Mutant mice with rod-specific VPS35 deletion exhibit retinal α-synuclein pathology-associated degeneration.

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.

MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1.

The primary cilium undergoes cell cycle-dependent assembly and disassembly. Dysregulated ciliary dynamics are associated with several pathological conditions called ciliopathies. Previous studies showed that the localization of phosphorylated Tctex-1 at Thr94 (T94) at the ciliary base critically regulates ciliary resorption by accelerating actin remodeling and ciliary pocket membrane endocytosis. Here, we show that microtubule-associated serine/threonine kinase family member 4 (MAST4) is localized at the primary cilium. Suppressing MAST4 blocks serum-induced ciliary resorption, and overexpressing MAST4 accelerates ciliary resorption. Tctex-1 binds to the kinase domain of MAST4, in which the R503 and D504 residues are key to MAST4-mediated ciliary resorption. The ciliary resorption and the ciliary base localization of phospho-(T94)Tctex-1 are blocked by the knockdown of MAST4 or the expression of the catalytic-inactive site-directed MAST4 mutants. Moreover, MAST4 is required for Cdc42 activation and Rab5-mediated periciliary membrane endocytosis during ciliary resorption. These results support that MAST4 is a novel kinase that regulates ciliary resorption by modulating the ciliary base localization of phospho-(T94)Tctex-1. MAST4 is a potential new target for treating ciliopathies causally by ciliary resorption defects.

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Chondroitin sulfate enhances the barrier function of basement membrane assembled by heparan sulfate.

Glycosaminoglycans are ubiquitously expressed polysaccharides that are attached to proteoglycans. Here, we showed that ablation of the heparan sulfate (HS) polymerase Ext1 in retinal progenitor cells did not affect initial progression of retinal angiogenesis, but it disrupted the pruning of blood vessels and establishment of arterioles and venules. In the absence of retinal HS, blood vessels were also vulnerable to high oxygen tension in early postnatal stages, which could be rescued by exogenous vascular endothelial growth factor (VEGF), consistent with the role of retinal HS in the fine-tuning of VEGF signaling. Furthermore, we observed that the retinal inner limiting membrane (ILM) was disrupted by deletion of Ext1 in a timing-specific manner, suggesting that retinal HS is required for the assembly but not the maintenance of the basement membrane. Lastly, we showed that further deletion of C4st1, a chondroitin sulfate (CS) sulfation enzyme, did not affect the assembly of the ILM but, when combined with Ext1 deletion, it aggravated the retinal permeability by disrupting the retinal glycocalyx. These results demonstrate an important role of CS and HS in establishing the barrier function of the extracellular matrix.

Visual pigment-deficient cones survive and mediate visual signaling despite the lack of outer segments.

Rhodopsin and cone opsins are essential for light detection in vertebrate rods and cones, respectively. It is well established that rhodopsin is required for rod phototransduction, outer segment disk morphogenesis, and rod viability. However, the roles of cone opsins are less well understood. In this study, we adopted a loss-of-function approach to investigate the physiological roles of cone opsins in mice. We showed that cones lacking cone opsins do not form normal outer segments due to the lack of disk morphogenesis. Surprisingly, cone opsin-deficient cones survive for at least 12 mo, which is in stark contrast to the rapid rod degeneration observed in rhodopsin-deficient mice, suggesting that cone opsins are dispensable for cone viability. Although the mutant cones do not respond to light directly, they maintain a normal dark current and continue to mediate visual signaling by relaying the rod signal through rod-cone gap junctions. Our work reveals a striking difference between the role of rhodopsin and cone opsins in photoreceptor viability.

Retinal pigment epithelium-specific CLIC4 mutant is a mouse model of dry age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation.

CLIC4 regulates late endosomal trafficking and matrix degradation activity of MMP14 at focal adhesions in RPE cells.

Dysregulation in the extracellular matrix (ECM) microenvironment surrounding the retinal pigment epithelium (RPE) has been implicated in the etiology of proliferative vitreoretinopathy and age-related macular degeneration. The regulation of ECM remodeling by RPE cells is not well understood. We show that membrane-type matrix metalloproteinase 14 (MMP14) is central to ECM degradation at the focal adhesions in human ARPE19 cells. The matrix degradative activity, but not the assembly, of the focal adhesion is regulated by chloride intracellular channel 4 (CLIC4). CLIC4 is co-localized with MMP14 in the late endosome. CLIC4 regulates the proper sorting of MMP14 into the lumen of the late endosome and its proteolytic activation in lipid rafts. CLIC4 has the newly-identified "late domain" motif that binds to MMP14 and to Tsg101, a component of the endosomal sorting complex required for transport (ESCRT) complex. Unlike the late domain mutant CLIC4, wild-type CLIC4 can rescue the late endosomal sorting defect of MMP14. Finally, CLIC4 knockdown inhibits the apical secretion of MMP2 in polarized human RPE monolayers. These results, taken together, demonstrate that CLIC4 is a novel matrix microenvironment modulator and a novel regulator for late endosomal cargo sorting. Moreover, the late endosomal sorting of MMP14 actively regulates its surface activation in RPE cells.

