions. Despite the obvious impact on human health, relatively little structural and biochemical information is available for this family. Here, we review recent structural and mechanistic work on the 5-phosphatases with a focus on OCRL, whose loss of function results in oculocerebrorenal syndrome of Lowe and Dent 2 disease. Studies of OCRL emphasize how the actions of 5-phosphatases rely on both intrinsic and extrinsic membrane recognition properties for full catalytic function. Additionally, structural analysis of missense mutations in the catalytic domain of OCRL provides insight into the phenotypic heterogeneity observed in Lowe syndrome and Dent disease.

The cellular and physiological functions of the Lowe syndrome protein OCRL1.

Phosphoinositide lipids play a key role in cellular physiology, participating in a wide array of cellular processes. Consequently, mutation of phosphoinositide-metabolizing enzymes is responsible for a growing number of diseases in humans. Two related disorders, oculocerebrorenal syndrome of Lowe (OCRL) and Dent-2 disease, are caused by mutation of the inositol 5-phosphatase OCRL1. Here, we review recent advances in our understanding of OCRL1 function. OCRL1 appears to regulate many processes within the cell, most of which depend upon coordination of membrane dynamics with remodeling of the actin cytoskeleton. Recently developed animal models have managed to recapitulate features of Lowe syndrome and Dent-2 disease, and revealed new insights into the underlying mechanisms of these disorders. The continued use of both cell-based approaches and animal models will be key to fully unraveling OCRL1 function, how its loss leads to disease and, importantly, the development of therapeutics to treat patients.

PIPs in neurological diseases.

Phosphoinositide (PIP) lipids regulate many aspects of cell function in the nervous system including receptor signalling, secretion, endocytosis, migration and survival. Levels of PIPs such as PI4P, PI(4,5)P2 and PI(3,4,5)P3 are normally tightly regulated by phosphoinositide kinases and phosphatases. Deregulation of these biochemical pathways leads to lipid imbalances, usually on intracellular endosomal membranes, and these changes have been linked to a number of major neurological diseases including Alzheimer's, Parkinson's, epilepsy, stroke, cancer and a range of rarer inherited disorders including brain overgrowth syndromes, Charcot-Marie-Tooth neuropathies and neurodevelopmental conditions such as Lowe's syndrome. This article analyses recent progress in this area and explains how PIP lipids are involved, to varying degrees, in almost every class of neurological disease. This article is part of a Special Issue entitled Brain Lipids.

OCRL controls trafficking through early endosomes via PtdIns4,5P2-dependent regulation of endosomal actin.

Mutations in the phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P(2)) 5-phosphatase OCRL cause Lowe syndrome, which is characterised by congenital cataracts, central hypotonia, and renal proximal tubular dysfunction. Previous studies have shown that OCRL interacts with components of the endosomal machinery; however, its role in endocytosis, and thus the pathogenic mechanisms of Lowe syndrome, have remained elusive. Here, we show that via its 5-phosphatase activity, OCRL controls early endosome (EE) function. OCRL depletion impairs the recycling of multiple classes of receptors, including megalin (which mediates protein reabsorption in the kidney) that are retained in engorged EEs. These trafficking defects are caused by ectopic accumulation of PtdIns4,5P(2) in EEs, which in turn induces an N-WASP-dependent increase in endosomal F-actin. Our data provide a molecular explanation for renal proximal tubular dysfunction in Lowe syndrome and highlight that tight control of PtdIns4,5P(2) and F-actin at the EEs is essential for exporting cargoes that transit this compartment.

A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1.

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like ß-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.

The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome.

Oculocerebral renal syndrome of Lowe (OCRL or Lowe syndrome), a severe X-linked congenital disorder characterized by congenital cataracts and glaucoma, mental retardation and kidney dysfunction, is caused by mutations in the OCRL gene. OCRL is a phosphoinositide 5-phosphatase that interacts with small GTPases and is involved in intracellular trafficking. Despite extensive studies, it is unclear how OCRL mutations result in a myriad of phenotypes found in Lowe syndrome. Our results show that OCRL localizes to the primary cilium of retinal pigment epithelial cells, fibroblasts and kidney tubular cells. Lowe syndrome-associated mutations in OCRL result in shortened cilia and this phenotype can be rescued by the introduction of wild-type OCRL; in vivo, knockdown of ocrl in zebrafish embryos results in defective cilia formation in Kupffer vesicles and cilia-dependent phenotypes. Cumulatively, our data provide evidence for a role of OCRL in cilia maintenance and suggest the involvement of ciliary dysfunction in the manifestation of Lowe syndrome.

The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module.

Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P(2) modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P(2) dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN.