Adam9-deficient retinal pigment epithelium pseudopods maintain photoreceptor outer segment renewal despite subretinal space expansion

Journal of Clinical Investigation · 2026-02-05 · 1 citations

Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9-knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE pseudopods extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

Molecular Components of Vesicle Cycling at the Rod Photoreceptor Ribbon Synapse

Advances in experimental medicine and biology · 2025-01-01

Addressing Challenges in Developing Treatments for Inherited Retinal Diseases: Recommendations From the Third Monaciano Symposium

Translational Vision Science & Technology · 2025-08-27 · 4 citations

Over the past decade, efforts focused on developing genetic therapies for inherited retinal diseases have advanced steadily to clinical trials and the development of a treatment, fueling optimism for the potential of precision medicines to provide safe and effective therapies for these rare conditions. Although several ongoing programs remain poised for success, numerous challenges have negatively impacted the ability to obtain regulatory approvals. The present position paper briefly summarizes recent advances and challenges in developing therapeutics for inherited retinal diseases, and presents a set of recommendations for moving the field forward. The priorities identified are discussed in terms of progress made and future needs, focusing on areas including patient support, disease mechanisms, outcome measures, and therapy approvals. A key point is the potential value of restructuring collaborative interactions into broadly resourced enterprises that are comprehensive in scope across critical areas of science, business, and medicine.

Unique ultrastructural organization of human rod photoreceptors

Communications Biology · 2025-01-16 · 6 citations

Rod and cone photoreceptor cells are specialized neurons responsible for transforming the information reaching the eyes in the form of photons into the language of neuronal activity. Rods are the most prevalent photoreceptor type, primarily responsible for light detection under conditions of limited illumination. Here we demonstrate that human rods have a morphological organization unique among all described species, whereby the cell soma extends alongside the light-sensitive outer segment compartment to form a structure we have termed the "accessory inner segment." These structures have two striking features: they are reinforced by a massive microtubular cytoskeleton and contain electron-dense adhesions that mediate their attachment to outer segments. Given that the spacing of human rod photoreceptors is sparser than in most other species, the accessory inner segment likely provides mechanical support to the closely apposed outer segment. This discovery expands our understanding of the human retina and directs future studies of human photoreceptor function in health and disease.

Neuronal compartmentalization results in “impoverished” axonal mitochondria

bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-29 · 5 citations

Abstract Mitochondria differ depending on their location within a neuron. Morphological heterogeneity between somatic, dendritic, and axonal mitochondria is well established. Emerging evidence suggests that further specialization is needed to meet the unique demands of different neuronal compartments. However, the molecular and functional diversity of mitochondria within a neuron remains poorly understood. Here, we utilized proteomics in MitoTag mice to profile somatodendritic and axonal mitochondria across four distinct neuron types, thereby generating a compendium of intracellular mitochondrial diversity. Combining proteomics, functional, and immunofluorescence analyses, we demonstrated that axonal mitochondria are not defined by the presence of unique proteins, but rather by the selective loss or preservation of specific pathways compared to their somatodendritic counterparts. This results in “impoverished” axonal mitochondria, which are characterized by diminished mtDNA expression and impaired oxidative phosphorylation yet retain other pathways, such as fatty acid metabolism. Bioinformatic analyses of multiomic data identified local translation as one mechanism underlying compartment-specific diversity. Together, these findings provide a comprehensive in vivo framework for understanding mitochondrial specialization across neuronal compartments.

Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse

Journal of Visualized Experiments · 2025-07-25

The conventional method of material delivery to the subretinal space in the mouse involves dual perforation of the neural retina, which causes extensive surgical damage. This leads to variability in the subsequent outcome measures of the visual function, such as electroretinogram (ERG) recordings or behavioral vision assays, which confound efficacy assessments of experimental therapeutics. To overcome these barriers, we optimized a transscleral minimally invasive subretinal injection technique in mice. In this technique, the superior fornix is accessed using a custom-made tungsten wire eyelid speculum to perform conjunctival peritomy and tenotomy. A pinpoint sclerotomy is made using a diamond knife without penetrating the retina, through which a fine glass needle is carefully inserted at a shallow angle. The payload is delivered to the subretinal space using a microinjection pump. Optical coherence tomography (OCT) is used to assess the size and position of the subretinal bleb immediately following injection. We compared the outcomes of this technique to the conventional transretinal method performed using a spring-loaded syringe with a 33G needle sclerotomy. ERG recordings indicated excellent preservation of retinal function in transsclerally-injected mice, comparable to that of uninjected control mice. In contrast, an appreciable ERG signal reduction was observed in the conventionally injected cohorts. In summary, we have optimized a minimally invasive technique for subretinal injection in mice. We demonstrate that this technique is a robust and efficient method for gene therapy administration that minimizes anatomic damage to the retina and has minimal impact on retinal function. This method lowers the threshold for the development of therapies targeting both the retinal pigmented epithelium and photoreceptors to treat a wide range of retinal and macular diseases.

