Publications

Purpose: This study aimed to investigate whether 40-Hz light flicker could modulate the expression of major histocompatibility complex class II (MHC-II) and enhance the clearance of amyloid-β (Aβ) deposition. Methods: We examined retinal MHC-II expression via RNA sequencing, immunofluorescence, and western blotting in mice 8 weeks, 9 months, and 18 to 20 months old. Retinal metabolic waste accumulation was induced by intravitreal and subretinal injections of Aβ oligomers. The impact of 40-Hz flicker on MHC-II expression, microglial activation, and retinal function was evaluated using immunofluorescence, western blotting, dot immunobinding assay, electroretinography, and optokinetic reflex (OKR) testing. Minocycline was used to inhibit microglial activity. Results: The 40-Hz light flicker upregulated MHC-II expression in the retinas of aged mice. MHC-II⁺ microglia accumulated along retinal veins and exhibited increased numbers and enlarged morphology in the subretinal space. Following intravitreal or subretinal Aβ injection, 40-Hz flicker enhanced microglial activation, further upregulated MHC-II expression, promoted Aβ clearance, and improved electroretinogram responses and OKR performance. These effects were abolished by minocycline treatment. Conclusions: We observed that 40-Hz light flicker enhances retinal microglial clearance of Aβ oligomers by upregulating MHC-II expression. These findings support 40-Hz light flicker as a non-invasive therapeutic strategy for age-related retinal disorders by promoting metabolic waste clearance.

Background: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival. Results: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia. Conclusions: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

Purpose: Age-related macular degeneration (AMD) is a major leading cause of visual impairment and blindness with no cure currently established. Cell replacement of RPE is discussed as a potential therapy for AMD. Previous studies were performed in animal models with severe limitations in recapitulating the disease progression. In detail, we describe the effect of systemic injection of sodium iodate in the mouse retina. We further evaluate the useful-ness of this animal model to analyze cell-specific effects following transplantation of human embryonic stem cell (hESC)-derived RPE cells. Methods: Morphologic, functional, and behavioral changes following sodium iodate injection were monitored by histology, gene expression analysis, electroretinography, and optokinetic head tracking. Human embryonic stem cell–derived RPE cells were transplanted 1 week after sodium iodate injection and experimental retinae were ana-lyzed 3 weeks later. Results: Injection of sodium iodate caused complete RPE cell loss, photoreceptor degeneration, and altered gene and protein expression in outer and inner nuclear layers. Retinal function was severely affected by day 3 and abol-ished from day 14. Following transplantation, donor hESC-derived RPE cells formed extensive monolayers that dis-played wild-type RPE cell morphology, organization, and function, including phagocytosis of host photoreceptor outer segments. Conclusions: Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors, and inner nu-clear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The avail-ability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polar-ized cell monolayers from donor hESC-derived RPE cell suspensions.

Dysfunctional cGMP signaling is implicated in multiple neurodegenerative diseases of the central nervous system (CNS), including glaucoma, an optic neuropathy and leading cause of irreversible blindness. Female mice lacking the alpha catalytic subunit of soluble guanylate cyclase (sGCα1^-/-), the active site that binds nitric oxide (NO) to produce cGMP, exhibit progressive retinal ganglion cell (RGC) degeneration with age. Yet, the role of sGC in age- and sex-dependent RGC function remains uncharacterized. We investigated how preventing NO binding to sGC influences RGC function in the context of aging and sex by combining bulk and single-cell RNA sequencing, Western blotting, mitochondrial ultrastructural analysis, visual acuity measurements, and in vivo measurements of retinal oxidative metabolism. We found that global sGCα1 deletion impairs visual function and RGC health in aging female mice, while male mice remained unaffected. Glucose uptake was significantly disrupted in female sGCα1^-/-retinas with age, and accompanied by reduced retinal expression of the glucose transporter, GLUT1. Aged sGCα1^-/- females also exhibited dysregulated retinal mitochondrial gene and protein expression and increased nitrosative stress localized to the RGC layer. RGC mitochondria in male mice increased in size with age, while female mitochondria did not. Furthermore, retinal metabolic analysis showed decreased oxygen consumption rate in aged female but not male sGCα1^-/-retinas, suggesting impaired oxidative metabolism. These findings reveal a potential sex-specific role for cGMP signaling in maintaining retinal metabolic integrity and RGC function with age. Our results point to a possible mechanistic link between impaired cGMP signaling and age-related retinal neurodegeneration in females, highlighting the sGC-cGMP signaling pathway as a promising therapeutic target for glaucoma and other CNS neurodegenerative diseases.

Purpose: This study aimed to investigate whether 40-Hz light flicker could modulate the expression of major histocompatibility complex class II (MHC-II) and enhance the clearance of amyloid-β (Aβ) deposition. Methods: We examined retinal MHC-II expression via RNA sequencing, immunofluorescence, and western blotting in mice 8 weeks, 9 months, and 18 to 20 months old. Retinal metabolic waste accumulation was induced by intravitreal and subretinal injections of Aβ oligomers. The impact of 40-Hz flicker on MHC-II expression, microglial activation, and retinal function was evaluated using immunofluorescence, western blotting, dot immunobinding assay, electroretinography, and optokinetic reflex (OKR) testing. Minocycline was used to inhibit microglial activity. Results: The 40-Hz light flicker upregulated MHC-II expression in the retinas of aged mice. MHC-II⁺ microglia accumulated along retinal veins and exhibited increased numbers and enlarged morphology in the subretinal space. Following intravitreal or subretinal Aβ injection, 40-Hz flicker enhanced microglial activation, further upregulated MHC-II expression, promoted Aβ clearance, and improved electroretinogram responses and OKR performance. These effects were abolished by minocycline treatment. Conclusions: We observed that 40-Hz light flicker enhances retinal microglial clearance of Aβ oligomers by upregulating MHC-II expression. These findings support 40-Hz light flicker as a non-invasive therapeutic strategy for age-related retinal disorders by promoting metabolic waste clearance.

We previously reported ocular phenotypes of 1-year-old 129S1/SvlmJ lysyl oxidase-like 1 null (Loxl1^-/-) mice. Here we sought to characterize age-dependent changes in C57BL/6J Loxl1^-/- mice in a longitudinal fashion. Retinal ganglion cell (RGC) function was assessed by electroretinography (ERG), and optic nerves were evaluated by histological analysis. Ocular biometric measurements were obtained by optical coherence tomography (OCT). We detected reduced RGC function, revealed by decreased amplitude and increased latency of ERG positive scotopic threshold responses (pSTRs) in Loxl1^-/- mice compared to age-matched wt mice. In addition, there is significant inter-eye asymmetry of RGC function, as well as age-related RGC function loss observed only in Loxl1^-/- mice. Histologically, we observed enlarged optic nerve areas in Loxl1^-/- mice compared to wt mice. Significant ocular biometric differences between two groups were detected, most notably, age-related axial elongation of the globe, accompanied by deepening of anterior chamber depth (ACD). Though eyes elongate with age in both groups, this is more pronounced in Loxl1^-/- mice, and the elongation of the globe correlated with decreased RGC function. The correlation of age-related reduction in RGC function with globe axial elongation may have implications for the association of axial myopia with glaucoma and aging in humans.

Aging is the major risk factor for neurodegeneration and is associated with structural and functional alterations in white matter. Myelin is particularly vulnerable to aging, resulting in white matter-associated microglia activation. Here we used pharmacological and genetic approaches to investigate microglial functions related to aging-associated changes in myelinated axons of mice. Our results reveal that maladaptive microglia activation promotes the accumulation of harmful CD8⁺ T cells, leading to the degeneration of myelinated axons and subsequent impairment of brain function and behavior. We characterize glial heterogeneity and aging-related changes in white matter by single-cell and spatial transcriptomics and reveal elaborate glial-immune interactions. Mechanistically, we show that the CXCL10-CXCR3 axis is crucial for the recruitment and retention of CD8⁺ T cells in aged white matter, where they exert pathogenic effects. Our results indicate that myelin-related microglia dysfunction promotes adaptive immune reactions in aging and identify putative targets to mitigate their detrimental impact.

Purpose: This study investigated the presence of beta-amyloid in the retina and optic nerve of 2-month-old mouse models of Alzheimer’s disease (AD) prior to the onset of clinical symptoms and compared the findings to those of age-matched controls. Methods: Visual acuity and contrast sensitivity were measured using an OptoDrum® in 2-month-old mouse models (5xFAD) and 2-month-old normal mice (C57BL/6). The retina and optic nerve of the experimental and control groups were stained fluorescently using an amyloid-beta antibody, and the level of amyloid-beta expression in the two groups was compared and analyzed histologically. Results: The 2-month-old mouse models (5xFAD) are at a stage prior to the characteristic spatial learning and cognitive impairments associated with AD. Visual acuity and contrast sensitivity did not differ between the experimental and control groups. Beta-amyloid was not observed in the retina or optic nerve of the control group or in the retina of the experimental group. In contrast to normal mice of the same age, beta-amyloid accumulated in the optic nerve in the experimental group, with a tendency for increased beta-amyloid accumulation in the optic nerve closer to the brain. Conclusions: In young AD mouse models prior to the onset of clinical symptoms, there were no differences in visual acuity, contrast sensitivity, or histological changes in the retina compared to controls. However, pathological changes in the accumulation of beta-amyloid in the optic nerve in the experimental group preceded other changes. These findings should aid the early diagnosis of AD in the future.

Background: Aging-induced decline in ciliary muscle function is an important factor in visual accommodative deficits in elderly adults. With this study, we provide an innovative investigation of the interaction between ciliary muscle aging and oxidative stress. Methods: Tricolor guinea pigs were used for the experiments in vivo and primary guinea pig ciliary smooth muscle cells were used for the experiments in vitro.Results: We enriched for genes associated with muscle-aging-lutein relationship using bioinformatics, including Nuclear factor-erythroid 2-related factor-2 (Nrf2), Glutathione Peroxidase (GPx) gene family, Superoxide Dismutase (SOD) gene family, NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase-1 (HO-1). After gavage to aged guinea pigs, lutein reduced Reactive Oxygen Species (ROS) and P21 levels in senescent ciliary muscle; lutein decreased refractive error and restored accommodation of the eye. In addition, lutein increased GPx, SOD, and Catalase (CAT) levels in serum; lutein increased GPx and CAT levels in ciliary bodies. Lutein regulated the expression of proteins such as Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and downstream proteins in senescent ciliary bodies. Similarly, guinea pig ciliary muscle cell senescence was associated with oxidative stress. In vitro, 100 μM lutein reversed the damage caused by 800 μM H2O2; it reduced Senescence-Associated β-galactosidase (SA-β-Gal) and ROS activites, cell apoptosis and cell migration. Also, lutein increased the expression of smooth muscle contractile proteins. Lutein also increased the expression of Nrf2, GPx2, NQO1 and HO-1, decreased the expression of Keap1. A reduction in Nrf2 activity led to a reduction in the ability of lutein to activate antioxidant enzymes in the cells, thus reducing its inhibitory effect on cell senescence. Conclusion: lutein improved resistance to oxidative stress in senescent ciliary muscle in vivo and in vitro by regulating the Keap1/Nrf2/Antioxidant Response Element pathway. We have innovatively demonstrated the molecular pharmacological mechanism by which lutein reverse age-related ciliary muscle systolic and diastolic deficits.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in non–image-forming visual functions. Given their significant loss observed in various ocular degenerative diseases at early stages, this study aimed to assess changes in both the morphology and associated behavioral functions of ipRGCs in mice between 6 (mature) and 12 (late adult) months old. The findings contribute to understanding the preservation of ipRGCs in late adults and their potential as a biomarker for early ocular degenerative diseases. Methods: Female and male C57BL/6J mice were used to assess the behavioral consequences of aging to mature and old adults, including pupillary light reflex, light aversion, visual acuity, and contrast sensitivity. Immunohistochemistry on retinal wholemounts from these mice was then conducted to evaluate ipRGC dendritic morphology in the ganglion cell layer (GCL) and inner nuclear layer (INL). Results: Morphological analysis showed that ipRGC dendritic field complexity was remarkably stable through 12 months old of age. Similarly, the pupillary light reflex, visual acuity, and contrast sensitivity were stable in mature and old adults. Although alterations were observed in ipRGC-independent light aversion distinct from the pupillary light reflex, aged wild-type mice continuously showed enhanced light aversion with dilation. No effect of sex was observed in any tests. Conclusions: The preservation of both ipRGC morphology and function highlights the potential of ipRGC-mediated function as a valuable biomarker for ocular diseases characterized by early ipRGC loss. The consistent stability of ipRGCs in mature and old adult mice suggests that detected changes in ipRGC-mediated functions could serve as early indicators or diagnostic tools for early-onset conditions such as Alzheimer's disease, Parkinson's disease, and diabetes, where ipRGC loss has been documented.

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.

Background: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival. Results: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia. Conclusions: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

Aging is a major risk factor for the development of nervous system functional decline, even in the absence of diseases or trauma. The axon–myelin units and synaptic terminals are some of the neural structures most vulnerable to aging-related deterioration, but the underlying mechanisms are poorly understood. In the peripheral nervous system, macrophages—important representatives of the innate immune system—are prominent drivers of structural and functional decline of myelinated fibers and motor endplates during aging. Similarly, in the aging central nervous system (CNS), microglial cells promote damage of myelinated axons and synapses. Here we examine the role of cytotoxic CD8+ T lymphocytes, a type of adaptive immune cells previously identified as amplifiers of axonal perturbation in various models of genetically mediated CNS diseases but understudied in the aging CNS. We show that accumulation of CD8+ T cells drives axon degeneration in the normal aging mouse CNS and contributes to age-related cognitive and motor decline. We characterize CD8+ T-cell population heterogeneity in the adult and aged mouse brain by single-cell transcriptomics and identify aging-related changes. Mechanistically, we provide evidence that CD8+ T cells drive axon degeneration in a T-cell receptor- and granzyme B-dependent manner. Cytotoxic neural damage is further aggravated by systemic inflammation in aged but not adult mice. We also find increased densities of T cells in white matter autopsy material from older humans. Our results suggest that targeting CD8+ CNS-associated T cells in older adults might mitigate aging-related decline of brain structure and function.

Tau is a microtubule-associated protein involved in Alzheimer's disease. However, little is known on its physiological function in the healthy central nervous system. Here, we observed that the expression of Tau isoforms was modulated by neuronal maturation and visual experience in the mouse retina and in the visual cortex. The visual function of wild-type (WT) and Tau knockout (KO) mice was evaluated using the optokinetic reflex (OKR), an innate visuomotor behavior, and by electroretinography. Visual tests did not reveal functional impairments in young adult and old Tau KO animals. Moreover, monocular deprivation (MD) was used to increase OKR sensitivity, a plasticity phenomenon depending on the visual cortex. MD-induced OKR sensitivity enhancement was significantly stronger in Tau KO than in WT mice suggesting that Tau restricts visual plasticity. In addition, human Tau expression did not affect visual function and plasticity in a mouse tauopathy model, relative to WT controls. Our results unveil a novel function for Tau in the adaptive mechanisms of plasticity operating in the adult brain subjected to sensory experience changes.

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Objective: The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. Methods: We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. Results: We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. Conclusions: We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

Purpose: This study aimed to investigate whether 40-Hz light flicker could modulate the expression of major histocompatibility complex class II (MHC-II) and enhance the clearance of amyloid-β (Aβ) deposition. Methods: We examined retinal MHC-II expression via RNA sequencing, immunofluorescence, and western blotting in mice 8 weeks, 9 months, and 18 to 20 months old. Retinal metabolic waste accumulation was induced by intravitreal and subretinal injections of Aβ oligomers. The impact of 40-Hz flicker on MHC-II expression, microglial activation, and retinal function was evaluated using immunofluorescence, western blotting, dot immunobinding assay, electroretinography, and optokinetic reflex (OKR) testing. Minocycline was used to inhibit microglial activity. Results: The 40-Hz light flicker upregulated MHC-II expression in the retinas of aged mice. MHC-II⁺ microglia accumulated along retinal veins and exhibited increased numbers and enlarged morphology in the subretinal space. Following intravitreal or subretinal Aβ injection, 40-Hz flicker enhanced microglial activation, further upregulated MHC-II expression, promoted Aβ clearance, and improved electroretinogram responses and OKR performance. These effects were abolished by minocycline treatment. Conclusions: We observed that 40-Hz light flicker enhances retinal microglial clearance of Aβ oligomers by upregulating MHC-II expression. These findings support 40-Hz light flicker as a non-invasive therapeutic strategy for age-related retinal disorders by promoting metabolic waste clearance.

Purpose: This study investigated the presence of beta-amyloid in the retina and optic nerve of 2-month-old mouse models of Alzheimer’s disease (AD) prior to the onset of clinical symptoms and compared the findings to those of age-matched controls. Methods: Visual acuity and contrast sensitivity were measured using an OptoDrum® in 2-month-old mouse models (5xFAD) and 2-month-old normal mice (C57BL/6). The retina and optic nerve of the experimental and control groups were stained fluorescently using an amyloid-beta antibody, and the level of amyloid-beta expression in the two groups was compared and analyzed histologically. Results: The 2-month-old mouse models (5xFAD) are at a stage prior to the characteristic spatial learning and cognitive impairments associated with AD. Visual acuity and contrast sensitivity did not differ between the experimental and control groups. Beta-amyloid was not observed in the retina or optic nerve of the control group or in the retina of the experimental group. In contrast to normal mice of the same age, beta-amyloid accumulated in the optic nerve in the experimental group, with a tendency for increased beta-amyloid accumulation in the optic nerve closer to the brain. Conclusions: In young AD mouse models prior to the onset of clinical symptoms, there were no differences in visual acuity, contrast sensitivity, or histological changes in the retina compared to controls. However, pathological changes in the accumulation of beta-amyloid in the optic nerve in the experimental group preceded other changes. These findings should aid the early diagnosis of AD in the future.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in non–image-forming visual functions. Given their significant loss observed in various ocular degenerative diseases at early stages, this study aimed to assess changes in both the morphology and associated behavioral functions of ipRGCs in mice between 6 (mature) and 12 (late adult) months old. The findings contribute to understanding the preservation of ipRGCs in late adults and their potential as a biomarker for early ocular degenerative diseases. Methods: Female and male C57BL/6J mice were used to assess the behavioral consequences of aging to mature and old adults, including pupillary light reflex, light aversion, visual acuity, and contrast sensitivity. Immunohistochemistry on retinal wholemounts from these mice was then conducted to evaluate ipRGC dendritic morphology in the ganglion cell layer (GCL) and inner nuclear layer (INL). Results: Morphological analysis showed that ipRGC dendritic field complexity was remarkably stable through 12 months old of age. Similarly, the pupillary light reflex, visual acuity, and contrast sensitivity were stable in mature and old adults. Although alterations were observed in ipRGC-independent light aversion distinct from the pupillary light reflex, aged wild-type mice continuously showed enhanced light aversion with dilation. No effect of sex was observed in any tests. Conclusions: The preservation of both ipRGC morphology and function highlights the potential of ipRGC-mediated function as a valuable biomarker for ocular diseases characterized by early ipRGC loss. The consistent stability of ipRGCs in mature and old adult mice suggests that detected changes in ipRGC-mediated functions could serve as early indicators or diagnostic tools for early-onset conditions such as Alzheimer's disease, Parkinson's disease, and diabetes, where ipRGC loss has been documented.

Tau is a microtubule-associated protein involved in Alzheimer's disease. However, little is known on its physiological function in the healthy central nervous system. Here, we observed that the expression of Tau isoforms was modulated by neuronal maturation and visual experience in the mouse retina and in the visual cortex. The visual function of wild-type (WT) and Tau knockout (KO) mice was evaluated using the optokinetic reflex (OKR), an innate visuomotor behavior, and by electroretinography. Visual tests did not reveal functional impairments in young adult and old Tau KO animals. Moreover, monocular deprivation (MD) was used to increase OKR sensitivity, a plasticity phenomenon depending on the visual cortex. MD-induced OKR sensitivity enhancement was significantly stronger in Tau KO than in WT mice suggesting that Tau restricts visual plasticity. In addition, human Tau expression did not affect visual function and plasticity in a mouse tauopathy model, relative to WT controls. Our results unveil a novel function for Tau in the adaptive mechanisms of plasticity operating in the adult brain subjected to sensory experience changes.

Aging is the major risk factor for neurodegeneration and is associated with structural and functional alterations in white matter. Myelin is particularly vulnerable to aging, resulting in white matter-associated microglia activation. Here we used pharmacological and genetic approaches to investigate microglial functions related to aging-associated changes in myelinated axons of mice. Our results reveal that maladaptive microglia activation promotes the accumulation of harmful CD8⁺ T cells, leading to the degeneration of myelinated axons and subsequent impairment of brain function and behavior. We characterize glial heterogeneity and aging-related changes in white matter by single-cell and spatial transcriptomics and reveal elaborate glial-immune interactions. Mechanistically, we show that the CXCL10-CXCR3 axis is crucial for the recruitment and retention of CD8⁺ T cells in aged white matter, where they exert pathogenic effects. Our results indicate that myelin-related microglia dysfunction promotes adaptive immune reactions in aging and identify putative targets to mitigate their detrimental impact.

Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8⁺ T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.

