Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.

The WAVE complex drives the morphogenesis of the photoreceptor outer segment cilium.

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

Absolute Quantification of Photoreceptor Outer Segment Proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of 20 key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio among all 20 proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

TMEM67, TMEM237, and Embigin in Complex With Monocarboxylate Transporter MCT1 Are Unique Components of the Photoreceptor Outer Segment Plasma Membrane.

The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment.

The Role of Peripherin-2/ROM1 Complexes in Photoreceptor Outer Segment Disc Morphogenesis.

The light-sensitive outer segment organelle of photoreceptor cells contains a stack of hundreds of flat, disc-shaped membranes called discs. The rims of these discs contain a photoreceptor-specific tetraspanin protein peripherin-2 (also known as rds or PRPH2). Mutations in the PRPH2 gene lead to a wide variety of inherited retinal degenerations in humans. The vast majority of these mutations occur within a large, intradiscal loop of peripherin-2, known as the D2 loop. The D2 loop mediates well-established intermolecular interactions of peripherin-2 molecules among themselves and a homologous protein ROM1. These interactions lead to the formation of large, highly ordered oligomers. In this chapter, we discuss the supramolecular organization of peripherin-2/ROM1 complexes and their contribution to the process of outer segment disc morphogenesis and enclosure.

Photoreceptor Disc Enclosure Is Tightly Controlled by Peripherin-2 Oligomerization.

Mutations in the PRPH2 gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened "disc" membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous "whorls" instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENT The light-sensitive photoreceptor outer segment contains a stack of flattened "disc" membranes that are surrounded, or "enclosed," by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism.

PRCD is essential for high-fidelity photoreceptor disc formation.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

Highly photostable fluorescent labeling of proteins in live cells using exchangeable coiled coils heterodimerization.

Fluorescent proteins are commonly used to label target proteins in live cells. However, the conventional approach based on covalent fusion of targeted proteins with fluorescent protein probes is limited by the slow rate of fluorophore maturation and irretrievable loss of fluorescence due to photobleaching. Here, we report a genetically encoded protein labeling system utilizing transient interactions of small, 21-28 residues-long helical protein tags (K/E coils, KEC). In this system, a protein of interest, covalently tagged with a single coil, is visualized through binding to a cytoplasmic fluorescent protein carrying a complementary coil. The reversible heterodimerization of KECs, whose affinity can be tuned in a broad concentration range from nanomolar to micromolar, allows continuous exchange and replenishment of the tag bound to a targeted protein with the entire cytosolic pool of soluble fluorescent coils. We found that, under conditions of partial illumination of living cells, the photostability of labeling with KECs exceeds that of covalently fused fluorescent probes by approximately one order of magnitude. Similarly, single-molecule localization microscopy with KECs provided higher labeling density and allowed a much longer duration of imaging than with conventional fusion to fluorescent proteins. We also demonstrated that this method is well suited for imaging newly synthesized proteins, because the labeling efficiency by KECs is not dependent on the rate of fluorescent protein maturation. In conclusion, KECs can be used to visualize various target proteins which are directly exposed to the cytosol, thereby enabling their advanced characterization in time and space.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood-retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENT Phospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood-retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

Cos2/Kif7 and Osm-3/Kif17 regulate onset of outer segment development in zebrafish photoreceptors through distinct mechanisms.

Zebrafish morphants of osm-3/kif17, a kinesin-2 family member and intraflagellar transport motor, have photoreceptor outer segments that are dramatically reduced in number and size. However, two genetic mutant lines, osm-3/kif17sa0119 and osm-3/kif17sa18340, reportedly lack any observable morphological outer segment defects. In this work, we use TALENs to generate an independent allele, osm-3/kif17mw405, and show that both osm-3/kif17sa0119 and osm-3/kif17mw405 have an outer segment developmental delay in both size and density that is fully recovered by 6 days post-fertilization. Additionally, we use CRISPRs to generate cos2/kif7mw406, a mutation in the kinesin-4 family member cos2/kif7 that has been implicated in controlling ciliary architecture and Hedgehog signaling to test whether it may be functioning redundantly with osm-3/kif17. We show that cos2/kif7mw406 has an outer segment developmental delay similar to the osm-3/kif17 mutants. Using a three-dimensional mathematical model of outer segments, we show that while cos2/kif7mw406 and osm-3/kif17mw405 outer segments are smaller throughout the first 6 days of development, the volumetric rates of outer segment morphogenesis are not different among wild-type, cos2/kif7mw406, and osm-3/kif17mw405 after 60hpf. Instead, our model suggests that cos2/kif7mw406 and osm-3/kif17mw405 impact outer segment morphogenesis through upstream events that that are different for each motor. In the case of cos2/kif7mw406 mutants, we show that early defects in Hedgehog signaling lead to a general, non-photoreceptor-specific delay of retinal neurogenesis, which in turn causes the secondary phenotype of delayed outer segment morphogenesis. In contrast, the osm-3/kif17mw405 outer segment morphogenesis delays are linked specifically to initial disc morphogenesis of photoreceptors rather than an upstream event. Further, we show that osm-3/kif17 mutant mice also exhibit a similarly delayed outer segment development, suggesting a role for osm-3/kif17 in early outer segment development that is conserved across species. In conclusion, we show that both osm-3/kif17 and cos2/kif7 have comparable outer segment developmental delays, although through independent mechanisms.

Kif17 phosphorylation regulates photoreceptor outer segment turnover.

BACKGROUND: KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. RESULTS: Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. CONCLUSIONS: Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

Downregulation of rhodopsin is an effective therapeutic strategy in ameliorating peripherin-2-associated inherited retinal disorders.

Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Absolute quantification of photoreceptor outer segment proteins.

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or 'discs', located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called 'incisures'. The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

Photoreceptor disc incisures form as an adaptive mechanism ensuring the completion of disc enclosure.

The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure.

ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims.

Visual signal transduction takes place within a stack of flattened membranous 'discs' enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims.

Visual signal transduction takes place within a stack of flattened membranous "discs" enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species.

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.