Publications

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 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.

Based on two major risk factors of glaucoma, elevated intraocular pressure (IOP) and senescence, two new series of nitric oxide (NO) donating dasatinib derivatives 1a-f, 2a-f were designed, synthesized, and biologically evaluated. The results demonstrated that the most active compound 2e effectively released NO and increased the concentration of 3',5'-cyclic guanosine monophosphate in human trabecular meshwork cells, as well as maintained senolytic activity. Topical administration of 2e in chronic ocular hypertension (COHT) glaucoma mice not only significantly eliminated senescent cells in retina but also exhibited potent retinal ganglion cells (RGCs) surviving, IOP lowering, and visual function protection activities, which were superior to those of dasatinib. Compared with younger adult mice, aged COHT mice resulted in more severe RGCs loss, while 2e demonstrated a greater capacity to improve RGCs survival. Our findings show that dual IOP lowering and senolytic functions could be a promising therapeutic strategy for glaucoma, particularly in older patients.

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.

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: 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.

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.

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: 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.

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.

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.

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.

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.

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.

Chromatin remodellers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), however, their functions during brain development are not fully understood. Here, we focused on Sifrim-Hitz-Weiss Syndrome (SIHIWES)-an intellectual disability disorder caused by mutations in the CHD4 chromodomain helicase gene. We utilized mouse genetics to excise the Chd4 ATPase/helicase domain-either constitutively, or conditionally in the developing telencephalon. Conditional heterozygotes exhibited no change in cortical size and cellular composition and had only subtle behavioral phenotypes. Telencephalon-specific conditional knockouts had marked reductions in cortical growth, reduced numbers of upper-layer neurons, and exhibited alterations in anxiety and repetitive behaviors. Despite the fact that whole-body heterozygotes exhibited comparable growth defects, they were unaffected in these behaviors, but instead exhibited female-specific alterations in learning and memory. These data reveal unexpected phenotypic divergence arising from differences in the spatiotemporal deployment of loss-of-function manipulations, underscoring the importance of context in chromatin remodeller function during neurodevelopment.

Regulation of gene expression is required for embryogenesis and maintenance of the highly specialized and diverse neuron populations of the retina. Chromatin remodelling proteins control gene expression by modifying chromatin structure and are essential for many biological processes including mammalian development. The ATP-dependent chromatin remodelling protein Snf2h is highly expressed in the central nervous system, and pathogenic variants that cause neurodevelopmental abnormalities in the human population have recently been identified. This work aims to characterize the effects of Snf2h loss in the retina. Snf2h retinal conditional knockout (cKO) mice were generated using Snf2h-floxed mice and Chx10-Cre retina-specific Cre driver lines to ablate the Snf2h protein from the retina at embryonic day 10.5. Visual function was assessed via optomotor response-based testing and full-field scotopic electroretinography, and histological changes were examined via immunohistochemistry. Disease progression was tracked at one, two, three, and six months of age. Snf2h cKO mice showed a significant decline in visual function and exhibited retinal neuron loss compared to wildtype control littermates at all time points assessed. This work shows that the chromatin remodelling protein Snf2h plays an essential role in the structure and function of the retina.

Mutations in the RYR1 gene are the most common cause of human congenital myopathies, and patients with recessive mutations are severely affected and often display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle (EOM) involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for Ryr1 mutations. Ex vivo force production in EOMs from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared with that observed in wild-type, single heterozygous mutant carriers or homozygous RyR1p.A4329D mice. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle-specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the EOM phenotype caused by the RyR1p.Q1970fsX16+p.A4329D compound heterozygous Ryr1 mutations is complex and due to a combination of modifications including a direct effect on the macromolecular complex involved in calcium release and indirect effects on the expression of myosin heavy chain isoforms.

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.

Cdr1as is the abundant circular RNA (circRNA) in human and vertebrate retinas. However, the role of Cdr1as in the retina remains unknown. In this study, we aimed to generate a Cdr1as knockout (KO) mouse model and investigate the retinal consequences of Cdr1as loss of function. Through in situ hybridization (ISH), we demonstrated that Cdr1as is mainly expressed in the inner retina. Using CRISPR/Cas9 targeting Cdr1as, we successfully generated KO mice. We carried out ocular examinations in the KO mice until postnatal day 500. Compared with the age-matched wild-type (WT) siblings, the KO mice displayed increased b-wave amplitude of photopic electrophysiological response and reduced vision contrast sensitivity. Through small RNA profiling of the retinas, we determined that miR-7 was downregulated, while its target genes were upregulated. Taken together, our results demonstrated for the first time that Cdr1as ablation led to a mild retinal consequence in mice, indicating that Cdr1as abundance is not indispensable for retinal development and maintenance.

