CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Perivascular neurons instruct 3D vascular lattice formation via neurovascular contact.

The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.

Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice.

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.

A Fine-Scale Functional Logic to Convergence from Retina to Thalamus.

Numerous well-defined classes of retinal ganglion cells innervate the thalamus to guide image-forming vision, yet the rules governing their convergence and divergence remain unknown. Using two-photon calcium imaging in awake mouse thalamus, we observed a functional arrangement of retinal ganglion cell axonal boutons in which coarse-scale retinotopic ordering gives way to fine-scale organization based on shared preferences for other visual features. Specifically, at the ∼6 µm scale, clusters of boutons from different axons often showed similar preferences for either one or multiple features, including axis and direction of motion, spatial frequency, and changes in luminance. Conversely, individual axons could "de-multiplex" information channels by participating in multiple, functionally distinct bouton clusters. Finally, ultrastructural analyses demonstrated that retinal axonal boutons in a local cluster often target the same dendritic domain. These data suggest that functionally specific convergence and divergence of retinal axons may impart diverse, robust, and often novel feature selectivity to visual thalamus.

Cyclic-Nucleotide- and HCN-Channel-Mediated Phototransduction in Intrinsically Photosensitive Retinal Ganglion Cells.

Non-image-forming vision in mammals is mediated primarily by melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, by far the best-studied subtype, melanopsin activates PLCß4 (phospholipase C-ß4) to open TRPC6,7 channels, mechanistically similar to phototransduction in fly rhabdomeric (microvillous) photoreceptors. We report here that, surprisingly, mouse M4-ipRGCs rely on a different and hitherto undescribed melanopsin-driven, ciliary phototransduction mechanism involving cyclic nucleotide as the second messenger and HCN channels rather than CNG channels as the ion channel for phototransduction. Even more surprisingly, within an individual mouse M2-ipRGC, this HCN-channel-dependent, ciliary phototransduction pathway operates in parallel with the TRPC6,7-dependent rhabdomeric pathway. These findings reveal a complex heterogeneity in phototransduction among ipRGCs and, more importantly, break a general dogma about segregation of the two phototransduction motifs, likely with strong evolutionary implications.

A Population Representation of Absolute Light Intensity in the Mammalian Retina.

Environmental illumination spans many log units of intensity and is tracked for essential functions that include regulation of the circadian clock, arousal state, and hormone levels. Little is known about the neural representation of light intensity and how it covers the necessary range. This question became accessible with the discovery of mammalian photoreceptors that are required for intensity-driven functions, the M1 ipRGCs. The spike outputs of M1s are thought to uniformly track intensity over a wide range. We provide a different understanding: individual cells operate over a narrow range, but the population covers irradiances from moonlight to full daylight. The range of most M1s is limited by depolarization block, which is generally considered pathological but is produced intrinsically by these cells. The dynamics of block allow the population to code stimulus intensity with flexibility and efficiency. Moreover, although spikes are distorted by block, they are regularized during axonal propagation.

Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity.

Mutations in SAMD7 cause autosomal-recessive macular dystrophy with or without cone dysfunction.

Sterile alpha motif domain containing 7 (SAMD7) is a component of the Polycomb repressive complex 1, which inhibits transcription of many genes, including those activated by the transcription factor Cone-Rod Homeobox (CRX). Here we report bi-allelic mutations in SAMD7 as a cause of autosomal-recessive macular dystrophy with or without cone dysfunction. Four of these mutations affect splicing, while another mutation is a missense variant that alters the repressive effect of SAMD7 on CRX-dependent promoter activity, as shown by in vitro assays. Immunostaining of human retinal sections revealed that SAMD7 is localized in the nuclei of both rods and cones, as well as in those of cells belonging to the inner nuclear layer. These results place SAMD7 as a gene crucial for human retinal function and demonstrate a significant difference in the role of SAMD7 between the human and the mouse retina.

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

Endothelial HIF2α suppresses retinal angiogenesis in neonatal mice by upregulating NOTCH signaling.

Prolyl hydroxylase domain (PHD) proteins are oxygen sensors that use intracellular oxygen as a substrate to hydroxylate hypoxia-inducible factor (HIF) α proteins, routing them for polyubiquitylation and proteasomal degradation. Typically, HIFα accumulation in hypoxic or PHD-deficient tissues leads to upregulated angiogenesis. Here, we report unexpected retinal phenotypes associated with endothelial cell (EC)-specific gene targeting of Phd2 (Egln1) and Hif2alpha (Epas1). EC-specific Phd2 disruption suppressed retinal angiogenesis, despite HIFα accumulation and VEGFA upregulation. Suppressed retinal angiogenesis was observed both in development and in the oxygen-induced retinopathy (OIR) model. On the other hand, EC-specific deletion of Hif1alpha (Hif1a), Hif2alpha, or both did not affect retinal vascular morphogenesis. Strikingly, retinal angiogenesis appeared normal in mice double-deficient for endothelial PHD2 and HIF2α. In PHD2-deficient retinal vasculature, delta-like 4 (DLL4, a NOTCH ligand) and HEY2 (a NOTCH target) were upregulated by HIF2α-dependent mechanisms. Inhibition of NOTCH signaling by a chemical inhibitor or DLL4 antibody partially rescued retinal angiogenesis. Taken together, our data demonstrate that HIF2α accumulation in retinal ECs inhibits rather than stimulates retinal angiogenesis, in part by upregulating DLL4 expression and NOTCH signaling.

Awakening adult neural stem cells: NOX signalling as a positive regulator of the quiescence-to-proliferation transition in the Xenopus retina.