Introduction: Optic neuritis (ON) is often an early sign of multiple sclerosis (MS), and recent studies show a link between HIF-1 pathway activation and inflammation. This study aimed to determine if inhibition of the HIF-1 pathway using the HIF-1a antagonist acriflavine (ACF) can reduce clinical progression and rescue the ocular phenotype in an experimental autoimmune encephalomyelitis (EAE) ON model. Methods: EAE-related ON was induced in 60 female C57BL/6J mice by immunization with MOG33-55, and 20 EAE mice received daily systemic injections of ACF at 5 mg/kg. Changes in the visual function and structure of ACF-treated EAE mice were compared to those of placebo-injected EAE mice and naïve control mice. Results: ACF treatment improved motor–sensory impairment along with preserving visual acuity and optic nerve function. Analysis of retinal ganglion cell complex also showed preserved thickness correlating with increased survival of retinal ganglion cells and their axons. Optic nerve cell infiltration and magnitude of demyelination were decreased in ACF-treated EAE mice. Subsequent in vitro studies revealed improvements not only attributed to the inhibition of HIF-1 but also to previously unappreciated interaction with the eIF2a/ATF4 axis in the unfolded protein response pathway. Discussion: This study suggests that ACF treatment is effective in an animal model of MS via its pleiotropic effects on the inhibition of HIF-1 and UPR signaling, and it may be a viable approach to promote rehabilitation in MS.

Background and objectives: Mechanisms of visual impairment in aquaporin 4 antibody (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody (MOG-IgG)-associated disorder (MOGAD) are incompletely understood. The respective impact of optic nerve demyelination and primary and secondary retinal neurodegeneration are yet to be investigated in animal models. Methods: Active MOG_35-55 experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6Jrj mice, and monoclonal MOG-IgG (8-18C5, murine), recombinant AQP4-IgG (rAb-53, human), or isotype-matched control IgG (Iso-IgG, human) was administered 10 days postimmunization. Mobility impairment was scored daily. Visual acuity by optomotor reflex and ganglion cell complex thickness (GCC, 3 innermost retinal layers) by optical coherence tomography (OCT) were longitudinally assessed. Histopathology of optic nerve and retina was investigated during presymptomatic, acute, and chronic disease phases for immune cells, demyelination, complement deposition, natural killer (NK) cell, AQP4, and astrocyte involvement, retinal ganglion cells (RGCs), and Müller cell activation. Groups were compared by nonparametric tests with a p value <0.05 indicating statistical significance. Results: Visual acuity decreased from baseline to chronic phase in MOG-IgG (mean ± standard error of the mean: 0.54 ± 0.01 to 0.46 ± 0.02 cycles/degree, p < 0.05) and AQP4-IgG EAE (0.54 ± 0.01 to 0.43 ± 0.02, cycles/degree, p < 0.05). Immune cell infiltration of optic nerves started in presymptomatic AQP4-IgG, but not in MOG-IgG EAE (5.85 ± 2.26 vs 0.13 ± 0.10 macrophages/region of interest [ROI] and 1.88 ± 0.63 vs 0.15 ± 0.06 T cells/ROI, both p < 0.05). Few NK cells, no complement deposition, and stable glial fibrillary acid protein and AQP4 fluorescence intensity characterized all EAE optic nerves. Lower GCC thickness (Spearman correlation coefficient r = -0.44, p < 0.05) and RGC counts (r = -0.47, p < 0.05) correlated with higher mobility impairment. RGCs decreased from presymptomatic to chronic disease phase in MOG-IgG (1,705 ± 51 vs 1,412 ± 45, p < 0.05) and AQP4-IgG EAE (1,758 ± 14 vs 1,526 ± 48, p < 0.01). Müller cell activation was not observed in either model. Discussion: In a multimodal longitudinal characterization of visual outcome in animal models of MOGAD and NMOSD, differential retinal injury and optic nerve involvement were not conclusively clarified. Yet optic nerve inflammation was earlier in AQP4-IgG-associated pathophysiology. Retinal atrophy determined by GCC thickness (OCT) and RGC counts correlating with mobility impairment in the chronic phase of MOG-IgG and AQP4-IgG EAE may serve as a generalizable marker of neurodegeneration.

Background: In the field of autoimmune demyelinating diseases, visual impairments have extensively been studied using the experimental autoimmune encephalomyelitis (EAE) mouse model, which is classically induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). However, this model does not involve B cells like its human analogs. New antigens have thus been developed to induce a B cell-dependent form of EAE that better mimics human diseases. Methods: The present study aimed to characterize the visual symptoms of EAE induced with such an antigen called bMOG. After the induction of EAE with bMOG in C57BL/6J mice, visual function changes were studied by electroretinography and optomotor acuity tests. Motor deficits were assessed in parallel with a standard clinical scoring method. Histological examinations and Western blot analyses allowed to follow retinal neuron survival, gliosis, microglia activation, opsin photopigment expression in photoreceptors and optic nerve demyelination. Disease effects on retinal gene expression were established by RNA sequencing. Results: We observed that bMOG EAE mice exhibited persistent loss of visual acuity, despite partial recovery of electroretinogram and motor functions. This loss was likely due to retinal inflammation, gliosis and synaptic impairments, as evi-denced by histological and transcriptomic data. Further analysis suggests that the M-cone photoreceptor pathway was also affected. Conclusion: Therefore, by documenting visual changes induced by bMOG and showing similarities to those seen in diseases such as multiple sclerosis and neuromyelitis optica, this study offers a new approach to test protective or restorative ophthalmic treatments.

Background and Objectives Myelin oligodendrocyte glycoprotein antibody–associated disorder (MOGAD) is a rare, autoimmune demyelinating CNS disorder, distinct from multiple sclerosis and neuromyelitis optica spectrum disor-der. Characterized by pathogenic immunoglobulin G (IgG) antibodies against MOG, a potential treatment strategy for MOGAD is to reduce circulating IgG levels, e.g., by interference with the IgG recycling pathway mediated by the neonatal Fc receptor (FcRn). Although the optic nerve is often detrimentally involved in MOGAD, the effect of FcRn blockade on the visual pathway has not been assessed. Our objective was to investigate effects of a monoclonal an-ti-FcRn antibody in murine MOG-IgG–associated experimental autoimmune encephalomyelitis (EAE). Methods We induced active MOG35-55 EAE in C57Bl/6 mice followed by the application of a monoclonal MOG-IgG (8-18C5) 10 days postimmunization (dpi). Animals were treated with either a specific monoclonal antibody against FcRn (α-FcRn, 4470) or an isotype-matched control IgG on 7, 10, and 13 dpi. Neurologic disability was scored daily on a 10-point scale. Visual acuity was assessed by optomotor reflex. Histopathologic hallmarks of disease were as-sessed in the spinal cord, optic nerve, and retina. Immune cell infiltration was visualized by immunohistochemistry, demyelination by Luxol fast blue staining and complement deposition and number of retinal ganglion cells by im-munofluorescence. Results In MOG-IgG–augmented MOG35-55 EAE, anti-FcRn treatment significantly attenuated neurologic disability over the course of disease (mean area under the curve and 95% confidence intervals (CIs): α-FcRn [n = 27], 46.02 [37.89–54.15]; isotype IgG [n = 24], 66.75 [59.54–73.96], 3 independent experiments), correlating with reduced amounts of demyelination and macrophage infiltration into the spinal cord. T- and B-cell infiltration and comple-ment deposition remained unchanged. Compared with isotype, anti-FcRn treatment prevented reduction of visual acuity over the course of disease (median cycles/degree and interquartile range: α-FcRn [n = 16], 0.50 [0.48–0.55] to 0.50 [0.48–0.58]; isotype IgG [n = 17], 0.50 [0.49–0.54] to 0.45 [0.39–0.51]). Discussion We show preserved optomotor response and ameliorated course of disease after anti-FcRn treatment in an experimental model using a monoclonal MOG-IgG to mimic MOGAD. Selectively targeting FcRn might represent a promising therapeutic approach in MOGAD.

Aging is a major risk factor for the development of nervous system functional decline, even in the absence of diseases or trauma. The axon–myelin units and synaptic terminals are some of the neural structures most vulnerable to aging-related deterioration, but the underlying mechanisms are poorly understood. In the peripheral nervous system, macrophages—important representatives of the innate immune system—are prominent drivers of structural and functional decline of myelinated fibers and motor endplates during aging. Similarly, in the aging central nervous system (CNS), microglial cells promote damage of myelinated axons and synapses. Here we examine the role of cytotoxic CD8+ T lymphocytes, a type of adaptive immune cells previously identified as amplifiers of axonal perturbation in various models of genetically mediated CNS diseases but understudied in the aging CNS. We show that accumulation of CD8+ T cells drives axon degeneration in the normal aging mouse CNS and contributes to age-related cognitive and motor decline. We characterize CD8+ T-cell population heterogeneity in the adult and aged mouse brain by single-cell transcriptomics and identify aging-related changes. Mechanistically, we provide evidence that CD8+ T cells drive axon degeneration in a T-cell receptor- and granzyme B-dependent manner. Cytotoxic neural damage is further aggravated by systemic inflammation in aged but not adult mice. We also find increased densities of T cells in white matter autopsy material from older humans. Our results suggest that targeting CD8+ CNS-associated T cells in older adults might mitigate aging-related decline of brain structure and function.

Histamine is best known for its role in allergies, but it could also be involved in autoimmune diseases such as multiple sclerosis. However, studies using experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis, have reported conflicting observations and suggest the implication of a nonclassical source of histamine. In this study, we demonstrate that neutrophils are the main producers of histamine in the spinal cord of EAE mice. To assess the role of histamine by taking into account its different cellular sources, we used CRISPR-Cas9 to generate conditional knockout mice for the histamine-synthesizing enzyme histidine decarboxylase. We found that ubiquitous and cell-specific deletions do not affect the course of EAE. However, neutrophil-specific deletion attenuates hypothermia caused by IgE-mediated anaphylaxis, whereas neuron-specific deletion reduces circadian activity. In summary, this study refutes the role of histamine in EAE, unveils a role for neutrophil-derived histamine in IgE-mediated anaphylaxis, and establishes a new mouse model to re-explore the inflammatory and neurologic roles of histamine.

Purpose: This study investigated the presence of beta-amyloid in the retina and optic nerve of 2-month-old mouse models of Alzheimer’s disease (AD) prior to the onset of clinical symptoms and compared the findings to those of age-matched controls. Methods: Visual acuity and contrast sensitivity were measured using an OptoDrum® in 2-month-old mouse models (5xFAD) and 2-month-old normal mice (C57BL/6). The retina and optic nerve of the experimental and control groups were stained fluorescently using an amyloid-beta antibody, and the level of amyloid-beta expression in the two groups was compared and analyzed histologically. Results: The 2-month-old mouse models (5xFAD) are at a stage prior to the characteristic spatial learning and cognitive impairments associated with AD. Visual acuity and contrast sensitivity did not differ between the experimental and control groups. Beta-amyloid was not observed in the retina or optic nerve of the control group or in the retina of the experimental group. In contrast to normal mice of the same age, beta-amyloid accumulated in the optic nerve in the experimental group, with a tendency for increased beta-amyloid accumulation in the optic nerve closer to the brain. Conclusions: In young AD mouse models prior to the onset of clinical symptoms, there were no differences in visual acuity, contrast sensitivity, or histological changes in the retina compared to controls. However, pathological changes in the accumulation of beta-amyloid in the optic nerve in the experimental group preceded other changes. These findings should aid the early diagnosis of AD in the future.