Astrocytes are the principle glial cells of the central nervous system and play an active role in maintaining proper metabolism in surrounding neurons. Because of their involvement in metabolic control, it is likely that their physiology changes in response to metabolic diseases such as diabetes and associated diabetic retinopathy. Here, we investigated whether microstructural changes in astrocyte morphology occur during the early stages of chronic hyperglycemia that may be indicative of early pathogenic programs. We used MORF3 mice in conjunction with streptozotocin-induced hyperglycemia to investigate the morphology of single retinal astrocytes at an early timepoint in diabetic disease. We report that astrocytes initiate a morphological remodeling program, which depends on both the glycemic background and the presence of intravitreal injury, to alter the amount of the neuronal-associated pad and bristle microstructural motifs. Additionally, hyperglycemia increases astrocyte uptake of cholera toxin B, possibly reflecting changes in glycolipid and glycoprotein biosynthesis. Chronic hyperglycemia coupled with intravitreal injection of cholera toxin B also causes extensive leukocyte infiltration into the retina. Our results have important clinical relevance as current therapies for diabetic retinopathy involve intravitreal injection of pharmaceuticals in individuals with often poorly controlled blood glucose levels.

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.

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.

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.

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.

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.

Based on two major risk factors of glaucoma, elevated intraocular pressure (IOP) and senescence, two new series of nitric oxide (NO) donating dasatinib derivatives 1a-f, 2a-f were designed, synthesized, and biologically evaluated. The results demonstrated that the most active compound 2e effectively released NO and increased the concentration of 3',5'-cyclic guanosine monophosphate in human trabecular meshwork cells, as well as maintained senolytic activity. Topical administration of 2e in chronic ocular hypertension (COHT) glaucoma mice not only significantly eliminated senescent cells in retina but also exhibited potent retinal ganglion cells (RGCs) surviving, IOP lowering, and visual function protection activities, which were superior to those of dasatinib. Compared with younger adult mice, aged COHT mice resulted in more severe RGCs loss, while 2e demonstrated a greater capacity to improve RGCs survival. Our findings show that dual IOP lowering and senolytic functions could be a promising therapeutic strategy for glaucoma, particularly in older patients.

Purpose: Exfoliation syndrome (XFS) is a systemic connective tissue disorder with elusive pathophysiology. We hypothesize that a mouse model with elastic fiber defects caused by lack of lysyl oxidase like 1 (LOXL1 encoded by Loxl1), combined with microfibril deficiency due to Fbn1 mutation (encoding fibrillin-1, Fbn1C1041G/+) will display ocular and systemic phenotypes of XFS. Methods: Loxl1-/- was crossed with Fbn1C1041G/+ to create double mutant (dbm) mice. Intraocular pressure (IOP), visual acuity (VA), electroretinogram (ERG), and biometry were characterized in 4 genotypes (wt, Fbn1C1041G/+, Loxl1-/-, dbm) at 16 weeks of age. Optic nerve (ON) area was measured by ImageJ, and axon counting was achieved by AxonJ. Deep whole-body phenotyping was performed in wt and dbm mice. Two-tailed Student t test was used for statistical analysis. Results: There was no difference in IOP between the 4 genotypes. VA was significantly reduced only in dbm mice. The majority of biometric parameters showed significant differences in all 3 mutant genotypes compared with wt, and dbm had exacerbated anomalies compared with single mutants. Dbm mice showed reduced retinal function and significantly enlarged ON area compared with wt. Dbm mice exhibited severe systemic phenotypes related to abnormal elastic fibers, such as pelvic organ prolapse and cardiovascular and pulmonary abnormalities. Conclusions: Ocular and systemic findings in dbm mice support functional overlap between fibrillin-1 and LOXL1, 2 prominent components of exfoliation material. Although no elevated IOP or reduction of axon numbers was detected in dbm mice at 16 weeks of age, their reduced retinal function and enlarged ON area indicate early retinal ganglion cell dysfunction. Dbm mice also provide insight on the link between XFS and systemic diseases in humans. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

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.

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.

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.

Glaucoma is a common neurodegenerative disorder characterized by retinal ganglion cell death, astrocyte reactivity in the optic nerve, and vision loss. Currently, lowering the intraocular pressure (IOP) is the first-line treatment, but adjuvant neuroprotective approaches would be welcome. Vitamin C possesses neuroprotective activities that are thought to be related to its properties as a co-factor of enzymes and its antioxidant effects. Here, we show that vitamin C promotes a neuroprotective phenotype and increases gene expression related to neurotropic factors, phagocytosis, and mitochondrial ATP production. This effect is dependent on the up-regulation of secreted phosphoprotein 1 (SPP1) in reactive astrocytes via the transcription factor E2F1. SPP1⁺ astrocytes in turn promote retinal ganglion cell survival in a mouse model of glaucoma. In addition, oral administration of vitamin C lowers the IOP in mice. This study identifies an additional neuroprotective pathway for vitamin C and suggests a potential therapeutic role of vitamin C in neurodegenerative diseases such as glaucoma.

Based on the synergistic therapeutic effect of nitric oxide (NO) and Rho-associated protein kinase (ROCK) inhibitors on glaucoma, a new group of NO-donating ripasudil derivatives RNO-1-RNO-6 was designed, synthesized, and biologically evaluated. The results demonstrated that the most active compound RNO-6 maintained potent ROCK inhibitory and NO releasing abilities, reversibly depolymerized F-actin, and suppressed mitochondrial respiration in human trabecular meshwork (HTM) cells. Topical administration of RNO-6 (0.26%) in chronic ocular hypertension glaucoma mice exhibited significant IOP lowering and visual function and retinal ganglion cell (RGC) protection activities, superior to an equal molar dose of ripasudil. RNO-6 could be a promising agent for glaucoma or ocular hypertension, warranting further investigation.

A reliable animal model providing chronic and persistent ocular hypertension and characteristic neurodegeneration is essential to recapitulate human glaucoma and understand the underlying pathophysiological mechanisms behind this disease. Many approaches have been tried to establish persistently elevated intraocular pressure (IOP), while no efficient model and no systematic evaluation has been widely accepted yet. Herein, we developed a novel approach to reliably induce persistent IOP elevation using an injectable hydrogel formulated by hyperbranched macromolecular poly(ethylene glycol) (HB-PEG) and thiolated hyaluronic acid (HA-SH) under physiological conditions and established a systematic system for model evaluation. By formulation screening, an appropriate hydrogel with proper mechanical property, non-swelling profile and cytocompatibility was selected for further experiment. By intracameral injection, a persistent IOP elevation over 50% above baseline was obtained and it led to progressive retinal ganglion cell loss and ganglion cell complex thickness reduction. The evaluation of the efficacy of the model was thoroughly analyzed by whole-mounts retina immunostaining, optical coherence tomography, and hematoxylin-eosin staining for histological changes and by electroretinography for visual function changes. The N35-P50 amplitude of the pattern electroretinography and the N2-P2 amplitude of the flash visual-evoked potential were decreased, while the scotopic electroretinography showed no statistically significant changes. The in situ-forming HB-PEG/HA-SH hydrogel system could be an appropriate strategy for developing a reliable experimental glaucoma model without any confounding factors. We expect this model would be conducive to the development of neuroprotective and neuro-regenerative therapies.

Neuroinflammation significantly contributes to the pathophysiology of several neurodegenerative diseases. This is also the case in glaucoma and may be a reason why many patients suffer from progressive vision loss despite max-imal reduction in intraocular pressure. Pioglitazone is an agonist of the peroxisome proliferator-activated receptor gamma (PPARγ) whose pleiotrophic activities include modulation of cellular energy metabolism and reduction in inflammation. In this study we employed the DBA2/J mouse model of glaucoma with chronically elevated intraocu-lar pressure to investigate whether oral low-dose pioglitazone treatment preserves retinal ganglion cell (RGC) sur-vival. We then used an inducible glaucoma model in C57BL/6J mice to determine visual function, pattern electro-retinographs, and tracking of optokinetic reflex. Our findings demonstrate that pioglitazone treatment does signifi-cantly protect RGCs and prevents axonal degeneration in the glaucomatous retina. Furthermore, treatment pre-serves and partially reverses vision loss in spite of continuously elevated intraocular pressure. These data suggest that pioglitazone may provide treatment benefits for those glaucoma patients experiencing continued vision loss.