The Late Endosomal Pathway Regulates the Ciliary Targeting of Tetraspanin Protein Peripherin 2.

Peripherin 2 (PRPH2) is a tetraspanin protein concentrated in the light-sensing cilium (called the outer segment) of the vertebrate photoreceptor. The mechanism underlying the ciliary targeting of PRPH2 and the etiology of cone dystrophy caused by PRPH2 mutations remain elusive. Here we show that the late endosome (LE) is the main waystation that critically sorts newly synthesized PRPH2 to the cilium. PRPH2 is expressed in the luminal membrane of the LE. We delineate multiple C-terminal motifs of PRPH2 that distinctively regulate its LE and ciliary targeting through ubiquitination and binding to ESCRT (Endosomal Sorting Complexes Required for Transport) component Hrs. Using the newly developed TetOn-inducible system in transfected male and female mouse cones in vivo, we show that the entry of nascent PRPH2 into the cone outer segment can be blocked by either cone dystrophy-causing C-terminal mutations of PRPH2, or by short-term perturbation of the LE or recycling endosomal traffic. These findings open new avenues of research to explore the biological role of the LE in the biosynthetic pathway and the etiology of cone dystrophy caused by PRPH2 mutations and/or malfunctions of the LE.SIGNIFICANCE STATEMENT Peripherin 2 (PRPH2) is a tetraspanin protein abundantly expressed in the light-sensing cilium, the outer segment, of the vertebrate photoreceptor. The mechanism underlying the ciliary transport of PRPH2 is unclear. The present study reveals a novel ciliary targeting pathway, in which the newly synthesized PRPH2 is first targeted to the lumen of the late endosome (LE) en route to the cilia. We deciphered the protein motifs and the machinery that regulates the LE trafficking of PRPH2. Using a novel TetOn-inducible system in transfected mouse cones, we showed that the LE pathway of PRPH2 is critical for its outer segment expression. A cone dystrophy-causing mutation impairs the LE and ciliary targeting of PRPH2, implicating the relevance of LE to cone/macular degenerative diseases.

Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines.

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The in vitro serum-stimulated ciliary assembly/disassembly assay has gained popularity in addressing the functions of the protein-of-interest in ciliary dynamics. Here, we describe a well-tested protocol for transfecting human retinal pigment epithelial cells (RPE-1) and performing ciliary assembly/disassembly assays on the transfected cells.

Nervous system development relies on endosomal trafficking.

Accumulating findings have begun to unveil the important role of the endosomal machinery in the nervous system development. Endosomes have been linked to the differential segregation of cell fate determining molecules in asymmetrically dividing progenitors during neurogenesis. Additionally, the precise removal and reinsertion of membrane components through endocytic trafficking regulates the spatial and temporal distribution of signaling receptors and adhesion molecules, which determine the morphology and motility of migrating neurons. Emerging evidence suggests that the role of the endosomal sorting adaptors is dependent upon cell type and developmental stage. The repertoire of the signaling receptors and/or adhesion molecules sorted by the endosome during these processes remains to be explored. In this commentary, we will briefly address the progress in this research field.

Tctex-1 controls ciliary resorption by regulating branched actin polymerization and endocytosis.

The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain. Furthermore, Tctex-1, originally identified as a cytoplasmic dynein light chain, has a dynein-independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex-1, actin, and dynamin. Mechanistically, Tctex-1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex-1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin-dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex-1-regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.

The Biology of Ciliary Dynamics.