Hereditary spastic paraplegias (HSPs) are genetically-determined, neurodegenerative disorders characterized by progressive weakness and spasticity of the lower limbs. Spastic paraplegia type 11 (SPG11) is a complicated form of HSP, which is caused by mutations in the SPG11 gene encoding spatacsin, a protein possibly involved in lysosomal reformation. Based on our previous studies demonstrating that secondary neuroinflammation can be a robust amplifier of various genetically-mediated diseases of both the central and peripheral nervous system, we here test the possibility that neuroinflammation may modify the disease outcome also in a mouse model for SPG11. Spg11-knockout (Spg11^-/-) mice develop early walking pattern and behavioral abnormalities, at least partially reflecting motor, and behavioral changes typical for patients. Furthermore, we detected a progressive increase in axonal damage and axonal spheroid formation in the white and grey matter compartments of the central nervous system of Spg11^-/- mice. This was accompanied by a concomitant substantial increase of secondary inflammation by cytotoxic CD8⁺ and CD4⁺ T-lymphocytes. We here provide evidence that disease-related changes can be ameliorated/delayed by the genetic deletion of the adaptive immune system. Accordingly, we provide evidence that repurposing clinically approved immunomodulators (fingolimod/FTY720 or teriflunomide), that are in use for treatment of multiple sclerosis (MS), also improve disease symptoms in mice, when administered in an early (before neural damage) or late (after/during neural damage) treatment regime. This work provides strong evidence that immunomodulation can be a therapeutic option for the still untreatable SPG11, including its typical neuropsychological features. This poses the question if inflammation is not only a disease amplifier in SPG11 but can act as a unifying factor also for other genetically mediated disorders of the CNS. If true, this may pave the way to therapeutic options in a wide range of still untreatable, primarily genetic, neurological disorders by repurposing approved immunomodulators.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in non–image-forming visual functions. Given their significant loss observed in various ocular degenerative diseases at early stages, this study aimed to assess changes in both the morphology and associated behavioral functions of ipRGCs in mice between 6 (mature) and 12 (late adult) months old. The findings contribute to understanding the preservation of ipRGCs in late adults and their potential as a biomarker for early ocular degenerative diseases. Methods: Female and male C57BL/6J mice were used to assess the behavioral consequences of aging to mature and old adults, including pupillary light reflex, light aversion, visual acuity, and contrast sensitivity. Immunohistochemistry on retinal wholemounts from these mice was then conducted to evaluate ipRGC dendritic morphology in the ganglion cell layer (GCL) and inner nuclear layer (INL). Results: Morphological analysis showed that ipRGC dendritic field complexity was remarkably stable through 12 months old of age. Similarly, the pupillary light reflex, visual acuity, and contrast sensitivity were stable in mature and old adults. Although alterations were observed in ipRGC-independent light aversion distinct from the pupillary light reflex, aged wild-type mice continuously showed enhanced light aversion with dilation. No effect of sex was observed in any tests. Conclusions: The preservation of both ipRGC morphology and function highlights the potential of ipRGC-mediated function as a valuable biomarker for ocular diseases characterized by early ipRGC loss. The consistent stability of ipRGCs in mature and old adult mice suggests that detected changes in ipRGC-mediated functions could serve as early indicators or diagnostic tools for early-onset conditions such as Alzheimer's disease, Parkinson's disease, and diabetes, where ipRGC loss has been documented.

Vision depends on accurate signal conduction from the retina to the brain through the optic nerve, an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells. The mammalian optic nerve, an important part of the central nervous system, cannot regenerate once it is injured, leading to permanent vision loss. To date, there is no clinical treatment that can regenerate the optic nerve and restore vision. Our previous study found that the mobile zinc (Zn²⁺) level increased rapidly after optic nerve injury in the retina, specifically in the vesicles of the inner plexiform layer. Furthermore, chelating Zn²⁺ significantly promoted axonal regeneration with a long-term effect. In this study, we conditionally knocked out zinc transporter 3 (ZnT3) in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines (VGAT^CreZnT3^fl/fl and VGLUT2^CreZnT3^fl/fl, respectively). We obtained direct evidence that the rapidly increased mobile Zn²⁺ in response to injury was from amacrine cells. We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury, improved retinal ganglion cell function, and promoted vision recovery. Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn²⁺ affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons, regulated the synaptic connection between amacrine cells and retinal ganglion cells, and affected the fate of retinal ganglion cells. These results suggest that amacrine cells release Zn²⁺ to trigger transcriptomic changes related to neuronal growth and survival in retinal ganglion cells, thereby influencing the synaptic plasticity of retinal networks. These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.

Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8⁺ T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.

Background and objectives: Mechanisms of visual impairment in aquaporin 4 antibody (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody (MOG-IgG)-associated disorder (MOGAD) are incompletely understood. The respective impact of optic nerve demyelination and primary and secondary retinal neurodegeneration are yet to be investigated in animal models. Methods: Active MOG_35-55 experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6Jrj mice, and monoclonal MOG-IgG (8-18C5, murine), recombinant AQP4-IgG (rAb-53, human), or isotype-matched control IgG (Iso-IgG, human) was administered 10 days postimmunization. Mobility impairment was scored daily. Visual acuity by optomotor reflex and ganglion cell complex thickness (GCC, 3 innermost retinal layers) by optical coherence tomography (OCT) were longitudinally assessed. Histopathology of optic nerve and retina was investigated during presymptomatic, acute, and chronic disease phases for immune cells, demyelination, complement deposition, natural killer (NK) cell, AQP4, and astrocyte involvement, retinal ganglion cells (RGCs), and Müller cell activation. Groups were compared by nonparametric tests with a p value <0.05 indicating statistical significance. Results: Visual acuity decreased from baseline to chronic phase in MOG-IgG (mean ± standard error of the mean: 0.54 ± 0.01 to 0.46 ± 0.02 cycles/degree, p < 0.05) and AQP4-IgG EAE (0.54 ± 0.01 to 0.43 ± 0.02, cycles/degree, p < 0.05). Immune cell infiltration of optic nerves started in presymptomatic AQP4-IgG, but not in MOG-IgG EAE (5.85 ± 2.26 vs 0.13 ± 0.10 macrophages/region of interest [ROI] and 1.88 ± 0.63 vs 0.15 ± 0.06 T cells/ROI, both p < 0.05). Few NK cells, no complement deposition, and stable glial fibrillary acid protein and AQP4 fluorescence intensity characterized all EAE optic nerves. Lower GCC thickness (Spearman correlation coefficient r = -0.44, p < 0.05) and RGC counts (r = -0.47, p < 0.05) correlated with higher mobility impairment. RGCs decreased from presymptomatic to chronic disease phase in MOG-IgG (1,705 ± 51 vs 1,412 ± 45, p < 0.05) and AQP4-IgG EAE (1,758 ± 14 vs 1,526 ± 48, p < 0.01). Müller cell activation was not observed in either model. Discussion: In a multimodal longitudinal characterization of visual outcome in animal models of MOGAD and NMOSD, differential retinal injury and optic nerve involvement were not conclusively clarified. Yet optic nerve inflammation was earlier in AQP4-IgG-associated pathophysiology. Retinal atrophy determined by GCC thickness (OCT) and RGC counts correlating with mobility impairment in the chronic phase of MOG-IgG and AQP4-IgG EAE may serve as a generalizable marker of neurodegeneration.

Myelin defects lead to neurological dysfunction in various diseases and in normal aging. Chronic neuroinflammation often contributes to axon-myelin damage in these conditions and can be initiated and/or sustained by perturbed myelinating glia. We have previously shown that distinct PLP1 mutations result in neurodegeneration that is largely driven by adaptive immune cells. Here we characterize CD8⁺ CNS-associated T cells in myelin mutants using single-cell transcriptomics and identify population heterogeneity and disease-associated changes. We demonstrate that early sphingosine-1-phosphate receptor modulation attenuates T cell recruitment and neural damage, while later targeting of CNS-associated T cell populations is inefficient. Applying bone marrow chimerism and utilizing random X chromosome inactivation, we provide evidence that axonal damage is driven by cytotoxic, antigen specific CD8⁺ T cells that target mutant myelinating oligodendrocytes. These findings offer insights into neural-immune interactions and are of translational relevance for neurological conditions associated with myelin defects and neuroinflammation.

The wiring of visual circuits requires that retinal neurons functionally connect to specific brain targets, a process that involves activity-dependent signaling between retinal axons and their postsynaptic targets. Vision loss in various ophthalmological and neurological diseases is caused by damage to the connections from the eye to the brain. How postsynaptic brain targets influence retinal ganglion cell (RGC) axon regeneration and functional reconnection with the brain targets remains poorly understood. Here, we established a paradigm in which the enhancement of neural activity in the distal optic pathway, where the postsynaptic visual target neurons reside, promotes RGC axon regeneration and target reinnervation and leads to the rescue of optomotor function. Furthermore, selective activation of retinorecipient neuron subsets is sufficient to promote RGC axon regeneration. Our findings reveal a key role for postsynaptic neuronal activity in the repair of neural circuits and highlight the potential to restore damaged sensory inputs via proper brain stimulation.

Aging is a major risk factor for the development of nervous system functional decline, even in the absence of diseases or trauma. The axon–myelin units and synaptic terminals are some of the neural structures most vulnerable to aging-related deterioration, but the underlying mechanisms are poorly understood. In the peripheral nervous system, macrophages—important representatives of the innate immune system—are prominent drivers of structural and functional decline of myelinated fibers and motor endplates during aging. Similarly, in the aging central nervous system (CNS), microglial cells promote damage of myelinated axons and synapses. Here we examine the role of cytotoxic CD8+ T lymphocytes, a type of adaptive immune cells previously identified as amplifiers of axonal perturbation in various models of genetically mediated CNS diseases but understudied in the aging CNS. We show that accumulation of CD8+ T cells drives axon degeneration in the normal aging mouse CNS and contributes to age-related cognitive and motor decline. We characterize CD8+ T-cell population heterogeneity in the adult and aged mouse brain by single-cell transcriptomics and identify aging-related changes. Mechanistically, we provide evidence that CD8+ T cells drive axon degeneration in a T-cell receptor- and granzyme B-dependent manner. Cytotoxic neural damage is further aggravated by systemic inflammation in aged but not adult mice. We also find increased densities of T cells in white matter autopsy material from older humans. Our results suggest that targeting CD8+ CNS-associated T cells in older adults might mitigate aging-related decline of brain structure and function.

