Crosstalk between the mTOR pathway and primary cilia in human diseases.

Autophagy is a fundamental catabolic process whereby excessive or damaged cytoplasmic components are degraded through lysosomes to maintain cellular homeostasis. Studies of mTOR signaling have revealed that mTOR controls biomass generation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Primary cilia, the assembly of which depends on kinesin molecular motors, serve as sensory organelles and signaling platforms. Given these pathways' central role in maintaining cellular and physiological homeostasis, a connection between mTOR and primary cilia signaling is starting to emerge in a variety of diseases. In this review, we highlight recent advances in our understanding of the complex crosstalk between the mTOR pathway and cilia and discuss its function in the context of related diseases.

Advances in Ophthalmic Optogenetics: Approaches and Applications.

Recent advances in optogenetics hold promise for vision restoration in degenerative eye diseases. Optogenetics refers to techniques that use light to control the cellular activity of targeted cells. Although optogenetics is a relatively new technology, multiple therapeutic options are already being explored in pre-clinical and phase I/II clinical trials with the aim of developing novel, safe, and effective treatments for major blinding eye diseases, such as glaucoma and retinitis pigmentosa. Optogenetic approaches to visual restoration are primarily aimed at replacing lost or dysfunctional photoreceptors by inserting light-sensitive proteins into downstream retinal neurons that have no intrinsic light sensitivity. Such approaches are attractive because they are agnostic to the genetic causes of retinal degeneration, which raises hopes that all forms of retinal dystrophic and degenerative diseases could become treatable. Optogenetic strategies can also have a far-reaching impact on translational research by serving as important tools to study the pathogenesis of retinal degeneration and to identify clinically relevant therapeutic targets. For example, the CRY-CIBN optogenetic system has been recently applied to animal models of glaucoma, suggesting a potential role of OCRL in the regulation of intraocular pressure in trabecular meshwork. As optogenetic strategies are being intensely investigated, it appears crucial to consider the opportunities and challenges such therapies may offer. Here, we review the more recent promising optogenetic molecules, vectors, and applications of optogenetics for the treatment of retinal degeneration and glaucoma. We also summarize the preliminary results of ongoing clinical trials for visual restoration.

Optogenetic Modulation of Intraocular Pressure in a Glucocorticoid-Induced Ocular Hypertension Mouse Model.

Purpose: Steroid-induced glaucoma is a common form of secondary open angle glaucoma characterized by ocular hypertension (elevated intraocular pressure [IOP]) in response to prolonged glucocorticoid exposure. Elevated IOP occurs with increased outflow resistance and altered trabecular meshwork (TM) function. Recently, we used an optogenetic approach in TM to regulate the 5-phosphatase, OCRL, which contributes to regulating PI(4,5)P2 levels. Here, we applied this system with the aim of reversing compromised outflow function in a steroid-induced ocular hypertension mouse model. Methods: Elevated IOP was induced by chronic subconjunctival dexamethasone injections in wild-type C57Bl/6j mice. AAV2 viruses containing optogenetic modules of cryptochrome 2 (Cry2)-OCRL-5ptase and CIBN-GFP were injected into the anterior chamber. Four weeks after viral expression and dexamethasone exposure, IOP was measured by tonometer and outflow facility was measured by perfusion apparatus. Human TM cells were treated with dexamethasone, stimulated by light and treated with rhodamine-phalloidin to analyze actin structure. Results: Dexamethasone treatment elevated IOP and decreased outflow facility in wild-type mice. Optogenetic constructs were expressed in the TM of mouse eyes. Light stimulation caused CRY2-OCRL-5ptase to translocate to plasma membrane (CIBN-CAAX-GFP) and cilia (CIBN-SSTR3-GFP) in TM cells, which rescued the IOP and outflow facility. In addition, aberrant actin structures formed by dexamethasone treatment were reduced by optogenetic stimulation in human TM cells in culture. Conclusions: Subcellular targeting of inositol phosphatases to remove PIP2 represents a promising strategy to reverse defective TM function in steroid-induced ocular hypertension. Translational Relevance: Targeted modulation of OCRL may be used to decrease steroid-induced elevated IOP.

OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring.

Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule-anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule-organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule-organizing centers and impaired microtubule-based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome-targeted PACT-SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients' cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.

Defective INPP5E distribution in NPHP1-related Senior-Loken syndrome.

BACKGROUND: Senior-Loken syndrome is a rare genetic disorder that presents with nephronophthisis and retinal degeneration, leading to end-stage renal disease and progressive blindness. The most frequent cause of juvenile nephronophthisis is a mutation in the nephronophthisis type 1 (NPHP1) gene. NPHP1 encodes the protein nephrocystin-1, which functions at the transition zone (TZ) of primary cilia. METHODS: We report a 9-year-old Senior-Loken syndrome boy with NPHP1 deletion, who presents with bilateral vision decrease and cystic renal disease. Renal function deteriorated to require bilateral nephrectomy and renal transplant. We performed immunohistochemistry, H&E staining, and electron microscopy on the renal sample to determine the subcellular distribution of ciliary proteins in the absence of NPHP1. RESULTS: Immunohistochemistry and electron microscopy of the resected kidney showed disorganized cystic structures with loss of cilia in renal tubules. Phosphoinositides have been recently recognized as critical components of the ciliary membrane and immunostaining of kidney sections for phosphoinositide 5-phosphatase, INPP5E, showed loss of staining compared to healthy control. Ophthalmic examination showed decreased electroretinogram consistent with early retinal degeneration. CONCLUSION: The decreased expression of INPP5E specifically in the primary cilium, coupled with disorganized cilia morphology, suggests a novel role of NPHP1 that it is involved in regulating ciliary phosphoinositide composition in the ciliary membrane of renal tubular cells.

