The Structural and Functional Integrity of Rod Photoreceptor Ribbon Synapses Depends on Redundant Actions of Dynamins 1 and 3.

Vertebrate vision begins with light absorption by rod and cone photoreceptors, which transmit signals from their synaptic terminals to second-order neurons: bipolar and horizontal cells. In mouse rods, there is a single presynaptic ribbon-type active zone at which the release of glutamate occurs tonically in the dark. This tonic glutamatergic signaling requires continuous exo- and endocytosis of synaptic vesicles. At conventional synapses, endocytosis commonly requires dynamins: GTPases encoded by three genes (Dnm1-3), which perform membrane scission. Disrupting endocytosis by dynamin deletions impairs transmission at conventional synapses, but the impact of disrupting endocytosis and the role(s) of specific dynamin isoforms at rod ribbon synapses are understood incompletely. Here, we used cell-specific knock-outs (KOs) of the neuron-specific Dnm1 and Dnm3 to investigate the functional roles of dynamin isoforms in rod photoreceptors in mice of either sex. Analysis of synaptic protein expression, synapse ultrastructure, and retinal function via electroretinograms (ERGs) showed that dynamins 1 and 3 act redundantly and are essential for supporting the structural and functional integrity of rod ribbon synapses. Single Dnm3 KO showed no phenotype, and single Dnm1 KO only modestly reduced synaptic vesicle density without affecting vesicle size and overall synapse integrity, whereas double Dnm1/Dnm3 KO impaired vesicle endocytosis profoundly, causing enlarged vesicles, reduced vesicle density, reduced ERG responses, synaptic terminal degeneration, and disassembly and degeneration of postsynaptic processes. Concurrently, cone function remained intact. These results show the fundamental redundancy of dynamins 1 and 3 in regulating the structure and function of rod ribbon synapses.

Molecular identification of wide-field amacrine cells in mouse retina that encode stimulus orientation.

Visual information processing is sculpted by a diverse group of inhibitory interneurons in the retina called amacrine cells. Yet, for most of the >60 amacrine cell types, molecular identities and specialized functional attributes remain elusive. Here, we developed an intersectional genetic strategy to target a group of wide-field amacrine cells (WACs) in mouse retina that co-express the transcription factor Bhlhe22 and the Kappa Opioid Receptor (KOR; B/K WACs). B/K WACs feature straight, unbranched dendrites spanning over 0.5 mm (∼15° visual angle) and produce non-spiking responses to either light increments or decrements. Two-photon dendritic population imaging reveals Ca 2+ signals tuned to the physical orientations of B/K WAC dendrites, signifying a robust structure-function alignment. B/K WACs establish divergent connections with multiple retinal neurons, including unexpected connections with non-orientation-tuned ganglion cells and bipolar cells. Our work sets the stage for future comprehensive investigations of the most enigmatic group of retinal neurons: WACs.

Apical CLC-2 in retinal pigment epithelium is crucial for survival of the outer retina.

Knockout of the chloride channel protein 2 (CLC-2; CLCN2) results in fast progressing blindness in mice. Retinal Pigment Epithelium (RPE) and photoreceptors undergo, in parallel, rapid, and profound morphological changes and degeneration. Immunohistochemistry and electron microscopy of the outer retina and electroretinography of the CLC-2 KO mouse demonstrated normal morphology at postnatal day 2, followed by drastic changes in RPE and photoreceptor morphology and loss of vision during the first postnatal month. To investigate whether the RPE or the photoreceptors are the primary cause of the degeneration, we injected lentiviruses carrying HA-tagged CLC-2 with an RPE-specific promotor in the subretinal space of CLC-2-KO mice at the time of eye opening. As expected, CLC-2-HA was expressed exclusively in RPE; strikingly, this procedure rescued the degeneration of both RPE and photoreceptors. Light response in transduced eyes was also recovered. Only a fraction of RPE was transduced with the lentivirus; however, the entire RPE monolayer appears healthy, even the RPE cells not expressing the CLC-2-HA. Surprisingly, in contrast with previous physiological observations that postulate that CLC-2 has a basolateral localization in RPE, our immunofluorescence experiments demonstrated CLC-2 has an apical distribution, facing the subretinal space and the photoreceptor outer segments. Our findings suggest that CLC-2 does not play the postulated role in fluid transport at the basolateral membrane. Rather, they suggest that CLC-2 performs a critical homeostatic role in the subretinal compartment involving a chloride regulatory mechanism that is critical for the survival of both RPE and photoreceptors.

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

Diffuse or hitch a ride: how photoreceptor lipidated proteins get from here to there.

Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment (OS) where vision begins, an inner segment (IS) where protein synthesis occurs and a synaptic terminal for signal transmission to second-order neurons. The OS is a large, modified primary cilium attached to the IS by a slender connecting cilium (CC), the equivalent of the transition zone (TZ). Daily renewal of ~10% of the OS requires massive protein biosynthesis in the IS with reliable transport and targeting pathways. Transport of lipidated ('sticky') proteins depends on solubilization factors, phosphodiesterase δ (PDEδ) and uncoordinated protein-119 (UNC119), and the cargo dispensation factor (CDF), Arf-like protein 3-guanosine triphosphate (ARL3-GTP). As PDE6 and transducin still reside prominently in the OS of PDEδ and UNC119 germline knockout mice, respectively, we propose the existence of an alternate trafficking pathway, whereby lipidated proteins migrate in rhodopsin-containing vesicles of the secretory pathway.

Expansion of Bacteriophages Is Linked to Aggravated Intestinal Inflammation and Colitis.

Bacteriophages are the most abundant members of the microbiota and have the potential to shape gut bacterial communities. Changes to bacteriophage composition are associated with disease, but how phages impact mammalian health remains unclear. We noted an induction of host immunity when experimentally treating bacterially driven cancer, leading us to test whether bacteriophages alter immune responses. Treating germ-free mice with bacteriophages leads to immune cell expansion in the gut. Lactobacillus, Escherichia, and Bacteroides bacteriophages and phage DNA stimulated IFN-γ via the nucleotide-sensing receptor TLR9. The resultant immune responses were both phage and bacteria specific. Additionally, increasing bacteriophage levels exacerbated colitis via TLR9 and IFN-γ. Similarly, ulcerative colitis (UC) patients responsive to fecal microbiota transplantation (FMT) have reduced phages compared to non-responders, and mucosal IFN-γ positively correlates with bacteriophage levels. Bacteriophages from active UC patients induced more IFN-γ compared to healthy individuals. Collectively, these results indicate that bacteriophages can alter mucosal immunity to impact mammalian health.

Insights into photoreceptor ciliogenesis revealed by animal models.

Photoreceptors are polarized neurons, with very specific subcellular compartmentalization and unique requirements for protein expression and trafficking. Each photoreceptor contains an outer segment, the site of photon capture that initiates vision, an inner segment that houses the biosynthetic machinery and a synaptic terminal for signal transmission to downstream neurons. Outer segments and inner segments are connected by a connecting cilium (CC), the equivalent of a transition zone (TZ) of primary cilia. The connecting cilium is part of the basal body/axoneme backbone that stabilizes the outer segment. This report will update the reader on late developments in photoreceptor ciliogenesis and transition zone formation, specifically in mouse photoreceptors, focusing on early events in photoreceptor ciliogenesis. The connecting cilium, an elongated and narrow structure through which all outer segment proteins and membrane components must traffic, functions as a gate that controls access to the outer segment. Here we will review genes and their protein products essential for basal body maturation and for CC/TZ genesis, sorted by phenotype. Emphasis is given to naturally occurring mouse mutants and gene knockouts that interfere with CC/TZ formation and ciliogenesis.

Binary Function of ARL3-GTP Revealed by Gene Knockouts.

UNC119 and PDEδ are lipid-binding proteins and are thought to form diffusible complexes with transducin-α and prenylated OS proteins, respectively, to mediate their trafficking to photoreceptor outer segments. Here, we investigate mechanisms of trafficking which are controlled by Arf-like protein 3 (Arl3), a small GTPase. The activity of ARL3 is regulated by a GEF (ARL13b) and a GAP (RP2). In a mouse germline knockout of RP2, ARL3-GTP is abundant as its intrinsic GTPase activity is extremely low. High levels of ARL3-GTP impair binding and trafficking of cargo to the outer segment. Germline knockout of ARL3 is embryonically lethal generating a syndromic ciliopathy-like phenotype. Retina- and rod-specific knockout of ARL3 allow to determine the precise mechanisms leading to photoreceptor degeneration. The knockouts reveal binary functions of ARL3-GTP as a key molecule in late-stage photoreceptor ciliogenesis and cargo displacement factor.

Rescue of cone function in cone-only Nphp5 knockout mouse model with Leber congenital amaurosis phenotype.

Purpose: Recessive mutations in the human IQCB1/NPHP5 gene are associated with Senior-Løken syndrome (SLS), a ciliopathy presenting with nephronophthisis and Leber congenital amaurosis (LCA). Nphp5-knockout mice develop LCA without nephronophthisis. Mutant rods rapidly degenerate while mutant cones survive for months. The purpose of this study was to reinitiate cone ciliogenesis in a Nphp5 -/-; Nrl -/- mouse with viral expression of full-length NPHP5 and rescue function. Methods: Nphp5 -/- mice were mated with Nrl -/- mice to generate Nphp5-/-; Nrl-/- double-knockouts. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were phenotyped with confocal microscopy from postnatal day 10 (P10) until 6 months of age. Nphp5-/-; Nrl-/- mice and Nphp5+/-; Nrl-/- controls were injected at P15 with self-complementary adenoassociated virus 8 (Y733F) (AAV8(Y733F)) expressing GRK1-FL-cNPHP5. Expression of mutant NPHP5 was verified with confocal microscopy and electroretinography (ERG). Results: In the Nphp5 -/- and cone-only Nphp5 -/-; Nrl -/- mice, cone outer segments did not form, but mutant cones continued to express cone pigments in the inner segments without obvious signs of cone cell death. The mutant cone outer nuclear layer (ONL) and the inner segments were stable for more than 6 months in the cone-only Nphp5 -/-; Nrl -/- retinas. Viral expression of NPHP5 initiated after eye opening showed that connecting cilia and RP1-positive axonemes were formed. Furthermore, cone pigments and other cone outer segment proteins (cone transducin and cone PDE6) were present in the nascent mutant cone outer segments, and rescued mutant cones exhibited a significant photopic b-wave (30% of Nphp5 +/-; Nrl -/- controls). Conclusions: Nphp5-/-; Nrl-/- cones persistently express cone pigments in the inner segments without obvious degeneration, providing an extended duration interval for viral gene expression. Viral expression of full-length NPHP5 initiates ciliogenesis between P15 and P60, and mutant cones are, in part, functional, encouraging future retina gene replacement therapy.

The guanine nucleotide exchange factor Arf-like protein 13b is essential for assembly of the mouse photoreceptor transition zone and outer segment.

Arf-like protein 13b (ARL13b) is a small GTPase that functions as a guanosine nucleotide exchange factor (GEF) for ARL3-GDP. ARL13b is located exclusively in photoreceptor outer segments (OS) presumably anchored to discs by palmitoylation, whereas ARL3 is an inner segment cytoplasmic protein. Hypomorphic mutations affecting the ARL13b G-domain inactivate GEF activity and lead to Joubert syndrome (JS) in humans. However, the molecular mechanisms in ARL13b mutation-induced Joubert syndrome, particularly the function of primary cilia, are still incompletely understood. Because Arl13b germline knockouts in mouse are lethal, we generated retina-specific deletions of ARL13b in which ARL3-GTP formation is impaired. In mouse retArl13b-/- central retina at postnatal day 6 (P6) and older, outer segments were absent, thereby preventing trafficking of outer segment proteins to their destination. Ultrastructure of postnatal day 10 (P10) central retArl13b-/- photoreceptors revealed docking of basal bodies to cell membranes, but mature transition zones and disc structures were absent. Deletion of ARL13b in adult mice via tamoxifen-induced Cre/loxP recombination indicated that axonemes gradually shorten and outer segments progressively degenerate. IFT88, essential for anterograde intraflagellar transport (IFT), was significantly reduced at tamArl13b-/- basal bodies, suggesting impairment of intraflagellar transport. AAV2/8 vector-mediated ARL13b expression in the retArl13b-/- retina rescued ciliogenesis.

Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation.

Null mutations in the human IQCB1/NPHP5 (nephrocystin-5) gene that encodes NPHP5 are the most frequent cause of Senior-Løken syndrome, a ciliopathy that is characterized by Leber congenital amaurosis and nephronophthisis. We generated germline Nphp5-knockout mice by placing a ß-Geo gene trap in intron 4, thereby truncating NPHP5 at Leu87 and removing all known functional domains. At eye opening, Nphp5-/- mice exhibited absence of scotopic and photopic electroretinogram responses, a phenotype that resembles Leber congenital amaurosis. Outer segment transmembrane protein accumulation in Nphp5-/- endoplasmic reticulum was evident as early as postnatal day (P)6. EGFP-CETN2, a centrosome and transition zone marker, identified basal bodies in Nphp5-/- photoreceptors, but without fully developed transition zones. Ultrastructure of P6 and 10 Nphp5-/- photoreceptors revealed aberrant transition zones of reduced diameter. Nphp5-/- photoreceptor degeneration was complete at 1 mo of age but was delayed significantly in Nphp5-/-;Nrl-/- (cone only) retina. Nphp5-/- mouse embryonic fibroblast developed normal cilia, and Nphp5-/- kidney histology at 1 yr of age showed no significant pathology. Results establish that nephrocystin-5 is essential for photoreceptor outer segment formation but is dispensable for kidney and mouse embryonic fibroblast ciliary formation.-Ronquillo, C. C., Hanke-Gogokhia, C., Revelo, M. P., Frederick, J. M., Jiang, L., Baehr, W. Ciliopathy-associated IQCB1/NPHP5 protein is required for mouse photoreceptor outer segment formation.