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring.

Local translation is rapidly regulated by extrinsic signals during neural wiring, but its control mechanisms remain elusive. Here we show that the extracellular cue Sema3A induces an initial burst in local translation that precisely controls phosphorylation of the translation initiation factor eIF2α via the unfolded protein response (UPR) kinase PERK. Strikingly, in contrast to canonical UPR signaling, Sema3A-induced eIF2α phosphorylation bypasses global translational repression and underlies an increase in local translation through differential activity of eIF2B mediated by protein phosphatase 1. Ultrasensitive proteomics analysis of axons reveals 75 proteins translationally controlled via the Sema3A-p-eIF2α pathway. These include proteostasis- and actin cytoskeleton-related proteins but not canonical stress markers. Finally, we show that PERK signaling is needed for directional axon migration and visual pathway development in vivo. Thus, our findings reveal a noncanonical eIF2 signaling pathway that controls selective changes in axon translation and is required for neural wiring.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Alternative oxidase blunts pseudohypoxia and photoreceptor degeneration due to RPE mitochondrial dysfunction.

Loss of mitochondrial electron transport complex (ETC) function in the retinal pigment epithelium (RPE) in vivo results in RPE dedifferentiation and progressive photoreceptor degeneration, and has been implicated in the pathogenesis of age-related macular degeneration. Xenogenic expression of alternative oxidases in mammalian cells and tissues mitigates phenotypes arising from some mitochondrial electron transport defects, but can exacerbate others. We expressed an alternative oxidase from Ciona intestinalis (AOX) in ETC-deficient murine RPE in vivo to assess the retinal consequences of stimulating coenzyme Q oxidation and respiration without ATP generation. RPE-restricted expression of AOX in this context is surprisingly beneficial. This focused intervention mitigates RPE mTORC1 activation, dedifferentiation, hypertrophy, stress marker expression, pseudohypoxia, and aerobic glycolysis. These RPE cell autonomous changes are accompanied by increased glucose delivery to photoreceptors with attendant improvements in photoreceptor structure and function. RPE-restricted AOX expression normalizes accumulated levels of succinate and 2-hydroxyglutarate in ETC-deficient RPE, and counteracts deficiencies in numerous neural retinal metabolites. These features can be attributed to the activation of mitochondrial inner membrane flavoproteins such as succinate dehydrogenase and proline dehydrogenase, and alleviation of inhibition of 2-oxyglutarate-dependent dioxygenases such as prolyl hydroxylases and epigenetic modifiers. Our work underscores the importance to outer retinal health of coenzyme Q oxidation in the RPE and identifies a metabolic network critical for photoreceptor survival in the context of RPE mitochondrial dysfunction.

Ultrafast terahertz Stark spectroscopy reveals the excited-state dipole moments of retinal in bacteriorhodopsin.

The photoinduced all-trans to 13-cis isomerization of the retinal Schiff base represents the ultrafast first step in the reaction cycle of bacteriorhodopsin (BR). Extensive experimental and theoretical work has addressed excited-state dynamics and isomerization via a conical intersection with the ground state. In conflicting molecular pictures, the excited state potential energy surface has been modeled as a pure S[Formula: see text] state that intersects with the ground state, or in a 3-state picture involving the S[Formula: see text] and S[Formula: see text] states. Here, the photoexcited system passes two crossing regions to return to the ground state. The electric dipole moment of the Schiff base in the S[Formula: see text] and S[Formula: see text] state differs strongly and, thus, its measurement allows for assessing the character of the excited-state potential. We apply the method of ultrafast terahertz (THz) Stark spectroscopy to measure electric dipole changes of wild-type BR and a BR D85T mutant upon electronic excitation. A fully reversible transient broadening and spectral shift of electronic absorption is induced by a picosecond THz field of several megavolts/cm and mapped by a 120-fs optical probe pulse. For both BR variants, we derive a moderate electric dipole change of 5 [Formula: see text] 1 Debye, which is markedly smaller than predicted for a neat S[Formula: see text]-character of the excited state. In contrast, S[Formula: see text]-admixture and temporal averaging of excited-state dynamics over the probe pulse duration gives a dipole change in line with experiment. Our results support a picture of electronic and nuclear dynamics governed by the interaction of S[Formula: see text] and S[Formula: see text] states in a 3-state model.

Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvß5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.

Excessive tubulin glutamylation leads to progressive cone-rod dystrophy and loss of outer segment integrity.

Mutations in Cytosolic Carboxypeptidase-like Protein 5 (CCP5) are associated with vision loss in humans. To decipher the mechanisms behind CCP5-associated blindness, we generated a novel mouse model lacking CCP5. In this model, we found that increased tubulin glutamylation led to progressive cone-rod dystrophy, with cones showing a more pronounced and earlier functional loss than rod photoreceptors. The observed functional reduction was not due to cell death, levels, or the mislocalization of major phototransduction proteins. Instead, the increased tubulin glutamylation caused shortened photoreceptor axonemes and the formation of numerous abnormal membranous whorls that disrupted the integrity of photoreceptor outer segments (OS). Ultimately, excessive tubulin glutamylation led to the progressive loss of photoreceptors, affecting cones more severely than rods. Our results highlight the importance of maintaining tubulin glutamylation for normal photoreceptor function. Furthermore, we demonstrate that murine cone photoreceptors are more sensitive to disrupted tubulin glutamylation levels than rods, suggesting an essential role for axoneme in the structural integrity of the cone outer segment. This study provides valuable insights into the mechanisms of photoreceptor diseases linked to excessive tubulin glutamylation.