Developmental distribution of primary cilia in the retinofugal visual pathway.

The mammalian visual system is composed of circuitry connecting sensory input from the retina to the processing core of the visual cortex. The two main retinorecipient brain targets, the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN), bridge retinal input and visual output. The primary cilium is a conserved organelle increasingly viewed as a critical sensor for the regulation of developmental and homeostatic pathways in most mammalian cell types. Moreover, cilia have been described as crucial for neurogenesis, neuronal maturation, and survival in the cortex and retina. However, cilia in the visual relay center remain to be fully described. In this study, we characterized the ciliation profile of the SC and dLGN and found that the overall number of ciliated cells declined during development. Interestingly, shorter ciliated cells in both regions were identified as neurons, whose numbers remained stable over time, suggesting that cilia retention is a critical feature for optimal neuronal function in SC and dLGN. Our study suggests that primary cilia are important for neuronal maturation and function in cells of the SC and dLGN.

Hexachloronaphthalene Induces Mitochondrial-Dependent Neurotoxicity via a Mechanism of Enhanced Production of Reactive Oxygen Species.

Hexachloronaphthalene (PCN67) is one of the most toxic among polychlorinated naphthalenes. Despite the known high bioaccumulation and persistence of PCN67 in the environment, it is still unclear to what extent exposure to these substances may interfere with normal neuronal physiology and lead to neurotoxicity. Therefore, the primary goal of this study was to assess the effect of PCN67 in neuronal in vitro models. Neuronal death was assessed upon PCN67 treatment using differentiated PC12 cells and primary hippocampal neurons. At 72 h postexposure, cell viability assays showed an IC50 value of 0.35 µg/ml and dose-dependent damage of neurites and concomitant downregulation of neurofilaments L and M. Moreover, we found that younger primary neurons (DIV4) were much more sensitive to PCN67 toxicity than mature cultures (DIV14). Our comprehensive analysis indicated that the application of PCN67 at the IC50 concentration caused necrosis, which was reflected by an increase in LDH release, HMGB1 protein export to the cytosol, nuclear swelling, and loss of homeostatic control of energy balance. The blockage of mitochondrial calcium uniporter partially rescued the cell viability, loss of mitochondrial membrane potential (ΔΨ m), and the overproduction of reactive oxygen species, suggesting that the underlying mechanism of neurotoxicity involved mitochondrial calcium accumulation. Increased lipid peroxidation as a consequence of oxidative stress was additionally seen for 0.1 µg/ml of PCN67, while this concentration did not affect ΔΨ m and plasma membrane permeability. Our results show for the first time that neuronal mitochondria act as a target for PCN67 and indicate that exposure to this drug may result in neuron loss via mitochondrial-dependent mechanisms.

Polycystin-1 regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling.

Mutations in PKD1 (encoding for polycystin-1 [PC1]) are found in 80%-85% of patients with autosomal dominant polycystic kidney disease (ADPKD). We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalized centrosomal RhoA signaling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganized in cystic cells derived from patients with PKD1 and was associated with an increase in total and centrosomal active RhoA and ROCK signaling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, -29, and -35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1-null cells using a centrosome-targeted proximity ligation assay and by dual immunofluorescence labeling. Finally, the ROCK inhibitor hydroxyfasudil reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a potentially novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signaling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo, indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.

Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation.

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.

Oculocerebrorenal syndrome of Lowe: Survey of ophthalmic presentations and management.

BACKGROUND: Lowe syndrome is a rare X-linked disease that is characterized by renal dysfunction, developmental delays, congenital cataracts and glaucoma. Mutations in the oculocerebral renal syndrome of Lowe (OCRL) gene are found in Lowe syndrome patients. Although loss of vision is a major concern for families and physicians who take care of Lowe syndrome children, definitive cause of visual loss is still unclear. Children usually present with bilateral dense cataracts at birth and glaucoma, which occurs in more than half of cases, either concurrently or following cataract surgery. MATERIALS AND METHODS: A retrospective review was conducted on the prevalence and characteristics of ocular findings among families of patients with Lowe syndrome with 137 uniquely affected individuals. RESULTS: Of 137 patients, all had bilateral congenital cataracts. Nystagmus was reported in 69.3% of cases, glaucoma in 54.7%, strabismus in 35.0%, and corneal scar in 18.2% of patients. Glaucoma was reported as the most common cause of blindness (46%) followed by corneal scars (41%). Glaucoma occurred in 54.7% of patients and affected both eyes in the majority of cases. Of these patients, 55% underwent surgery for glaucoma, while the remaining patients used medications to control their eye pressure. Timolol and latanoprost were the most commonly used medications. Although trabeculectomy and goniotomy are commonly used for pressure management, aqueous tube shunts had the best outcomes. CONCLUSION: Ocular manifestations in individuals with Lowe syndrome and carriers with OCRL mutation are reported which may help familiarize clinicians with the ocular manifestations and management of a rare and complex syndrome.

Loss of OCRL increases ciliary PI(4,5)P2 in Lowe oculocerebrorenal syndrome.

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.