Targeting neuroinflammation in models for infantile and juvenile forms of neuronal ceroid lipofuscinosis (NCL, CLN disease) with the clinically established immunomodulators fingolimod and teriflunomide significantly attenuates the neurodegenerative phenotype when applied preventively, i.e. before the development of substantial neural damage and clinical symptoms. Here, we show that in a mouse model for the early onset and rapidly progressing CLN1 form, more complex clinical phenotypes like disturbed motor coordination and impaired visual acuity are also ameliorated by immunomodulation. Moreover, we show that the disease outcome can be attenuated even when fingolimod and teriflunomide treatment starts after disease onset, i.e. when neurodegeneration is ongoing and clinical symptoms are detectable. In detail, treatment with either drug led to a reduction in T-cell numbers and microgliosis in the CNS, although not to the same extent as upon preventive treatment. Pharmacological immunomodulation was accompanied by a reduction of axonal damage, neuron loss and astrogliosis in the retinotectal system and by reduced brain atrophy. Accordingly, the frequency of myoclonic jerks and disturbed motor coordination were attenuated. Overall, disease alleviation was remarkably substantial upon therapeutic treatment with both drugs, although less robust than upon preventive treatment. To test the relevance of putative immune-independent mechanisms of action in this model, we treated CLN1 mice lacking mature T- and B-lymphocytes. Immunodeficient CLN1 mice showed, as previously reported, an improved neurological phenotype in comparison with genuine CLN1 mice which could not be further alleviated by either of the drugs, reflecting a predominantly immune-related therapeutic mechanism of action. The present study supports and strengthens our previous view that repurposing clinically approved immunomodulators may alleviate the course of CLN1 disease in human patients, even though diagnosis usually occurs when symptoms have already emerged.

Background/Objectives: Retinitis pigmentosa is a degenerative retinal disease and a major cause of inherited blindness globally. The pro-survival kinase AKT is downregulated in degenerating photoreceptors in retinitis pigmentosa, and its activation has shown neuroprotective effects in retinitis pigmentosa and other neurodegenerative disorders. In this study, we evaluated the therapeutic potential of SC79, a pharmaceutical AKT activator, in two mouse models of retinitis pigmentosa, rd1.GFP and RhoP23H.GFP. Methods: SC79 was administered intravitreally at postnatal day 12 (P12) and analysis was conducted at P16. Results: SC79 at 10 µM was well tolerated in wildtype mice, with no reduction in retinal function or thickness. In rd1.GFP mice, SC79 partially preserved peripheral outer nuclear layer (ONL) thickness, improved rod photoreceptor-driven optomotor contrast sensitivity responses, and improved cone photoreceptor morphology. Immunohistochemistry of retinal sections indicated AKT-related protein expression changes in both sham and SC79-treated rd1.GFP retinas, with sham injections leading to decreases in this pathway and SC79 injections restoring this back to uninjected protein levels or higher, indicating the damage from intravitreal injections can induce AKT-related protein expression changes. In RhoP23H.GFP mice, changes to the visual response from the therapeutic effects of SC79 were not detectable. An increased dosage of SC79 at 100 µM was evaluated in wildtype mice and showed no major toxic effects, although it did not confer neuroprotective benefits in either disease model. Conclusions: These results demonstrate the potential therapeutic effect of AKT pathway modulation for preserving photoreceptors in recessive retinitis pigmentosa, with further optimisation of treatment delivery required.

Dysfunctional cGMP signaling is implicated in multiple neurodegenerative diseases of the central nervous system (CNS), including glaucoma, an optic neuropathy and leading cause of irreversible blindness. Female mice lacking the alpha catalytic subunit of soluble guanylate cyclase (sGCα1^-/-), the active site that binds nitric oxide (NO) to produce cGMP, exhibit progressive retinal ganglion cell (RGC) degeneration with age. Yet, the role of sGC in age- and sex-dependent RGC function remains uncharacterized. We investigated how preventing NO binding to sGC influences RGC function in the context of aging and sex by combining bulk and single-cell RNA sequencing, Western blotting, mitochondrial ultrastructural analysis, visual acuity measurements, and in vivo measurements of retinal oxidative metabolism. We found that global sGCα1 deletion impairs visual function and RGC health in aging female mice, while male mice remained unaffected. Glucose uptake was significantly disrupted in female sGCα1^-/-retinas with age, and accompanied by reduced retinal expression of the glucose transporter, GLUT1. Aged sGCα1^-/- females also exhibited dysregulated retinal mitochondrial gene and protein expression and increased nitrosative stress localized to the RGC layer. RGC mitochondria in male mice increased in size with age, while female mitochondria did not. Furthermore, retinal metabolic analysis showed decreased oxygen consumption rate in aged female but not male sGCα1^-/-retinas, suggesting impaired oxidative metabolism. These findings reveal a potential sex-specific role for cGMP signaling in maintaining retinal metabolic integrity and RGC function with age. Our results point to a possible mechanistic link between impaired cGMP signaling and age-related retinal neurodegeneration in females, highlighting the sGC-cGMP signaling pathway as a promising therapeutic target for glaucoma and other CNS neurodegenerative diseases.

Photoreceptor degeneration is a leading cause of untreatable sight loss. Previously, we showed that human pluripotent stem cell-derived cone photoreceptors (hCones) can rescue retinal function in the Rd1 mouse model of rod-cone dystrophy. However, retinal degenerations display markedly different severities and concomitant remodeling of the remaining retina; for photoreceptor replacement therapy to be broadly effective, it must work for a variety of disease phenotypes. Here, we sought to rescue the Aipl1^-/- model of Leber congenital amaurosis, a particularly fast, severe condition. After transplantation of hCones, host cone bipolar cells underwent extensive remodeling and formed nascent synaptic-like connections. Electrophysiological recordings showed robust rescue of light-evoked activity across visually relevant photopic intensities, and treated mice exhibited visually evoked optokinetic head-tracking behavior. Thus, human cone photoreceptor replacement therapy is feasible even in very severe cases of retinal dystrophy, offering promise as a disease-agnostic therapy in Leber congenital amaurosis (LCA) and in other advanced retinal degenerations.

Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration worldwide. It is characterized by the sequential death of rod and cone photoreceptors, the cells responsible for night and daylight vision, respectively. Although the expression of most RP genes occurs only in rods, there is a secondary degeneration of cones. One possible mechanism of cone death is metabolic dysregulation. Photoreceptors are highly metabolically active, consuming large quantities of glucose and producing substantial amounts of lactate. The retinal pigment epithelium (RPE) mediates the transport of glucose from the blood to photoreceptors and, in turn, removes lactate, which can influence the rate of consumption of glucose by the RPE. One model for metabolic dysregulation in RP suggests that following the death of rods, lactate levels are substantially diminished causing the RPE to withhold glucose, resulting in nutrient deprivation for cones. Here, we present adeno-associated viral vector-mediated delivery of monocarboxylate transporter 2 (MCT2, Slc16a7) into the eye, with expression limited to RPE cells, with the aim of promoting lactate uptake from the blood and encouraging the passage of glucose to cones. We demonstrate prolonged survival and function of cones in rat and mouse RP models, revealing a possible gene-agnostic therapy for preserving vision in RP. We also present the use of fluorescence lifetime imaging-based biosensors for lactate and glucose within the eye. Using this technology, we show changes to lactate and glucose levels within MCT2-expressing RPE, suggesting that cone survival is impacted by changes in RPE metabolism.

Background: Multiple Sulfatase Deficiency (MSD) is a rare inherited lysosomal storage disorder characterized by loss of function mutations in the SUMF1 gene that manifests as a severe pediatric neurological disease. There are no available targeted therapies for MSD. Methods: We engineered a viral vector (AAV9/SUMF1) to deliver working copies of the SUMF1 gene and tested the vector in Sumf1 knock out mice that generally display a median lifespan of 10 days. Mice were injected as pre-symptomatic neonates via intracerebroventricular administration, or as post-symptomatic juveniles via intrathecal alone or combination intrathecal and intravenous delivery. Cohorts were assessed for survival, behavioral outcomes, and post-mortem for sulfatase activity. Results: We show that treatment of neonates extends survival up to 1-year post-injection. Importantly, delivery of SUMF1 through cerebral spinal fluid at 7 days of age alleviates MSD symptoms. The treated mice show wide distribution of the SUMF1 gene, no signs of toxicity or neuropathy, improved vision and cardiac function, and no behavioral deficits. One-year post treatment, tissues show increased sulfatase activity, indicating functional SUMF1. Further, a GLP toxicology study conducted in rats demonstrates favorable overall safety of this approach. Conclusions: These preclinical studies highlight the potential of our AAV9/SUMF1 vector, the design of which is directly translatable for clinical use, as a gene replacement therapy for MSD patients.

Minocycline, a broad-spectrum tetracycline antibiotic, has been shown to possess anti-inflammatory and antioxidative effects in various neurodegenerative diseases. However, its specific effects on retinitis pigmentosa (RP) have not been thoroughly investigated. Therefore, the objective of this study was to explore the potential role of minocycline in treating RP. In this investigation, we used rd1 to explore the antioxidant effect of minocycline in RP. Minocycline therapy effectively restored retinal function and structure in rd1 mice at 14 days postnatal. Additionally, minocycline inhibited the activation of microglia. Moreover, RNA sequencing analysis revealed a significant downregulation in the expression of mitochondrial genes within the retina of rd1 mice. Further KEGG and GO pathway analysis indicated impaired oxidative phosphorylation and electron transport chain processes. TEM confirmed the presence of damaged mitochondria in photoreceptors, while JC-1 staining demonstrated a decrease in mitochondrial membrane potential, accompanied by an increase in mitochondrial reactive oxygen species (ROS) levels. However, treatment with minocycline successfully reversed the abnormal expression of mitochondrial genes and reduced the levels of mitochondrial ROS, thereby providing protection against photoreceptor degeneration. Collectively, minocycline demonstrated the ability to rescue photoreceptor cells in RP by effectively modulating mitochondrial homeostasis and subsequently inflammation. These findings hold significant implications for the development of potential therapeutic strategies for RP.

Optogenetics is a promising approach for restoring vision to the blind after photoreceptor degeneration. The ability to restore vision through AAV-mediated delivery of light-sensitive proteins, especially channelrhodopsins, into retinal ganglion cells has been extensively demonstrated in animal models. For clinical application, knowledge of viral dose-dependent functional efficacy is desired. In this study, using a triple-knockout blind mouse model and a highly light-sensitive channelrhodopsin variant, we evaluated viral dose-dependent vision restoration through retinal ganglion cell expression by using optomotor behavioral assays. Our results show that both the restored light sensitivity and visual acuity reached peak levels at a medial viral dose of 10⁸ vg. With increasing dose, transduction efficiency continued to increase while protein expression peaked at the dose of ~10⁹ vg and declined at higher doses. Also, a significant increase in retinal gliosis and inflammatory responses started at the dose of ~10⁹ vg, and a marked increase was observed at the dose of ~10¹⁰. These results provide valuable insights into viral dose design for clinical studies.

Retinal ischaemia/reperfusion (I/R) injury is a common cause of retinal ganglion cell (RGC) apoptosis and axonal degeneration, resulting in irreversible visual impairment. However, there are no available neuroprotective and neurorestorative therapies for retinal I/R injury, and more effective therapeutic approaches are needed. The role of the myelin sheath of the optic nerve after retinal I/R remains unknown. Here, we report that demyelination of the optic nerve is an early pathological feature of retinal I/R and identify sphingosine-1-phosphate receptor 2 (S1PR2) as a therapeutic target for alleviating demyelination in a model of retinal I/R caused by rapid changes in intraocular pressure. Targeting the myelin sheath via S1PR2 protected RGCs and visual function. In our experiment, we observed early damage to the myelin sheath and persistent demyelination accompanied by S1PR2 overexpression after injury. Blockade of S1PR2 by the pharmacological inhibitor JTE-013 reversed demyelination, increased the number of oligodendrocytes, and inhibited microglial activation, contributing to the survival of RGCs and alleviating axonal damage. Finally, we evaluated the postoperative recovery of visual function by recording visual evoked potentials and assessing the quantitative optomotor response. In conclusion, this study is the first to reveal that alleviating demyelination by inhibiting S1PR2 overexpression may be a therapeutic strategy for retinal I/R-related visual impairment.