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.

Objective: Traumatic optic neuropathy (TON) causes partial or complete blindness because death of irreplaceable retinal ganglion cells (RGCs). Neuroprotective functions of erythropoietin (EPO) in the nervous system have been considered by many studies investigating effectiveness of this cytokine in various retinal disease models. It has been found that changes in retinal neurons under conditions of glial cells are effective in vision loss, therefore, the present study hypothesized that EPO neuroprotective effect could be mediated through glial cells in TON model. Materials and methods: In this experiment study, 72 rats were assessed in the following groups: intact and optic nerve crush which received either the 4000 IU EPO or saline. Visual evoked potential and optomotor response and RGC number were assessed and regenerated axons evaluated by anterograde test. Cytokines gene expression changes were compared by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Density of astrocytes cells, assessed by fluorescence intensity, in addition, possible cytotoxic effect of EPO was measured on mouse astrocyte culture in vitro. Results: in vitro data showed that EPO was not toxic for mouse astrocytes. Intravenous injection of EPO improved vision, in terms of visual behavioral tests. RGCs protection was more than two times in EPO, compared to the vehicle group. More regenerated axons were determined by anterograde tracing in the EPO group compared to the vehicle. Moreover, GFAP immunostaining showed while the intensity of reactive astrocytes was increased in injured retina, systemic EPO decreased it. In the treatment group, expression of GFAP was down-regulated, while CNTF was upregulated as assessed by qRT-PCR in the 60^th day post-crush. Conclusion: Our study showed that systemic administration of EPO can protect degenerating RGCs. Indeed, exogenous EPO exerted neuroprotective and neurotrophic functions by reducing reactive astrocytic gliosis. Therefore, reduction of gliosis by EPO may be considered as therapeutic targets for TON.

A rapidly approaching dark object evokes an evolutionarily conserved fear response in both vertebrates and invertebrates, young to old. A looming visual stimulus mimics an approaching object and triggers a similarly robust fear response in mice, resulting in freeze and flight. However, the retinal neural pathway responsible for this innate response has not been fully understood. We first explored a variety of visual stimuli that reliably induced these innate responses, and found that a looming stimulus with 2-d acclimation consistently evoked fear responses. Because the fear responses were triggered by the looming stimulus with moving edges, but not by a screen flipping from light to dark, we targeted the starburst amacrine cells (SACs), crucial neurons for retinal motion detection. We used intraocular injection of diphtheria toxin (DT) in mutant mice expressing diphtheria toxin receptors (DTR) in SACs. The looming-evoked fear responses disappeared in half of the DT-injected mice, and the other mice still exhibited the fear responses. The optomotor responses (OMRs) were reduced or eliminated, which occurred independent of the disappearance of the fear responses. A histologic examination revealed that ON SACs were reduced in both mouse groups preserved or absent fear responses. In contrast, the number of OFF SACs was different among two groups. The OFF SACs were relatively preserved in mice exhibiting continued fear responses, whereas they were ablated in mice lacking fear response to looming stimulation. These results indicate that OFF SACs and the direction-selective pathway in the retina play a role in looming-induced fear behaviors.

Purpose: To characterize postnatal ocular pathology in a Ndufs4^−/− mouse model of complex I deficiency using noninvasive retinal imaging and visual testing. Methods: Ndufs4^−/− mice and wild-type (WT) littermates were analyzed at 3, 5, and 7 weeks postnatal. Retinal morphology was visualized by optical coherence tomography (OCT). OCT images were analyzed for changes in retinal thickness and reflectivity profiles. Visual function was assessed by electroretinogram (ERG) and optomotor reflex (OMR). Results: Ndufs4^−/− animals have normal OCT morphology at weaning and develop inner plexiform layer atrophy over weeks 5 to 7. Outer retinal layers show hyporeflectivity of the external limiting membrane (ELM) and photoreceptor ellipsoid zone (EZ). Retinal function is impaired at 3 weeks, with profound deficits in b-wave, a-wave, and oscillatory potential amplitudes. The b-wave and oscillatory potential implicit times are delayed, but the a-wave implicit time is unaffected. Ndufs4^−/− animals have normal OMR at 3 weeks and present with increasing acuity and contrast OMR deficits at 5 and 7 weeks. Physiological thinning of inner retinal layers, attenuation of ELM reflectivity, and attenuation of ERG b- and a-wave amplitudes occur in WT C57BL/6 littermates between weeks 3 and 7. Conclusions: Noninvasive ocular imaging captures early-onset retinal degeneration in Ndufs4^−/− mice and is a tractable approach for investigating retinal pathology subsequent to complex I deficiency. Translational relevance: Ophthalmic imaging captures clinically relevant measures of retinal disease in a fast-progressing mouse model of complex I deficiency consistent with human Leigh syndrome.