Inpp5e is crucial for photoreceptor outer segment maintenance

Journal of Cell Science · 2025-01-28 · 6 citations

In humans, inositol polyphosphate-5-phosphatase E (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi, mislocalized rhodopsin and an accumulation of extracellular vesicles. In addition, knockout cells showed disruption of the actin network. Together, these data demonstrate that Inpp5e plays a crucial role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

RPE pseudopods maintain photoreceptor outer segment renewal despite subretinal space expansion in <i>Adam9</i> knockout mice

bioRxiv (Cold Spring Harbor Laboratory) · 2025-12-19

Abstract Vision begins in the outer segment compartment of photoreceptor cells, which is constantly renewed through the addition of membrane material at its base and ingestion of mature membranes at its tip by the retinal pigment epithelium (RPE). The close apposition of outer segments to the RPE is believed to be critical for maintaining this renewal process. Yet, in several retinal diseases, expansion of the subretinal space separating photoreceptors from the RPE does not immediately impact photoreceptor functionality. Here, we analyzed outer segment function and renewal in the Adam9 knockout mouse characterized by a major expansion of the subretinal space. Surprisingly, photoreceptor-RPE separation affected neither the sensitivity of photoreceptor light-responses nor the normal rate of outer segment renewal in this mouse prior to the onset of photoreceptor degeneration. The latter is achieved through the formation of elongated RPE “pseudopods” extending across the enlarged subretinal space to ingest outer segment tips. This work suggests that pseudopod formation may underlie the persistence of photoreceptor function in human diseases accompanied by photoreceptor-RPE separation, such as vitelliform macular dystrophy or age-related macular degeneration associated with subretinal drusenoid deposits.

The mouse neonatal small intestine is regionally specialized for protein absorption and transepithelial transport

Development · 2025-11-10 · 1 citations

In neonates, gastric protein digestion is limited, requiring specialized mechanisms for intestinal protein absorption. While neonatal enterocytes are thought to mediate endocytosis, degradation and transcytosis of dietary proteins, whether these activities are spatially segregated and their molecular basis are unknown. Here, we combine in vivo and ex vivo cargo transport assays with transcriptomic and genetic approaches in mice to uncover distinct roles for jejunal and ileal neonatal enterocytes. We show that the jejunum is highly active in transepithelial transport of intact proteins, whereas the ileum specializes in their lysosomal degradation. Although both regions express similar endocytic receptors, structural and transcriptional analyses uncover divergent endolysosomal programs. Single-cell RNA sequencing reveals that jejunal and ileal enterocytes emerge from a similar progenitor pool but diverge transcriptionally. Moreover, ileal enterocytes share features with lysosome-rich enterocytes in zebrafish, suggesting evolutionary conservation. Conditional loss of Dab2 disrupts protein, but not antibody, transcytosis, supporting distinct uptake routes for nutritional and immune cargos. These findings show regional and functional specialization of enterocytes during early postnatal development, and underscore conserved protein absorption mechanisms in vertebrates.

Peripherin-2 and ROM1 Incorporate Directly Into the Rims of Enclosing Photoreceptor Discs Without Accumulating in the Nascent Disc Lamellae

Investigative Ophthalmology & Visual Science · 2025-08-13 · 2 citations

Purpose: Oligomeric complexes of peripherin-2 and ROM1 support the rim structure of membrane discs stacked inside the light-sensitive outer segment of vertebrate photoreceptor cells. We investigated the route by which peripherin-2 and ROM1 reach their destination within the disc rims. We addressed two possible mechanisms: first, whether these proteins accumulate within the lamellae of newly forming discs prior to their enclosure, after which they incorporate into the rims, or second, whether they incorporate directly into the rims of discs undergoing the process of enclosure. Methods: Subcellular localization of endogenous and myc-tagged peripherin-2, ROM1, and the cyclic nucleotide-gated (CNG) channel in rod photoreceptors of wild type mice was analyzed by their co-immunostaining with prominin-1, a protein marker labeling the expanding edges of nascent discs. Myc-tagged peripherin-2 was introduced into rods by adeno-associated virus transduction. The preservation of outer segment ultrastructure in immunostained tissue was confirmed by electron microscopy. Results: We observed a minimal overlap between immunostaining of peripherin-2/ROM1 and prominin-1. Whereas prominin-1 was primarily detected at the base of the outer segment, where nascent discs are formed, peripherin-2 and ROM1 were observed more distally at a location where discs undergo enclosure. The CNG channel subunits were similarly not detected in the lamellae of newly forming discs but were robustly stained in the plasma membrane enclosing mature outer segments. Conclusions: Our data confirm the previously established strong affinity of peripherin-2 for highly curved disc rims and demonstrate that ROM1 exhibits a similar preference; neither protein significantly accumulates in disc lamellae prior to enclosure.