Phototransduction is mediated by distinct types of G protein cascades in different animal taxa: bilateral invertebrates typically utilise the Gαq pathway whereas vertebrates typically utilise the Gαt(i/o) pathway. By contrast, photoreceptors in jellyfish (Cnidaria) utilise the Gαs intracellular pathway, similar to olfactory transduction in mammals¹. How this habitually slow pathway has adapted to support dynamic vision in jellyfish remains unknown. Here we study a light-sensing protein (rhodopsin) from the box jellyfish Carybdea rastonii and uncover a mechanism that dramatically speeds up phototransduction: an uninterrupted G protein-coupled receptor - G protein complex. Unlike known G protein-coupled receptors (GPCRs), this rhodopsin constitutively binds a single downstream Gαs partner to enable G-protein activation and inactivation within tens of milliseconds. We use this GPCR in a viral gene therapy to restore light responses in blind mice.

Retinitis pigmentosa, an inherited form of retinal degeneration, is characterized by a progressive loss of rods and subsequent degeneration of cones, leading to blindness. However, the remaining neural portion of the retina (bipolar and ganglion cells) remains anatomically and functionally intact for an extended time. A possible treatment to restore the light sensitivity of the retina consists of rendering the remaining retinal cells photosensitive using optogenetic tools like, for example, Opto-mGluR6, a light-sensitive mGluR6 receptor. We have previously demonstrated that AAV vector-mediated expression of Opto-mGluR6 in ON-bipolar cells restores visual function in otherwise blind mice. However, classical gene supplementation therapy still suffers from high off-target expression rates and uncontrollable target gene expression levels that may lead to either cytotoxicity or lack of functional restoration. To address these issues and achieve cell-specific and endogenously controlled Opto-mGluR6 expression, we employed the CRISPR/Cas technology—in particular, homology-independent targeted integration (HITI) and microhomology-dependent targeted integration (MITI)—to knock-in the Opto-mGluR6 gene behind the ON-bipolar cell-specific GRM6 promoter. We compared four Cas systems in vitro and show that SpCas9 for HITI and LbCpf1 for MITI are well suited to promoting knock-in. As AAV2-mediated ON-bipolar cell transduction resulted in inefficiency, we evaluated Exo-AAVs as delivery vehicles and found Exo-AAV1 efficient for targeting ON-bipolar cells. We demonstrate that intravitreal injection of Exo-AAV1 carrying vectors that promote MITI significantly improved visual acuity in otherwise blind rd1 mice. We conclude by confirming and providing a qualitative evaluation of the MITI-mediated knock-in in the correct genomic locus.

Optogenetic gene therapies to restore vision are in clinical trials. Whilst current clinical approaches target the ganglion cells, the output neurons of the retina, new molecular tools enable efficient targeting of the first order retinal interneurons, the bipolar cells, with the potential to restore a higher quality of vision. Here we investigate retinal signaling and behavioral vision in blind mice treated with bipolar cell targeted optogenetic gene therapies. All tested tools, including medium-wave opsin, Opto-mGluR6, and two new melanopsin based chimeras restored visual acuity and contrast sensitivity. The best performing opsin was a melanopsin-mGluR6 chimera, which in some cases restored visual acuities and contrast sensitivities that match wild-type animals. Light responses from the ganglion cells were robust with diverse receptive-field types, inferring elaborate inner retinal signaling. Our results highlight the potential of bipolar cell targeted optogenetics to recover high-level vision in human patients with end-stage retinal degenerations.

One of the critical prerequisites for the successful development of retinal prostheses is understanding the physio-logical features of retinal ganglion cells (RGCs) in the different stages of retinal degeneration (RD). This study used our custom-made rd10 mice, C57BL/6-Pde6bem1(R560C)Dkl/Korl mutated on the Pde6b gene in C57BL/6J mouse with the CRISPR/Cas9-based gene-editing method. We selected the postnatal day (P) 45, P70, P140, and P238 as representa-tive ages for RD stages. The optomotor response measured the visual acuity across degeneration stages. At P45, the rd10 mice exhibited lower visual acuity than wild-type (WT) mice. At P140 and older, no optomotor response was observed. We classified RGC responses to the flashed light into ON, OFF, and ON/OFF RGCs via in vitro multichannel recording. With degeneration, the number of RGCs responding to the light stimulation decreased in all three types of RGCs. The OFF response disappeared faster than the ON response with older postnatal ages. We elicited RGC spikes with electrical stimulation and analyzed the network-mediated RGC response in the rd10 mice. Across all postnatal ages, the spikes of rd10 RGCs were less elicited by pulse amplitude modulation than in WT RGCs. The ratio of RGCs showing multiple peaks of spike burst increased in older ages. The electrically evoked RGC spikes by the pulse amplitude modulation differ across postnatal ages. Therefore, degeneration stage-dependent stimulation strategies should be considered for developing retinal prosthesis and successful vision restoration.

Optogenetic gene therapy holds promise to restore high-quality vision in blind patients and recently reached clinical trials. Although the ON-bipolar cells, the first retinal interneurons, make the most attractive targets for optogenetic vision restoration, they have remained inaccessible to human gene therapy due to the lack of a robust cell-specific promoter. We describe the design and functional evaluation of 770En_454P(hGRM6), a human GRM6 gene-derived, short promoter that drives strong and highly specific expression in both the rod- and cone-type ON-bipolar cells of the human retina. Expression also in cone-type ON-bipolar cells is of importance, since the cone-dominated macula mediates high-acuity vision and is the primary target of gene therapies. 770En_454P(hGRM6)-driven middle-wave opsin expression in ON-bipolar cells achieved lasting restoration of high visual acuity in the rd1 mouse model of late retinal degeneration. The new promoter enables precise manipulation of the inner retinal network and paves the way for clinical application of gene therapies for high-resolution optogenetic vision restoration, raising hopes of significantly improving the life quality of people suffering from blindness.

Testing optokinetic head or eye movements is an established method to determine visual performance of laboratory animals, including chickens, guinea pigs, mice, or fish. It is based on the optokinetic reflex which causes the ani-mals to track a drifting stripe pattern with eye and head movements. We have developed an improved version of the optomotor test with better control over the stimulus parameters, as well as a high degree of automation. The stripe pattern is presented on computer monitors surrounding the animal. By tracking the head position of freely moving animals in real time, the visual angle under which the stripes of the pattern appeared was kept constant even for changing head positions. Furthermore, an algorithm was developed for automated evaluation of the track-ing performance of the animal. Comparing the automatically determined behavioral score with manual assessment of the animals’ tracking behavior confirmed the reliability of our methodology. As an example, we reproduced the known contrast sensitivity function of wild type mice. Furthermore, the progressive decline in visual performance of a mouse model of retinal degeneration, rd10, was demonstrated.

Highly myopic cataract (HMC) is a leading cause of blindness among the working-age individuals, with its pathogenesis poorly understood. This study aimed to elucidate the role of ferroptosis in HMC development as well as the underlying mechanisms. In HMC lens epithelia, levels of Fe2+ and lipid peroxidation were found elevated, with increased vulnerability towards ferroptosis as revealed by transmission electron microscopy. Mechanistically, RNA sequencing of HMC lens epithelial samples identified up-regulated expression of discoidin domain receptor tyrosine kinase 2 (DDR2) as a key factor, which could enhance ferroptosis sensitivity via the Src-Hippo pathway. Specifically, DDR2 interacted with Src kinase, leading to the nuclear translocation of homologous transcriptional regulators (yes-associated protein 1 [YAP1] and WW domain containing transcription regulator 1 [WWTR1]) of the Hippo pathway, which altered the expression level of ferroptosis-related genes. Notably, highly myopic eyes of mice exhibited higher sensitivity to RSL3, a ferroptosis inducer, manifested as more severe nuclear lens opacities both in vitro and in vivo compared with the contralateral control eyes, which could be alleviated by inhibitors of either ferroptosis or DDR2. Altogether, these findings highlighted the role of DDR2 in mediating ferroptosis in HMC formation, providing a novel insight for therapeutic interventions.

This study aimed to investigate the role of quercetin in myopic ciliary muscle remodeling through the regulation of SIRT1 expression. To this end, a randomized controlled trial was conducted using negative lens-induced myopic (LIM) guinea pig models for durations of 4 weeks and 6 weeks, with subjects administered quercetin (QUE) or SRT1720. Changes in axial lengths and refractive errors were measured at 4 and 6 weeks. Subsequently, protein expression (e.g., SIRT1, KEAP1, NRF2, MMP-2, and TIMP-2) in ciliary muscles was analyzed by Western blotting, while the expression of inflammatory cytokines (e.g., TNF-α, IL-6, and IL-10) was quantified via ELISA, and ciliary muscle morphologies were assessed using hematoxylin and eosin (H&E) staining and optical coherence tomography (OCT). Calcium fluxes, reactive oxygen species (ROS) levels, and mitochondrial membrane potentials (ΔΨm) were measured via noninvasive microtests and flow cytometry. Compared with the LIM group, the QUE and SIRT1 activator (SRT1720) intervention groups presented significantly reduced axial elongation amounts and refractive errors (P < 0.01), along with thickened ciliary muscles and improved fiber alignments. Additionally, the calcium influx and ROS levels were significantly reduced, and the ΔΨm levels were partially restored. Furthermore, Western blotting revealed upregulated SIRT1, NRF2, and TIMP-2 expression but downregulated KEAP1 and MMP-2 expression in the intervention groups, and ELISA revealed decreased inflammatory responses. In conclusion,quercetin could play a protective role by increasing the expression of SIRT1, which in turn alleviated KEAP1/NRF2-induced oxidative stress and mitigated NF-κB-driven inflammatory damage, thereby ameliorating ciliary muscle remodeling and potentially delaying the progression of myopia.

Background/Objectives: Retinitis pigmentosa is a degenerative retinal disease and a major cause of inherited blindness globally. The pro-survival kinase AKT is downregulated in degenerating photoreceptors in retinitis pigmentosa, and its activation has shown neuroprotective effects in retinitis pigmentosa and other neurodegenerative disorders. In this study, we evaluated the therapeutic potential of SC79, a pharmaceutical AKT activator, in two mouse models of retinitis pigmentosa, rd1.GFP and RhoP23H.GFP. Methods: SC79 was administered intravitreally at postnatal day 12 (P12) and analysis was conducted at P16. Results: SC79 at 10 µM was well tolerated in wildtype mice, with no reduction in retinal function or thickness. In rd1.GFP mice, SC79 partially preserved peripheral outer nuclear layer (ONL) thickness, improved rod photoreceptor-driven optomotor contrast sensitivity responses, and improved cone photoreceptor morphology. Immunohistochemistry of retinal sections indicated AKT-related protein expression changes in both sham and SC79-treated rd1.GFP retinas, with sham injections leading to decreases in this pathway and SC79 injections restoring this back to uninjected protein levels or higher, indicating the damage from intravitreal injections can induce AKT-related protein expression changes. In RhoP23H.GFP mice, changes to the visual response from the therapeutic effects of SC79 were not detectable. An increased dosage of SC79 at 100 µM was evaluated in wildtype mice and showed no major toxic effects, although it did not confer neuroprotective benefits in either disease model. Conclusions: These results demonstrate the potential therapeutic effect of AKT pathway modulation for preserving photoreceptors in recessive retinitis pigmentosa, with further optimisation of treatment delivery required.