The cilium is an evolutionally conserved apical membrane protrusion that senses and transduces diverse signals to regulate a wide range of cellular activities. The cilium is dynamic in length, structure, and protein composition. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases. The cilium undergoes cell-cycle-dependent assembly and disassembly, with ciliary resorption linked with G1-S transition and cell-fate choice. In the resting cell, the cilium remains sensitive to environmental cues for remodeling during tissue homeostasis and repair. Recent findings further reveal an interplay between the cilium and extracellular vesicles and identify bioactive cilium-derived vesicles, posing a previously unrecognized role of cilia for sending signals. The photoreceptor outer segment is a notable dynamic cilium. A recently discovered protein transport mechanism in photoreceptors maintains light-regulated homeostasis of ciliary length.

SARA regulates neuronal migration during neocortical development through L1 trafficking.

Emerging evidence suggests that endocytic trafficking of adhesion proteins plays a crucial role in neuronal migration during neocortical development. However, molecular insights into these processes remain elusive. Here, we study the early endosomal protein Smad anchor for receptor activation (SARA) in the developing mouse brain. SARA is enriched at the apical endfeet of radial glia of the neocortex. Although SARA knockdown did not lead to detectable neurogenic phenotypes, SARA-suppressed neurons exhibited impaired orientation and migration across the intermediate zone. Mechanistically, we show that SARA knockdown neurons exhibit increased surface expression of the L1 cell adhesion molecule. Neurons ectopically expressing L1 phenocopy the migration and orientation defects caused by SARA knockdown and display increased contact with neighboring neurites. L1 knockdown effectively rescues SARA suppression-induced phenotypes. SARA knockdown neurons eventually overcome their migration defect and enter later into the cortical plate. Nevertheless, these neurons localize at more superficial cortical layers than their control counterparts. These results suggest that SARA regulates the orientation, multipolar-to-bipolar transition and the positioning of cortical neurons via modulating surface L1 expression.

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon. Analyses of Tulp1 knock-out mice demonstrate that Tulp1 is essential to keep endocytic proteins enriched at the periactive zone and to maintain high levels of endocytic activity close to the synaptic ribbon. Moreover, we have discovered a novel interaction between Tulp1 and the synaptic ribbon protein RIBEYE, which is important to maintain synaptic ribbon integrity. The current findings suggest a new model for Tulp1-mediated localization of the endocytic machinery at the periactive zone of ribbon synapses and offer a new rationale and mechanism for vision loss associated with genetic defects in Tulp1.

CLIC4 regulates apical exocytosis and renal tube luminogenesis through retromer- and actin-mediated endocytic trafficking.

Chloride intracellular channel 4 (CLIC4) is a mammalian homologue of EXC-4 whose mutation is associated with cystic excretory canals in nematodes. Here we show that CLIC4-null mouse embryos exhibit impaired renal tubulogenesis. In both developing and developed kidneys, CLIC4 is specifically enriched in the proximal tubule epithelial cells, in which CLIC4 is important for luminal delivery, microvillus morphogenesis, and endolysosomal biogenesis. Adult CLIC4-null proximal tubules display aberrant dilation. In MDCK 3D cultures, CLIC4 is expressed on early endosome, recycling endosome and apical transport carriers before reaching its steady-state apical membrane localization in mature lumen. CLIC4 suppression causes impaired apical vesicle coalescence and central lumen formation, a phenotype that can be rescued by Rab8 and Cdc42. Furthermore, we show that retromer- and branched actin-mediated trafficking on early endosome regulates apical delivery during early luminogenesis. CLIC4 selectively modulates retromer-mediated apical transport by negatively regulating the formation of branched actin on early endosomes.

A dynein independent role of Tctex-1 at the kinetochore.

Dynein light chains are accessory subunits of the cytoplasmic dynein complex, a minus-end directed microtubule motor. Here, we demonstrate that the dynein light chain Tctex-1 associates with unattached kinetochores and is essential for accurate chromosome segregation. Tctex-1 knockdown in cells does not affect the localization and function of dynein at the kinetochore, but produces a prolonged mitotic arrest with a few misaligned chromosomes, which are subsequently missegregated during anaphase. This function is independent of Tctex-1's association with dynein. The kinetochore localization of Tctex-1 is independent of the ZW10-dynein pathway, but requires the Ndc80 complex. Thus, our findings reveal a dynein independent role of Tctex-1 at the kinetochore to enhance the stability of kinetochore-microtubule attachment.

Light regulates the ciliary protein transport and outer segment disc renewal of mammalian photoreceptors.

The outer segment (OS) of the rod photoreceptor is a light-sensing cilium containing ~1,000 membrane-bound discs. Each day, discs constituting the distal tenth of the OS are shed, whereas nascent discs are formed at the base of the OS through the incorporation of molecules transported from the inner segment. The mechanisms regulating these processes remain elusive. Here, we show that rhodopsin preferentially enters the OS in the dark. Photoexcitation of post-Golgi rhodopsins retains them in the inner segment. Disc-rim protein peripherin2/rds enters the OS following a rhythm complementary to that of rhodopsin. Light-dark cycle-regulated protein trafficking serves as a mechanism to segregate rhodopsin-rich and peripherin2/rds-rich discs into alternating stacks, which are flanked by characteristic cytoplasmic pockets. This periodic cytostructure divides the OS into approximately ten fractions, each containing discs synthesized in a single day. This mechanism may explain how the rod photoreceptor balances the quantity of discs added and removed daily.

Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods.

BACKGROUND: Cilia are vital to various cellular and sensory functions. The pathway by which ciliary membrane proteins translocate through the transition zone is not well understood. Direct morphological characterization of ciliary cargoes in transit remains lacking. In the vertebrate photoreceptor, rhodopsin is synthesized and transported from the inner segment to the disc membranes of the outer segment (OS), which is a modified cilium. To date, the membrane topology of the basal OS and the mechanisms by which rhodopsin is transported through the transition zone (i.e., connecting cilium) and by which nascent disc membranes are formed remain controversial. RESULTS: Using an antibody recognizing its cytoplasmic C-terminus, we localize rhodopsin on both the plasma membrane and lumen of the connecting cilium by immuno-electron microscopy (EM). We also use transmission EM to visualize the electron-dense enzymatic products derived from the rhodopsin-horseradish peroxidase (HRP) fusion in transfected rodent rods. In the connecting cilium, rhodopsin is not only expressed in the plasma membrane but also in the lumen on two types of membranous carriers, long smooth tubules and small, coated, filament-bound vesicles. Additionally, membrane-bound rhodopsin carriers are also found in close proximity to the nascent discs at the basal OS axoneme and in the distal inner segment. This topology-indicative HRP-rhodopsin reporter shows that the nascent basalmost discs and the mature discs have the same membrane topology, with no indication of evagination or invagination from the basal OS plasma membranes. Serial block face and focus ion beam scanning EM analyses both indicate that the transport carriers enter the connecting cilium lumen from either the basal body lumen or cytoplasmic space between the axonemal microtubules and the ciliary plasma membrane. CONCLUSIONS: Our results suggest the existence of multiple ciliary gate entry pathways in rod photoreceptors. Rhodopsin is likely transported across the connecting cilium on the plasma membrane and through the lumens on two types of tubulovesicular carriers produced in the inner segment. Our findings agree with a previous model that rhodopsin carriers derived from the cell body may fuse directly onto nascent discs as they grow and mature.

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates.

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy. Our experiments directly demonstrate that rhodopsin-GFP traffics through apical recycling endosomes (AREs) that bear the small GTPase Rab11a before arriving at the apical membrane. Expression of dominant-negative Rab11a drastically reduced apical delivery of rhodopsin-GFP and caused its missorting to the basolateral membrane. Surprisingly, functional inhibition of dynamin-2 trapped rhodopsin-GFP at AREs and caused aberrant accumulation of coated vesicles on AREs, suggesting a previously unrecognized role for dynamin-2 in the scission of apical carrier vesicles from AREs. A second set of experiments, using a unique method to carry out total internal reflection fluorescence microscopy (TIRFM) from the apical side, allowed us to visualize the fusion of rhodopsin-GFP carrier vesicles, which occurred randomly all over the apical plasma membrane. Furthermore, two-color TIRFM showed that Rab11a-mCherry was present in rhodopsin-GFP carrier vesicles and was rapidly released upon fusion onset. Our results provide direct evidence for a role of AREs as a post-Golgi sorting hub in the biosynthetic route of polarized epithelia, with Rab11a regulating cargo sorting at AREs and carrier vesicle docking at the apical membrane.

The roles of evolutionarily conserved functional modules in cilia-related trafficking.

Cilia are present across most eukaryotic phyla and have diverse sensory and motility roles in animal physiology, cell signalling and development. Their biogenesis and maintenance depend on vesicular and intraciliary (intraflagellar) trafficking pathways that share conserved structural and functional modules. The functional units of the interconnected pathways, which include proteins involved in membrane coating as well as small GTPases and their accessory factors, were first experimentally associated with canonical vesicular trafficking. These components are, however, ancient, having been co-opted by the ancestral eukaryote to establish the ciliary organelle, and their study can inform us about ciliary biology in higher organisms.