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.

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.

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 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.

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.

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.

The Jimpy mutant mouse has a point mutation in the proteolipid protein gene (plp1). The resulting misfolding of the protein leads to oligodendrocyte death, myelin destruction, and failure to produce adequately myelinated axons in the central nervous system (CNS). It is not known how the absence of normal myelination during development influences neural function. We characterized the Jimpy mouse retina to find out whether lack of myelination in the optic nerve during development has an effect on normal functioning and morphology of the retina. Optokinetic reflex measurements showed that Jimpy mice had, in general, a functional visual system. Both PLP1 antibody staining and reverse transcriptase-polymerase chain reaction for plp1 mRNA showed that plp1 is not expressed in the wild-type retina. However, in the optic nerve, plp1 is normally expressed, and consequently, in Jimpy mutant mice, myelination of axons in the optic nerve was mostly absent. Nevertheless, neither axon count nor axon ultrastructure in the optic nerve was affected. Physiological recordings of ganglion cell activity using microelectrode arrays revealed a decrease of stimulus-evoked activity at mesopic light levels. Morphological analysis of the retina did not show any significant differences in the gross morphology, such as thickness of retinal layers or cell number in the inner and outer nuclear layer. The cell bodies in the inner nuclear layer, however, were larger in the peripheral retina of Jimpy mutant mice. Antibody labeling against cell type-specific markers showed that the number of rod bipolar and horizontal cells was increased in Jimpy mice. In conclusion, whereas the Jimpy mutation has dramatic effects on the myelination of retinal ganglion cell axons, it has moderate effects on retinal morphology and function.

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.

Purpose: This study aims to investigate the effect of quercetin (QUE) on scleral remodeling by inhibiting the PERK-EIF2α signaling pathway and to evaluate its potential role in slowing myopia. Methods: Lens-induced myopia (LIM) guinea pigs were obtained and treated with QUE. After 4 and 6 weeks of treatments, ocular biological measurements were conducted. Hematoxylin and eosin (H&E) staining was used to observe the changes in scleral morphology and thickness, and Masson staining was used to examine scleral collagen fiber arrangement. Quantitative PCR (qPCR) and Western bolt were utilized to detect the mRNA and protein expression of PERK, EIF2α, MMP-2, TIMP-2, and collagen I in the scleral tissues. Calcium ion flow in each group was measured using noninvasive micro-test technology, and reactive oxygen species levels were detected by flow cytometry. Results: Compared with the LIM group, the ocular measurements showed that the refractive errors and axial length of the eyes were significantly reduced in the LIM + QUE group (P < 0.01). H&E and Masson staining showed that sclera in the LIM + QUE group was thickened, collagen was dense, and the fiber gap was reduced. In the LIM + QUE group, the expression levels of PERK, EIF2α, and MMP-2 were decreased, whereas the expression levels of TIMP-2 and collagen I were increased. Calcium release and reactive oxygen species (ROS) in the LIM + QUE group were decreased. Conclusions: Quercetin ameliorates scleral remodeling in myopic guinea pigs by inhibiting the PERK-EIF2α signaling pathway, thereby alleviating the progression of myopia. These findings provide new experimental evidence for the potential application of quercetin in myopia prevention and treatment.

Introduction: To establish a high myopia model in C57BL/6J mice with monocular form deprivation myopia (FDM) and investigate its ocular structure pathological trajectory. Methods: Healthy 3-week-old C57BL/6J mice were divided into an FDM group (n = 36) and a control group (n = 24). The left eyes of the FDM group were patched, while the right eyes served as controls. Biometric parameters and fundus morphology were assessed at baseline and after 4, 8, and 12 weeks of form deprivation. Results: Significant differences were observed in the deprived eyes, including longer axial length, higher refractive power, deeper vitreous chambers, thinner retina, choroid, and sclera, and smaller scleral fibers' diameters under a transmission electron microscope. Retinal vascular area proportion in covered eyes decreased significantly (P < 0.05), with a decline rate of 11% from weeks 4 to 8 and a faster decline of 19% from weeks 8 to 12, while this proportion increased significantly in control eyes. Discussion: This study successfully induced a high myopia model in mice with long-term form deprivation. The axial length grew dramatically in FDM in the first 8 weeks, while the pathological progress of the fundus accelerated from weeks 8 to 12.

Pathological myopia (PM) is one of the leading causes of blindness, especially in Asia. To identify the genetic risk factors of PM, a two-stage genome-wide association study (GWAS) and replication analysis in East Asian populations is conducted. The analysis identified LILRB2 in 19q13.42 as a new candidate locus for PM. The increased protein expression of LILRB2/Pirb (mouse orthologous protein) in PM patients and myopia mouse models is validated. It is further revealed that the increase in LILRB2/Pirb promoted fatty acid synthesis and lipid accumulation, leading to the destruction of choroidal function and the development of PM. This study revealed the association between LILRB2 and PM, uncovering the molecular mechanism of lipid metabolism disorders leading to the pathogenesis of PM due to LILRB2 upregulation.

Myopia presents a formidable challenge, especially in children, where clinical management involves the use of atropine and 7-methylxanthine, necessitating multiple doses, strong side effects and encountering suboptimal compliance due to children's resistance. In response to these issues, our research proposes a self-generated electricity-driven drug delivery system (EDDDS) designed for precision myopia treatment. The EDDDS intelligently releases therapeutic drugs, such as atropine, at ocular tissues using drug-coated nanogenerators, aiming to effectively slow down the progression of myopia. This system combines nanotechnology and smart drug delivery mechanisms to achieve controlled and responsive drug administration. The nanogenerators embedded in the EDDDS generate electric fields that trigger drug release upon closing eyes conditions, providing targeted treatment. Simultaneously, the weak voltage can effectively relieve the ciliary muscle, offering additional benefits for myopia management. In vitro and in vivo experiments have demonstrated the efficacy and potential of the EDDDS in precisely modulating myopia progression. The proposed EDDDS holds significant promise as an advanced and high-precision therapeutic approach for myopia management. Its potential to address the limitations of current treatments makes it a promising avenue for future research and clinical applications.  

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.

Purpose: The aim of this study was to assess the functional and clinical impact of intravitreal administration of a neutralizing anti-IL-6 antibody in the treatment of experimental autoimmune uveitis (EAU) in mice. Methods: EAU was induced in 17 female B10.RIII mice by administering Inter-Photoreceptor-Binding-Protein (IRBP) in complete Freund's adjuvant, followed by a boost with Pertussis toxin. Intravitreal injections of anti-Interleukin (IL)-6 antibody were administered on days 10, 13, and 16 after EAU induction (day 0) into the randomized treatment eye, with an isotype antibody similarly injected into the fellow control eye. Visual acuity was assessed using the optomotor reflex via OptoDrum, and clinical scoring was performed via fundus imaging (utilizing 6 EAU grades) in a single-blinded manner on days 0, 10, 13, 16, and 18. Results: Uveitis developed in all 17 mice. Significantly higher visual acuity was observed in treated eyes compared to control eyes on days 13, 16, and 18. The most pronounced effect was noted on days 16 and 18 (p < 0.001). On days 13, 16, and 18 the number of eyes with lower EAU-score was significantly higher in the treatment group, with the most notable effect observed on day 18 (p < 0.003). Conclusion: Intravitreal administration of anti-IL-6 treatment notably mitigates experimental autoimmune uveitis in mice, both functionally and clinically. Further investigations are warranted to assess the potential of intravitreal anti-IL-6 therapy as a treatment option for non-infectious uveitis in humans.

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.

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.

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: Chronic inflammation significantly contributes to photoreceptor death in blinding retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that act as key proinflammatory factors. We recently found the first-generation BET inhibitor JQ1 alleviated sodium iodate-induced retinal degeneration by suppressing cGAS-STING innate immunity. Here, we investigated the effects and mechanism of dBET6, a proteolysis‑targeting chimera (PROTAC) small molecule that selectively degrades BET by the ubiquitin‒proteasome system, in light-induced retinal degeneration. Methods: Mice were exposed to bright light to induce retinal degeneration, and the activation of cGAS-STING was determined by RNA-sequencing and molecular biology. Retinal function, morphology, photoreceptor viability and retinal inflammation were examined in the presence and absence of dBET6 treatment. Results: Intraperitoneal injection of dBET6 led to the rapid degradation of BET protein in the retina without detectable toxicity. dBET6 improved retinal responsiveness and visual acuity after light damage (LD). dBET6 also repressed LD-induced retinal macrophages/microglia activation, Müller cell gliosis, photoreceptor death and retinal degeneration. Analysis of single-cell RNA-sequencing results revealed cGAS-STING components were expressed in retinal microglia. LD led to dramatic activation of the cGAS-STING pathway, whereas dBET6 suppressed LD-induced STING expression in reactive macrophages/microglia and the related inflammatory response. Conclusions: This study indicates targeted degradation of BET by dBET6 exerts neuroprotective effects by inhibiting cGAS-STING in reactive retinal macrophages/microglia, and is expected to become a new strategy for treatment of retinal degeneration.

Wolfram syndrome 1 (WS1) is a rare genetic disorder caused by mutations in the WFS1 gene leading to a wide spectrum of clinical dysfunctions, among which blindness, diabetes, and neurological deficits are the most prominent. WFS1 encodes for the endoplasmic reticulum (ER) resident transmembrane protein wolframin with multiple functions in ER processes. However, the WFS1-dependent etiopathology in retinal cells is unknown. Herein, we showed that Wfs1 mutant mice developed early retinal electrophysiological impairments followed by marked visual loss. Interestingly, axons and myelin disruption in the optic nerve preceded the degeneration of the retinal ganglion cell bodies in the retina. Transcriptomics at pre-degenerative stage revealed the STAT3-dependent activation of proinflammatory glial markers with reduction of the homeostatic and pro-survival factors glutamine synthetase and BDNF. Furthermore, label-free comparative proteomics identified a significant reduction of the monocarboxylate transport isoform 1 (MCT1) and its partner basigin that are highly enriched on retinal glia and myelin-forming oligodendrocytes in optic nerve together with wolframin. Loss of MCT1 caused a failure in lactate transfer from glial to neuronal cell bodies and axons leading to a chronic hypometabolic state. Thus, this bioenergetic impairment is occurring concurrently both within the axonal regions and cell bodies of the retinal ganglion cells, selectively endangering their survival while impacting less on other retinal cells. This metabolic dysfunction occurs months before the frank RGC degeneration suggesting an extended time-window for intervening with new therapeutic strategies focused on boosting retinal and optic nerve bioenergetics in WS1.

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.

Acute optic neuritis (ON) is a common cause of vision loss and is often associated with multiple sclerosis (MS). Cholesterol recycling has been identified as a key limiting factor in recovery after demyelination events. Thus, the purpose of our study was to determine if the augmentation of cholesterol transport by gentisic acid (GA) benefits retinal ganglion cell (RGC) development and myelination in organoid systems and enables the recovery of the ocular phenotype upon systemic GA treatment in a MOG-induced experimental autoimmune encephalomyelitis (EAE) ON model. The retinal organoids treated with GA demonstrate an accelerated maturation when compared to the conventionally derived organoids, which was evidenced by the improved organization of Brn3a-GFP⁺RGC and increased synaptogenesis. A GA supplementation in brain organoids leads to a 10-fold increase in NG2 and Olig2 expression. Weekly GA injections of EAE mice significantly lessened motor-sensory impairment, protected amplitudes in pattern electroretinogram recordings, and preserved visual acuity over the study period of 56 days. Furthermore, GA-treated EAE mice revealed diminished GCL/IPL complex thinning when compared to the untreated EAE mice. An optic nerve histopathology revealed less severe grades of demyelination in the GA-treated EAE cohort and fewer infiltrating cells were observed. Interventions to improve cholesterol homeostasis may be a viable approach to promoting the rehabilitation of MS patients.

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.