Background: The ciliary muscle, a critical intraocular smooth muscle, plays a potent role in ocular accommodation. Investigating the potential detrimental effects on the ciliary muscle during prolonged contraction and precise mechanism underlying the cell damage hold significance in treating ciliary muscle dysfunction. The effect and mechanism of vitamin E (VitE), an antioxidant, in mitigating these adverse effects of prolonged contraction remains to be thoroughly elucidated. Methods: A guinea pig model of prolonged contraction of the ciliary muscle was established through topical administration of carbachol, and primary ciliary muscle cells isolated from guinea pigs were used for in vitro experiments. Results: The ophthalmic examination results demonstrated that prolonged contraction of the ciliary muscle impaired accommodative function in guinea pigs. This condition may lead to disrupted cellular migration, intracellular adenosine triphosphate depletion, decreased mitochondrial membrane potential, and reactive oxygen species accumulation. Further examination revealed that carbachol induced darker-stained mitochondrial membranes and diminished cristae density, consistent with morphological features of ferroptosis. Moreover, sustained cell contraction modulated specific contraction-related proteins (α-smooth muscle actin), concurrently decreased the expression of antioxidant proteins (glutathione peroxidase, superoxide dismutase, catalase, and GSH) in both tissue specimens and cells, culminating in ferroptosis in vivo and in vitro experiments. Our findings demonstrated that pretreatment with VitE alleviated oxidative injury and mitigated ferroptosis. Additionally, knocking down acetyl-coenzyme A thiosterase 7 attenuated the beneficial effect of VitE. Conclusions: These findings collectively indicated that VitE presented a viable approach to ameliorate oxidative stress and ferroptosis induced by prolonged contraction of the ciliary muscle via activating acetyl-coenzyme A thiosterase 7.

Retinitis pigmentosa (RP) is the most common cause of inherited retinal degeneration worldwide. It is characterized by the sequential death of rod and cone photoreceptors, the cells responsible for night and daylight vision, respectively. Although the expression of most RP genes occurs only in rods, there is a secondary degeneration of cones. One possible mechanism of cone death is metabolic dysregulation. Photoreceptors are highly metabolically active, consuming large quantities of glucose and producing substantial amounts of lactate. The retinal pigment epithelium (RPE) mediates the transport of glucose from the blood to photoreceptors and, in turn, removes lactate, which can influence the rate of consumption of glucose by the RPE. One model for metabolic dysregulation in RP suggests that following the death of rods, lactate levels are substantially diminished causing the RPE to withhold glucose, resulting in nutrient deprivation for cones. Here, we present adeno-associated viral vector-mediated delivery of monocarboxylate transporter 2 (MCT2, Slc16a7) into the eye, with expression limited to RPE cells, with the aim of promoting lactate uptake from the blood and encouraging the passage of glucose to cones. We demonstrate prolonged survival and function of cones in rat and mouse RP models, revealing a possible gene-agnostic therapy for preserving vision in RP. We also present the use of fluorescence lifetime imaging-based biosensors for lactate and glucose within the eye. Using this technology, we show changes to lactate and glucose levels within MCT2-expressing RPE, suggesting that cone survival is impacted by changes in RPE metabolism.

Background: Impairment of the inner blood-retinal barrier (iBRB) leads to various blinding diseases including diabetic retinopathy (DR). The cGAS-STING pathway has emerged as a driving force of cardiovascular destruction, but its impact on the neurovascular system is unclear. Here, we show that cGAMP, the endogenous STING agonist, causes iBRB breakdown and retinal degeneration thorough P2RX7-mediated transport into microglia. Methods: Extracellular cGAMP and STING pathway were determined in tissue samples from patients with proliferative DR (PDR) and db/db diabetic mice. Histological, molecular, bioinformatic and behavioral analysis accessed effects of cGAMP on iBRB. Single-cell RNA sequencing identified the primary retinal cell type responsive to cGAMP. Specific inhibitors and P2RX7-deficienct mice were used to evaluate P2RX7' role as a cGAMP transporter. The therapeutic effects of P2RX7 inhibitor were tested in db/db mice. Results: cGAMP was detected in the aqueous humor of patients with PDR and elevated in the vitreous humor with STING activation in db/db mouse retinas. cGAMP administration led to STING-dependent iBRB breakdown and neuron degeneration. Microglia were the primary cells responding to cGAMP, essential for cGAMP-induced iBRB breakdown and visual impairment. The ATP-gated P2RX7 transporter was required for cGAMP import and STING activation in retinal microglia. Contrary to previous thought that mouse P2RX7 nonselectively transports cGAMP only at extremely high ATP concentrations, human P2RX7 directly binds to cGAMP and activates STING under physiological conditions. Clinically, cGAMP-induced microglial signature was recapitulated in fibrovascular membranes from patients with PDR, with P2RX7 being predominantly expressed in microglia. Inhibiting P2RX7 reduced cGAMP-STING activation, protected iBRB and improved neuron survival in diabetic mouse retinas. Conclusions: Our study reveals a mechanism for cGAMP-mediated iBRB breakdown and suggests that targeting microglia and P2RX7 may mitigate the deleterious effects of STING activation in retinal diseases linked to iBRB impairment.

Background: Aging-induced decline in ciliary muscle function is an important factor in visual accommodative deficits in elderly adults. With this study, we provide an innovative investigation of the interaction between ciliary muscle aging and oxidative stress. Methods: Tricolor guinea pigs were used for the experiments in vivo and primary guinea pig ciliary smooth muscle cells were used for the experiments in vitro.Results: We enriched for genes associated with muscle-aging-lutein relationship using bioinformatics, including Nuclear factor-erythroid 2-related factor-2 (Nrf2), Glutathione Peroxidase (GPx) gene family, Superoxide Dismutase (SOD) gene family, NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and Heme Oxygenase-1 (HO-1). After gavage to aged guinea pigs, lutein reduced Reactive Oxygen Species (ROS) and P21 levels in senescent ciliary muscle; lutein decreased refractive error and restored accommodation of the eye. In addition, lutein increased GPx, SOD, and Catalase (CAT) levels in serum; lutein increased GPx and CAT levels in ciliary bodies. Lutein regulated the expression of proteins such as Nrf2, Kelch-like ECH-associated protein 1 (Keap1), and downstream proteins in senescent ciliary bodies. Similarly, guinea pig ciliary muscle cell senescence was associated with oxidative stress. In vitro, 100 μM lutein reversed the damage caused by 800 μM H2O2; it reduced Senescence-Associated β-galactosidase (SA-β-Gal) and ROS activites, cell apoptosis and cell migration. Also, lutein increased the expression of smooth muscle contractile proteins. Lutein also increased the expression of Nrf2, GPx2, NQO1 and HO-1, decreased the expression of Keap1. A reduction in Nrf2 activity led to a reduction in the ability of lutein to activate antioxidant enzymes in the cells, thus reducing its inhibitory effect on cell senescence. Conclusion: lutein improved resistance to oxidative stress in senescent ciliary muscle in vivo and in vitro by regulating the Keap1/Nrf2/Antioxidant Response Element pathway. We have innovatively demonstrated the molecular pharmacological mechanism by which lutein reverse age-related ciliary muscle systolic and diastolic deficits.

Receptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear. This study demonstrates that RIP1 inhibition protects retinal ganglion cells (RGCs) in preclinical glaucoma models. Genetic inactivation of RIP1 improves RGC survival and preserves retinal function in the preclinical glaucoma models of optic nerve crush (ONC) and ischemia-reperfusion injury (IRI). In addition, the involvement of necroptosis in ONC and IRI glaucoma models was examined by utilizing RIP1 kinase-dead (RIP1-KD), RIP3 knockout (RIP3-KO), and MLKL knockout (MLKL-KO) mice. The number of RGCs, retinal thickness, and visual acuity were rescued in RIP1-kinase-dead (RIP1-KD) mice in both models, while wild-type (WT) mice experienced significant retinal thinning, RGC loss, and vision impairment. RIP3-KO and MLKL-KO mice showed moderate protective effects in the IRI model and limited in the ONC model. Furthermore, we confirmed that a glaucoma causative mutation in optineurin, OPTN-E50K, sensitizes cells to RIP1-mediated inflammatory cell death. RIP1 inhibition reduces RGC death and axonal degeneration following IRI in mice expressing OPTN-WT and OPTN-E50K variant mice. We demonstrate that RIP1 inactivation suppressed microglial infiltration in the RGC layer following glaucomatous damage. Finally, this study highlights that human glaucomatous retinas exhibit elevated levels of TNF and RIP3 mRNA and microglia infiltration, thus demonstrating the role of neuroinflammation in glaucoma pathogenesis. Altogether, these data indicate that RIP1 plays an important role in modulating neuroinflammation and that inhibiting RIP1 activity may provide a neuroprotective therapy for glaucoma.

Minocycline, a broad-spectrum tetracycline antibiotic, has been shown to possess anti-inflammatory and antioxidative effects in various neurodegenerative diseases. However, its specific effects on retinitis pigmentosa (RP) have not been thoroughly investigated. Therefore, the objective of this study was to explore the potential role of minocycline in treating RP. In this investigation, we used rd1 to explore the antioxidant effect of minocycline in RP. Minocycline therapy effectively restored retinal function and structure in rd1 mice at 14 days postnatal. Additionally, minocycline inhibited the activation of microglia. Moreover, RNA sequencing analysis revealed a significant downregulation in the expression of mitochondrial genes within the retina of rd1 mice. Further KEGG and GO pathway analysis indicated impaired oxidative phosphorylation and electron transport chain processes. TEM confirmed the presence of damaged mitochondria in photoreceptors, while JC-1 staining demonstrated a decrease in mitochondrial membrane potential, accompanied by an increase in mitochondrial reactive oxygen species (ROS) levels. However, treatment with minocycline successfully reversed the abnormal expression of mitochondrial genes and reduced the levels of mitochondrial ROS, thereby providing protection against photoreceptor degeneration. Collectively, minocycline demonstrated the ability to rescue photoreceptor cells in RP by effectively modulating mitochondrial homeostasis and subsequently inflammation. These findings hold significant implications for the development of potential therapeutic strategies for RP.

The ciliary muscle constitutes a crucial element in refractive regulation. Investigating the pathophysiological mechanisms within the ciliary muscle during excessive contraction holds significance in treating ciliary muscle dysfunction. A guinea pig model of excessive contraction of the ciliary muscle induced by drops pilocarpine was employed, alongside the primary ciliary muscle cells was employed in in vitro experiments. The results of the ophthalmic examination showed that pilocarpine did not significantly change refraction and axial length during the experiment, but had adverse effects on the regulatory power of the ciliary muscle. The current data reveal notable alterations in the expression profiles of hypoxia inducible factor 1 (HIF-1α), ATP2A2, P53, α-SMA, Caspase-3, and BAX within the ciliary muscle of animals subjected to pilocarpine exposure, alongside corresponding changes observed in cultured cells treated with pilocarpine. Augmented levels of ROS were detected in both tissue specimens and cells, culminating in a significant increase in cell apoptosis in in vivo and in vitro experiments. Further examination revealed that pilocarpine induced an increase in intracellular Ca²⁺ levels and disrupted MMP, as evidenced by mitochondrial swelling and diminished cristae density compared to control conditions, concomitant with a noteworthy decline in antioxidant enzyme activity. However, subsequent blockade of Ca²⁺ channels in cells resulted in downregulation of HIF-1α, ATP2A2, P53, α-SMA, Caspase-3, and BAX expression, alongside ameliorated mitochondrial function and morphology. The inhibition of Ca²⁺ channels presents a viable approach to mitigate ciliary cells damage and sustain proper ciliary muscle function by curtailing the mitochondrial damage induced by excessive contractions.

Fluorescent proteins (FPs) have been widely used to investigate cellular and molecular interactions and trace biological events in many applications. Some of the FPs have been demonstrated to cause undesirable cellular damage by light-induced ROS production in vivo or in vitro. However, it remains unknown if one of the most popular FPs, tdTomato, has similar effects in neuronal cells. In this study, we discovered that tdTomato expression led to unexpected retinal dysfunction and ultrastructural defects in the transgenic mouse retina. The retinal dysfunction mainly manifested in the reduced photopic electroretinogram (ERG) responses and decreased contrast sensitivity in visual acuity, caused by mitochondrial damages characterized with cellular redistribution, morphological modifications and molecular profiling alterations. Taken together, our findings for the first time demonstrated the retinal dysfunction and ultrastructural defects in the retinas of tdTomato-transgenic mice, calling for a more careful design and interpretation of experiments involved in FPs.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

The purpose of this study was to examine the effect of a blast exposure generated from a shock tube on retinal ganglion cell (RGC) function and structure. Mice were exposed to one of three blast conditions using a shock tube; a single blast wave of 20 PSI, a single blast wave of 30 PSI, or three blast waves of 30 PSI given on three consecutive days with a one-day inter-blast interval. The structure and function of the retina were analyzed using the pattern electroretinogram (PERG), the optomotor reflex (OMR), and optical coherence tomography (OCT). The in vivo parameters were examined at baseline, and then again 1-week, 4-weeks, and 16-weeks following blast exposure. The number of surviving RGCs was quantified at the end of the study. Analysis of mice receiving a 20 PSI injury showed decreased PERG and OMR responses 16-weeks post blast, without evidence of changed retinal thickness or RGC death. Mice subjected to a 30 PSI injury showed decreased PERG responses 4 weeks and 16 weeks after injury, without changes in the retinal thickness or RGC density. Mice subjected to 30 PSI X 3 blast exposures had PERG deficits 1-week and 4-weeks post exposure. There was also significant change in retinal thickness 1-week and 16-weeks post blast exposure. Mice receiving 30 PSI X 3 blast injuries had regional loss of RGCs in the central retina, but not in the mid-peripheral or peripheral retina. Overall, this study has shown that increasing the number of blast exposures and the intensity leads to earlier functional loss of RGCs. We have also shown regional RGC loss only when using the highest blast intensity and number of blast injuries.

Retinal neurovascular injuries are a leading cause of vision loss in young adults presenting unmet therapeutic needs. Neurovascular injuries damage homeostatic communication between endothelial, pericyte, glial, and neuronal cells through signaling pathways that remain to be established. To understand the mechanisms that contribute to neuronal death, we use a mouse model of retinal vein occlusion (RVO). Using this model, we previously discovered that after vascular damage, there was non-apoptotic activation of endothelial caspase-9 (EC Casp9); knock-out of EC Casp9 led to a decrease in retinal edema, capillary ischemia, and neuronal death. In this study, we aimed to explore the role of EC Casp9 in vision loss and inflammation. We found that EC Casp9 is implicated in contrast sensitivity decline, induction of inflammatory cytokines, and glial reactivity. One of the noted glial changes was increased levels of astroglial cl-caspase-6, which we found to be activated cell intrinsically by astroglial caspase-9 (Astro Casp9). Lastly, we discovered that Astro Casp9 contributes to capillary ischemia and contrast sensitivity decline after RVO (P-RVO). These findings reveal specific endothelial and astroglial non-apoptotic caspase-9 roles in inflammation and neurovascular injury respectively; and concomitant relevancy to contrast sensitivity decline.

Purpose: The purpose of this study was to examine the role of the immune system and its influence on chronic retinal ganglion cell (RGC) dysfunction following blast-mediated traumatic brain injury (bTBI). Methods: C57BL/6J and B6.129S7-Rag1^tm1Mom/J (Rag^-/-) mice were exposed to one blast injury of 140 kPa. A separate cohort of C57BL/6J mice was exposed to sham-blast. Four weeks following bTBI mice were euthanized, and splenocytes were collected. Adoptive transfer (AT) of splenocytes into naïve C57BL/6J recipient mice was accomplished via tail vein injection. Three groups of mice were analyzed: those receiving AT of splenocytes from C57BL/6J mice exposed to blast (AT-TBI), those receiving AT of splenocytes from C57BL/6J mice exposed to sham (AT-Sham), and those receiving AT of splenocytes from Rag^-/- mice exposed to blast (AT-Rag^-/-). The visual function of recipient mice was analyzed with the pattern electroretinogram (PERG), and the optomotor response (OMR). The structure of the retina was evaluated using optical coherence tomography (OCT), and histologically using BRN3A-antibody staining. Results: Analysis of the PERG showed a decreased amplitude two months post-AT that persisted for the duration of the study in AT-TBI mice. We also observed a significant decrease in the retinal thickness of AT-TBI mice two months post-AT compared to sham, but not at four or six months post-AT. The OMR response was significantly decreased in AT-TBI mice 5- and 6-months post-AT. BRN3A staining showed a loss of RGCs in AT-TBI and AT-Rag^-/- mice. Conclusion: These results suggest that the immune system contributes to chronic RGC dysfunction following bTBI.

Retinal ischemia-reperfusion injury (IRI) is one of the main pathogenic mechanisms of glaucoma, which are largely unknown, including neuroinflammation and neuronal death in the pathological process. In our previous studies, mesenchymal stem cells (MSCs) have been reported to play anti-inflammatory and neuroprotective roles. Addition-ally, conditioned culture medium (CM) of MSCs stimulated by TNF-α have achieved better antiallergic effects in an experimental allergic conjunctivitis mouse model. However, there is an urgent need for cell-free therapy approach-es, like exosomes, to reduce the side effects of autoimmunity. The present study aimed to elucidate the pathways involving TNF-α-stimulated gingival MSC (GMSC)-exosomes (TG-exos), in modulating inflammatory microglia and alleviating apoptosis. In this study, exosomes from the CM of GMSCs were isolated by ultracentrifugation and were injected into the vitreous of mice. The results showed that intraocular injection of TG-exos into mice with IRI nota-bly reduced inflammation and cell loss than that with G-exos (GMSC-exosomes). Similar results were observed in vitro. Additionally, with the microRNA (miR) arrays, it was found that miR-21-5p acted as a crucial factor in TG-exos for neuroprotection and anti-inflammation. Following target prediction and dual-luciferase assay suggested that miR-21-5p played a role by combining with programmed cell death 4 (PDCD4), which was regulated by the long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) as a competing endogenous RNA (ceRNA). This study demonstrates a new therapeutic pathway for neuroprotection against IRI by delivering miR-21-5p-enriched exosomes through MEG3/miR-21-5p/PDCD4 axis and paves the way for the establishment of a cell-free therapeutic approach for glaucoma.

Targeting neuroinflammation in models for infantile and juvenile forms of neuronal ceroid lipofuscinosis (NCL, CLN disease) with the clinically established immunomodulators fingolimod and teriflunomide significantly attenuates the neurodegenerative phenotype when applied preventively, i.e. before the development of substantial neural damage and clinical symptoms. Here, we show that in a mouse model for the early onset and rapidly progressing CLN1 form, more complex clinical phenotypes like disturbed motor coordination and impaired visual acuity are also ameliorated by immunomodulation. Moreover, we show that the disease outcome can be attenuated even when fingolimod and teriflunomide treatment starts after disease onset, i.e. when neurodegeneration is ongoing and clinical symptoms are detectable. In detail, treatment with either drug led to a reduction in T-cell numbers and microgliosis in the CNS, although not to the same extent as upon preventive treatment. Pharmacological immunomodulation was accompanied by a reduction of axonal damage, neuron loss and astrogliosis in the retinotectal system and by reduced brain atrophy. Accordingly, the frequency of myoclonic jerks and disturbed motor coordination were attenuated. Overall, disease alleviation was remarkably substantial upon therapeutic treatment with both drugs, although less robust than upon preventive treatment. To test the relevance of putative immune-independent mechanisms of action in this model, we treated CLN1 mice lacking mature T- and B-lymphocytes. Immunodeficient CLN1 mice showed, as previously reported, an improved neurological phenotype in comparison with genuine CLN1 mice which could not be further alleviated by either of the drugs, reflecting a predominantly immune-related therapeutic mechanism of action. The present study supports and strengthens our previous view that repurposing clinically approved immunomodulators may alleviate the course of CLN1 disease in human patients, even though diagnosis usually occurs when symptoms have already emerged.

Purpose: Age-related macular degeneration (AMD) is a major leading cause of visual impairment and blindness with no cure currently established. Cell replacement of RPE is discussed as a potential therapy for AMD. Previous studies were performed in animal models with severe limitations in recapitulating the disease progression. In detail, we describe the effect of systemic injection of sodium iodate in the mouse retina. We further evaluate the useful-ness of this animal model to analyze cell-specific effects following transplantation of human embryonic stem cell (hESC)-derived RPE cells. Methods: Morphologic, functional, and behavioral changes following sodium iodate injection were monitored by histology, gene expression analysis, electroretinography, and optokinetic head tracking. Human embryonic stem cell–derived RPE cells were transplanted 1 week after sodium iodate injection and experimental retinae were ana-lyzed 3 weeks later. Results: Injection of sodium iodate caused complete RPE cell loss, photoreceptor degeneration, and altered gene and protein expression in outer and inner nuclear layers. Retinal function was severely affected by day 3 and abol-ished from day 14. Following transplantation, donor hESC-derived RPE cells formed extensive monolayers that dis-played wild-type RPE cell morphology, organization, and function, including phagocytosis of host photoreceptor outer segments. Conclusions: Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors, and inner nu-clear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The avail-ability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polar-ized cell monolayers from donor hESC-derived RPE cell suspensions.