Arf-like Protein 3 (ARL3) Regulates Protein Trafficking and Ciliogenesis in Mouse Photoreceptors.

Arf-like protein 3 (ARL3) is a ubiquitous small GTPase expressed in ciliated cells of plants and animals. Germline deletion ofArl3in mice causes multiorgan ciliopathy reminiscent of Bardet-Biedl or Joubert syndromes. As photoreceptors are elegantly compartmentalized and have cilia, we probed the function of ARL3 (ADP-ribosylation factor (Arf)-like 3 protein) by generating rod photoreceptor-specific (prefix(rod)) and retina-specific (prefix(ret))Arl3deletions. In predegenerate(rod)Arl3(-/-)mice, lipidated phototransduction proteins showed trafficking deficiencies, consistent with the role of ARL3 as a cargo displacement factor for lipid-binding proteins. By contrast,(ret)Arl3(-/-)rods and cones expressing Cre recombinase during embryonic development formed neither connecting cilia nor outer segments and degenerated rapidly. Absence of cilia infers participation of ARL3 in ciliogenesis and axoneme formation. Ciliogenesis was rescued, and degeneration was reversed in part by subretinal injection of adeno-associated virus particles expressing ARL3-EGFP. The conditional knock-out phenotypes permitted identification of two ARL3 functions, both in the GTP-bound form as follows: one as a regulator of intraflagellar transport participating in photoreceptor ciliogenesis and the other as a cargo displacement factor transporting lipidated protein to the outer segment. Surprisingly, a farnesylated inositol polyphosphate phosphatase only trafficked from the endoplasmic reticulum to the Golgi, thereby excluding it from a role in photoreceptor cilia physiology.

The Function of Arf-like Proteins ARL2 and ARL3 in Photoreceptors.

Arf-like proteins (ARLs) are ubiquitously expressed small G proteins of the RAS superfamily. In photoreceptors, ARL2 and ARL3 participate in the trafficking of lipidated membrane-associated proteins and colocalize in the inner segment with UNC119A and PDEδ. UNC119A and PDEδ are acyl- and prenyl-binding proteins, respectively, involved in trafficking of acylated (transducin-α subunit, nephrocystin NPHP3) and prenylated proteins (GRK1, PDE6). Germline Arl3 knockout mice do not survive beyond postnatal day 21 and display ciliary defects in multiple organs (kidney, liver and pancreas) as well as retinal degeneration. Conditional knockouts will be necessary to delineate mechanisms of protein transport in retina disease.

Mistrafficking of prenylated proteins causes retinitis pigmentosa 2.

The retinitis pigmentosa 2 polypeptide (RP2) functions as a GTPase-activating protein (GAP) for ARL3 (Arf-like protein 3), a small GTPase. ARL3 is an effector of phosphodiesterase 6 Δ (PDE6D), a prenyl-binding protein and chaperone of prenylated protein in photoreceptors. Mutations in the human RP2 gene cause X-linked retinitis pigmentosa (XLRP) and cone-rod dystrophy (XL-CORD). To study mechanisms causing XLRP, we generated an RP2 knockout mouse. The Rp2h(-/-) mice exhibited a slowly progressing rod-cone dystrophy simulating the human disease. Rp2h(-/-) scotopic a-wave and photopic b-wave amplitudes declined at 1 mo of age and continued to decline over the next 6 mo. Prenylated PDE6 subunits and G-protein coupled receptor kinase 1 (GRK1) were unable to traffic effectively to the Rp2h(-/-) outer segments. Mechanistically, absence of RP2 GAP activity increases ARL3-GTP levels, forcing PDE6D to assume a predominantly "closed" conformation that impedes binding of lipids. Lack of interaction disrupts trafficking of PDE6 and GRK1 to their destination, the photoreceptor outer segments. We propose that hyperactivity of ARL3-GTP in RP2 knockout mice and human patients with RP2 null alleles leads to XLRP resembling recessive rod-cone dystrophy.