ABSTRACT: 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-?, TGF-?, 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.
Project description: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.
Project description:Glaucoma is a group of progressive optic neuropathies that cause irreversible vision loss. Although elevated intraocular pressure (IOP) is associated with the development and progression of glaucoma, the mechanisms for its regulation are not well understood. Here, we have designed CIBN/CRY2-based optogenetic constructs to study phosphoinositide regulation within distinct subcellular compartments. We show that stimulation of CRY2-OCRL, an inositol 5-phosphatase, increases aqueous humor outflow and lowers IOP in vivo, which is caused by a calcium-dependent actin rearrangement of the trabecular meshwork cells. Phosphoinositide stimulation also rescues defective aqueous outflow and IOP in a Lowe syndrome mouse model but not in IFT88fl/fl mice that lack functional cilia. Thus, our study is the first to use optogenetics to regulate eye pressure and demonstrate that tight regulation of phosphoinositides is critical for aqueous humor homeostasis in both normal and diseased eyes.
Project description:Lowe syndrome is a rare X-linked disorder characterized by bilateral congenital cataracts and glaucoma, mental retardation, and proximal renal tubular dysfunction. Mutations in OCRL, an inositol polyphosphate 5-phosphatase that dephosphorylates PI(4,5)P2, cause Lowe syndrome. Previously we showed that OCRL localizes to the primary cilium, which has a distinct membrane phospholipid composition, but disruption of phosphoinositides in the ciliary membrane is poorly understood. Here, we demonstrate that cilia from Lowe syndrome patient fibroblasts exhibit increased levels of PI(4,5)P2 and decreased levels of PI4P. In particular, subcellular distribution of PI(4,5)P2 build-up was observed at the transition zone. Accumulation of ciliary PI(4,5)P2 was pronounced in mouse embryonic fibroblasts (MEFs) derived from Lowe syndrome mouse model as well as in Ocrl-null MEFs, which was reversed by reintroduction of OCRL. Similarly, expression of wild-type OCRL reversed the elevated PI(4,5)P2 in Lowe patient cells. Accumulation of sonic hedgehog protein in response to hedgehog agonist was decreased in MEFs derived from a Lowe syndrome mouse model. Together, our findings show for the first time an abnormality in ciliary phosphoinositides of both human and mouse cell models of Lowe syndrome.
Project description:Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm's canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer's vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.
Project description:An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca(2+) influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca(2+)-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension.
Project description:Mutations in the OCRL1 gene result in the oculocerebrorenal syndrome of Lowe, with symptoms including congenital bilateral cataracts, glaucoma, renal failure, and neurological impairments. OCRL1 encodes an inositol polyphosphate 5-phosphatase which preferentially dephosphorylates phosphatidylinositide 4,5 bisphosphate (PI(4,5)P2). We have identified two novel mutations in two unrelated Lowe syndrome patients with congenital glaucoma. Novel deletion mutations are detected at c.739-742delAAAG in Lowe patient 1 and c.1595-1631del in Lowe patient 2. End stage glaucoma in patient 2 resulted in the enucleation of the eye, which on histology demonstrated corneal keloid, fibrous infiltration of the angle, ectropion uvea, retinal gliosis, and retinal ganglion cell loss. We measured OCRL protein levels in patient keratinocytes and found that Lowe 1 patient cells had significantly reduced OCRL protein as compared to the control keratinocytes. Genotype-phenotype correlation of OCRL1 mutations associated with congenital glaucoma revealed clustering of missense and deletion mutations in the 5-phosphatase domain and the RhoGAP-like domain. In conclusion, we report novel OCRL1 mutations in Lowe syndrome patients and the corresponding histopathologic analysis of one patient's ocular pathology.
Project description:Normally, trabecular meshwork (TM) and Schlemm's canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model. These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM-like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open-angle glaucoma patients, providing a novel alternative treatment option.
Project description:Glaucoma is a major cause of blindness and is frequently associated with elevated intraocular pressure. The trabecular meshwork (TM), the tissue that primarily regulates intraocular pressure, is known to have reduced cellularity in glaucoma. Thus, stem cells, if properly delivered to the TM, may offer a novel therapeutic option for intraocular pressure control in glaucoma patients. For this purpose, targeted delivery of stem cells to the TM is desired. Here, we used magnetic nanoparticles (Prussian blue nanocubes [PBNCs]) to label mesenchymal stem cells and to magnetically steer them to the TM following injection into the eye's anterior chamber. PBNC-labeled stem cells showed increased delivery to the TM vs. unlabeled cells after only 15-minute exposure to a magnetic field. Further, PBNC-labeled mesenchymal stem cells could be delivered to the entire circumference of the TM, which was not possible without magnetic steering. PBNCs did not affect mesenchymal stem cell viability or multipotency. We conclude that this labeling approach allows for targeted, relatively high-efficiency delivery of stem cells to the TM in clinically translatable time-scales, which are necessary steps towards regenerative medicine therapies for control of ocular hypertension in glaucoma patients.
Project description:Skeletal homeostasis requires the continued replenishment of the bone forming osteoblast from a mesenchymal stem cell (MSC) population, a process that has been shown to be mechanically regulated. However, the mechanisms by which a biophysical stimulus can induce a change in biochemical signaling, mechanotransduction, is poorly understood. As a precursor to loading-induced bone formation, deciphering the molecular mechanisms of MSC osteogenesis is a critical step in developing novel anabolic therapies. Therefore, in this study we characterize the expression of the mechanosensitive calcium channel Transient Receptor Potential subfamily V member 4 (TRPV4) in MSCs and demonstrate that TRPV4 localizes to areas of high strain, specifically the primary cilium. We demonstrate that TRPV4 is required for MSC mechanotransduction, mediating oscillatory fluid shear induced calcium signaling and early osteogenic gene expression. Furthermore, we demonstrate that TRPV4 can be activated pharmacologically eliciting a response that mirrors that seen with mechanical stimulation. Lastly, we show that TRPV4 localization to the primary cilium is functionally significant, with MSCs with defective primary cilia exhibiting an inhibited osteogenic response to TRPV4 activation. Collectively, this data demonstrates a novel mechanism of stem cell mechanotransduction, which can be targeted therapeutically, and further highlights the critical role of the primary cilium in MSC biology.
Project description:Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Müller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4?-PDD and GSK1016790A elevated [Ca2+]i in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca2+](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca2+](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca2+ -free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca2+]i and augmented excitability. Excessive Ca2+ influx through TRPV4 predisposes RGCs to activation of Ca2+ -dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure.