Myopia is the most prevalent eye disease globally with currently no safe and accessible treatments available. Bone morphogenetic protein 2 (BMP2) is located in multiple eye tissues and is crucial for eye development and differentiation. Nonetheless, the precise roles of BMP2 in myopia still unclear. In this investigation, we observed a marked decrease in scleral BMP2 levels in form-deprivation myopia (FDM) guinea pigs. Subconjunctival injections of exogenous BMP2 recombinant protein could partially counteract the decline in choroidal blood perfusion (ChBP) and myopia development. Subsequent investigations revealed that BMP2 alleviates the myopia-induced reduction in scleral Collagen type I alpha 1 expression and the elevation of scleral hypoxia-inducible factor-1α (HIF-1α) levels. In an in vitro hypoxia model, the inhibition of peroxisome proliferator-activated receptor γ (PPARγ) counteracts the influence of BMP2 on scleral extracellular matrix (ECM) remodeling in human scleral fibroblasts. In conclusion, our findings demonstrate that BMP2 inhibits myopia development and regulates scleral ECM remodeling through the enhancement of PPARγ, highlighting BMP2 as a viable and easily obtainable candidate for myopia management.

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

The function of rod photoreceptors as dim light photon detectors depends critically on the molecular properties of their visual pigment, rhodopsin. The structure of rhodopsin has evolved under selective pressure to light conditions of different spectral composition and overall intensity. One notable example is the switch of mammalian species from nocturnal to diurnal environments. Comparison of the rhodopsins of the nocturnal mouse and the diurnal human reveals high sequence similarity, with only 18 distinct amino acids. Here, we examined the role of one of these, mouse phenylalanine (F) vs. human leucine (L) at position 88, in modulating the molecular properties of rhodopsin and the function of rods by generating an F88L rhodopsin knock-in mouse. Our detailed in vitro analysis of the physicochemical properties of this mutant F88L rhodopsin showed a higher conformational stability and more efficient chromophore regeneration compared to the WT mouse pigment. We also found that the decay of metarhodopsin II in the F88L mutant occurred significantly faster than in the WT. However, despite these molecular changes, the visual function of knock-in mutant mice carrying the F88L mutation was not significantly altered by this amino acid change. These findings demonstrate the role of the F88L evolutionary switch in enhancing the stability and regeneration of rhodopsin towards visual function in diurnal human rods over nocturnal mouse rods. Our results provide new insights into the molecular evolution of rhodopsin in vertebrates.

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

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

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

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

The propagation of signals from the retina to the brain is crucial for proper visual and non-visual behavior. This signal propagation is mediated by the output neurons of the retina; the retinal ganglion cell (RGC). The development of RGCs is a complex process that relies on multiple stages of overlapping but distinct developmental programs, with hierarchal transcriptional changes facilitating broad RGC pathfinding and guidance, then further specificity generated by a mixture of transcriptional regulation, activity-dependent mechanisms, and paracrine signaling. The understanding of RGC development is critical to work that seeks to regenerate the retina following injury or disease. In this thesis, I explored two distinct methods of RGC development and the classes of RGCs that are generated. Firstly, I explored the role of a novel transcription factor POU3F1, demonstrating its critical role in the formation of contralaterally projecting retinal ganglion cells. Secondly, I explored the role of glial cell exocytosis in retinal development, where I found it was crucial for the proper targeting of axons to the circadian centers of the brain and for the proper development of circadian photoentrainment. Taken together this thesis provides a comprehensive overview of two stages of development.

Circadian photoentrainment depends on intrinsically photosensitive retinal ganglion cells (ipRGCs), which convey environmental light information to the hypothalamus. While the anatomy and function of mature ipRGCs are well characterized, the mechanisms guiding their developmental integration into the circadian circuit remain unclear. Here, using transsynaptic rabies tracing in mice, we show that Müller glia are primary upstream partners of developing hypothalamus-projecting ipRGCs, suggesting a role for glia-neuron communication in the assembly of the photoentrainment pathway. We further show that disrupting SNARE function specifically in Müller glia during early postnatal development impairs ATP release, induces ipRGC hyper-responsiveness to light, and results in defective photoentrainment. In contrast, visual acuity and the pupillary light reflex, which are mediated by different RGC/ipRGC subtypes, remain unaffected. These results indicate a critical and circuit-specific role for Müller glia in shaping the development and function of the retinal pathway underlying circadian photoentrainment.

Background: Myopia has reached epidemic proportions globally with limited therapeutic targets. This study elucidated the pivotal role of retinal epitranscriptomic regulation in myopia pathogenesis, aiming to identify novel therapeutic targets for myopia control. Methods: The study enrolled 38 myopic patients scheduled for refractive surgery to analyze peripheral blood N6-methyladenosine (m6A) levels. In parallel, we established a murine form-deprivation myopia (FDM) model to examine retinal m6A dynamics and Alkylation repair homolog protein 5 (ALKBH5) expression. Pharmacological interventions using the ALKBH5 inhibitor DDO-2728 and Extracellular signal-related kinases 1 and 2 (ERK1/2) activator Ro67-7476 were administered intravitreally. To genetically validate the role of ALKBH5, ALKBH5-knockout (ALKBH5-KO) mice were subjected to the FDM model. Comprehensive assessments included refractive measurements, retinal structure analysis, and molecular profiling of inflammatory and extracellular matrix (ECM) markers. Statistical analyses employed Pearson's correlation for clinical data and ANOVA with Tukey's post hoc tests for experimental comparisons. Results: Clinical data revealed significant inverse correlations between blood m6A levels and myopia severity (refraction: r = 0.435, p < 0.01; axial length: r = -0.338, p < 0.05). FDM retinas exhibited progressive m6A reduction and specific upregulation of ALKBH5 in the ganglion cell layer (GCL), with no changes observed in other m6A regulators. Mechanistic studies demonstrated that intravitreal injection of the selective ALKBH5 inhibitor DDO-2728 (2.5 mM) effectively attenuated myopia progression, mitigated retinal thinning, suppressed inflammatory mediators, and modulated early ECM dysregulation through specific suppression of ERK1/2 hyperphosphorylation. Crucially, genetic ablation of ALKBH5 recapitulated the protective phenotype of the inhibitor, conferring significant resistance to myopia development and associated retinal pathology. Importantly, co-administration of the ERK1/2 activator Ro 67-7476 completely abolished DDO-2728's protective effects. Conclusions: Peripheral blood m6A reduction correlates with human myopia severity. Retinal ALKBH5 upregulation drives myopia progression through ERK1/2 hyperactivation. Both pharmacological inhibition and genetic deletion of ALKBH5 effectively protect against myopia, attenuate inflammatory responses, and mitigate ECM dysregulation, mechanistically linked to the suppression of ERK1/2 signaling. These findings collectively identify ALKBH5 as a novel therapeutic target and DDO-2728 as a promising strategy for myopia control.

Focusing on the mTOR-HIF-1α signaling pathway, this study investigated the mechanism through which the traditional Chinese medicine compound Mingshi Formula delays the progression of form-deprivation myopia (FDM) in guinea pigs. The guinea pigs were divided into the normal control group (NC), FDM group, Mingshi Formula low-dose group (FDM + Low), medium-dose group (FDM + Medium), high-dose group (FDM + High), and MTOR inhibitor group (FDM + RapaLink-1). The guinea pig model of FDM was established by applying 3D-printed hoods modified by a latex balloon with 60% light transmission for 4 weeks. Refractive error changes were monitored using a refractometer. Axial length was quantitatively analyzed using A-scan ultrasound, choroidal thickness was measured with SD-OCT, and structural changes of the choroid and sclera were observed after hematoxylin-eosin (HE) staining. At the molecular level, the expression levels of the mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), HIF-1α, LDHA, PKM2, MMP2, Collagen I, and α-SMA in the sclera were measured by RT-qPCR and Western blotting. The spatial distribution characteristics of mTOR, p-mTOR, HIF-1α, and Collagen I were verified via immunofluorescence techniques. The results demonstrated that the Mingshi Formula significantly decreased myopic refractive error and axial length (p < 0.01), increased choroidal thickness (p < 0.05), downregulated the gene and protein expression of mTOR, p-mTOR, HIF-1α, LDHA, PKM2, MMP2, and α-SMA in response to hypoxia, and upregulated the expression of Collagen I compared to the FDM group. We demonstrated that MingShi formula modulates the mTOR/HIF-1α signaling axis to ameliorate scleral hypoxic metabolic homeostasis, regulate Collagen synthesis, inhibit aberrant extracellular matrix remodeling, and ultimately delay myopia progression.

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

Glaucoma remains a major cause of irreversible blindness worldwide, driving the need for preclinical models that accurately replicate human disease and support the development of novel treatment methods. Intracameral microbead injection is a widely used method to induce ocular hypertension and mimic the pathophysiology of glaucoma. Most studies using this model focus on retinal ganglion cell (RGC) loss and occasionally pattern electroretinography (pERG) for functional assessment of RGC. In our study, we broaden model characterization by incorporating behavioral, functional, and morphological assessments using in vivo monitoring. In addition, we explored how behavioral, functional, and morphological read-outs correlated with intraocular pressure (IOP) in this model. Spectral-domain optical coherence tomography (SD-OCT) was used for quantitative evaluation of inner retinal layer (IRL) thickness. PERG was recorded to assess RGC function. Optomotor tracking was performed to evaluate visual acuity (VA) and contrast sensitivity (CS). After 4 weeks, the animals were euthanized, and retinal wholemounts with optic nerves were collected for RGC quantification and axon assessment. As expected, we found a significant thinning of IRL as confirmed by SD-OCT imaging. Additionally, pERG recordings revealed diminished RGC function, evidenced by reduced amplitude, whereas optomotor testing detected significant declines in VA and CS. Significant correlations were found between IOP and all parameters. The magnetic microbead-induced ocular hypertension model successfully mimicked structural and functional damage of RGCs, with early visual function declines linked to elevated IOP. Strong correlations between structural and functional parameters enhanced the translational relevance of this model in exploring new neuroprotective and regenerative therapies.

ROSAH (retinal dystrophy, optic nerve edema, splenomegaly, anhidrosis, and headache) syndrome is a rare genetic disease caused by variants in alpha-kinase 1 (ALPK1) resulting in downstream pro-inflammatory signaling mediated by the TIFA/TRAF6/NF-κB pathway. Here, we report the design of an ALPK1 inhibitor, DF-003, with pharmacokinetic properties suitable for daily oral dosing. In biochemical assays, DF-003 potently inhibits human ALPK1 (IC₅₀ = 1.5 nM) and the ROSAH disease-causing mutant ALPK1[T237M] (IC₅₀ = 16 nM). When tested against a panel of 394 human kinases, DF-003 exhibits ≥860-fold selectivity over the closest kinase. In cell-based assays, DF-003 suppresses inflammatory cytokine signaling mediated both by wild-type ALPK1 and the disease-causing ALPK1[T237M] mutant. Using mice heterozygous for wild-type human ALPK1 and ALPK1^T237M established to model ROSAH syndrome that exhibit retinal microglial infiltration, astrocyte activation, and inflammatory cytokine upregulation in the retina, optic nerve, and cortex, we show that orally administered DF-003 is sufficient to inhibit these inflammatory phenotypes.

Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is one of the significant risk factors in glaucoma; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. A high-fidelity CRISPR-Cas13 (hfCas13X) system is employed to selectively target and knock down Cx43 expression in macroglia. The findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in macroglia using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases.

Genetic predisposition has been increasingly reported in patients with high myopia. A previous study reported that a deleterious mutation in cathepsin H (CTSH) gene causes high myopia. However, the phenotypic and mechanistic characteristics of Ctsh-deficient mice remain unknown. In this study, we generated a Ctsh knockout mouse model using CRISPR/Cas9, and confirmed the abolishment of Ctsh by Sanger sequencing. In the mouse model, myopic shift was measured by photorefraction and axial elongation was detected by magnetic resonance imaging (MRI). Retinal function detected by electroretinogram (ERG) indicated the scotopic responses of knockout mice were reduced, and slight retinal thinning was observed using optical coherence tomography (OCT). In addition, ribonucleic acid sequencing (RNA-seq) and real-time polymerase chain reaction (RT-PCR) demonstrated gene expression changes in the retinas of knockout mice. Our results indicated that Ctsh plays an important role in emmetropization and that its loss-of-function leads to myopia development.

Interaction between Müller cells and microglia aggravates neuroinflammation, resulting in retinal ganglion cell (RGC) death in glaucoma. Here, we investigated how tumor necrosis factor-alpha (TNF-α) produced by activated microglia mediates the crosstalk between Müller cells and microglia and impacts RGC injury in a chronic ocular hypertension (COH) glaucoma model. In COH retinas, elevated TNF-α induced the activation of Müller cells and microglia, and recruited microglia to the ganglion cell layer. Co-culture with Müller cells enhanced TNF-α-induced microglial activation, migration, and proliferation. Both in vivo and in vitro experiments confirmed that chemokine C-C motif ligand 2 (CCL2), primarily released from Müller cells, mediated the TNF-α-induced effects on microglia in COH retinas. Knockdown of CCL2 attenuated RGC damage and vision loss. Our results demonstrate that TNF-α released from microglia induces the secretion of CCL2 from Müller cells, thus inducing microglial activation and migration, exacerbating retinal neuroinflammation and RGC injury in glaucoma.

Photorefraction is seven-decade old technology that allows objective and non-invasive estimation of the eye’s refractive error using retro-illuminated photographs of the pupil. A special variant of this technique—eccentric infrared photorefraction—has now become the technology of choice for the screening of uncorrected refractive errors and other amblyogenic factors in the pediatric population. Eccentric infrared photorefraction is also the preferred measurement tool for scientific investigations on the physiology and pathobiology of the near-triadic reflex (accommodation, convergence and pupil miosis). Broadly, this technique uses an extended near-infrared light source that is mounted eccentric to the camera aperture to illuminate the retina. The optically double-pass reflected light generates a retro-illuminated luminance profile across the pupil, the gradient of which may be used to estimate the eye’s refractive power using standard calibration techniques. The present review summarizes the origins of photorefraction and then delves into the optical principles of eccentric infrared photorefraction, its properties, calibration techniques and associated pitfalls. The review also discusses the present and future use-cases of this technology. Overall, this review provides the basics of photorefraction for early readers of this topic and offers some practical guidelines for the moderate to advanced users of this technique.

The loss of integrity of the blood retina barrier (BRB) is a key pathological hallmark of vision-threatening complications in diabetic retinopathy (DR). Although DR is considered a microvascular disease, mounting evidence from mouse models and patients show that inflammation is closely connected with microvasculopathy. Inflammatory responses during retinal pathophysiology are often orchestrated by microglia, resident innate immune cells of the retina. However, the precise role of microglia activity during DR pathogenesis remains elusive. Here, we used an anti-PDGFRβ antibody and inducible endothelial cell-specific PDGFB-KO during postnatal development of retinal vasculature to reproduce a key feature of DR pathology in mice. In addition, we applied a minocycline therapy to modulate retinal inflammation. Postnatal depletion of pericytes or loss of PDGFB in retinal vessels altered BRB integrity and triggered secretion of angiogenic and inflammatory factors with concomitant microglia reactivity, which was sustained in retinas of adult mice. Microglia reactivity was accompanied by upregulation of disease-associated genes. Notably, minocycline attenuated the cycle of inflammatory responses in young and mature retinas, thereby preserving retinal vascular and structural integrity in mice. Together, our findings suggest that immunomodulation of microglia-driven inflammatory responses preserves retinal vasculature and maintains BRB integrity in 2 different mouse models of human DR.

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

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

Myopia, a predominant cause of visual impairment, is often associated with scleral extracellular matrix (ECM) remodeling and axial elongation. Currently, effective therapeutic strategies for addressing scleral ECM remodeling remain limited, necessitating the development of new treatments. Quercetin, a natural flavonoid, has been shown to alleviate ECM remodeling. However, its hydrophobic nature limits clinical application. To address this, we developed a quercetin-loaded exosome delivery system (Exo-Que) to enhance quercetin bioavailability and investigated its effects and mechanisms in myopia prevention. This system exhibited excellent aqueous solubility, enhanced corneal permeability, and prolonged precorneal retention, enabling low-dose administration with significant efficacy. In the initial phase of treatment, Exo-Que showed a pronounced myopia prevention effect, with reductions of 58.41 % in refractive error progression and 38.46 % in axial length growth after two weeks of treatment. As the treatment duration extended to four weeks, its therapeutic efficacy remained robust, achieving reductions of 59.97 % and 35.85 %, respectively. The therapeutic efficacy of Exo-Que was comparable to that of the 0.1 % atropine group (at two weeks, reducing 59.07 % and 35.9 %, respectively; at four weeks, 59.84 % and 37.74 %, respectively). Mechanistically, Exo-Que inhibited the activation of the IRE1-XBP1, PERK-eIF2, and ATF6 pathways, alleviating endoplasmic reticulum stress. Furthermore, it suppressed ferroptosis by modulating GRP78-ACSL4 and GRP78-GPX4 protein interactions, thus mitigating ECM remodeling and slowing myopia progression. Besides, Exo-Que showed excellent biosafety in both in vitro and in vivo studies. Collectively, these results highlight the promising therapeutic potential of Exo-Que for myopia prevention.

Keywords: EAE; RGC; electrophysiology; fatty acids; ketogenic diet; multiple sclerosis; optic neuritis; saturated fats.

Purpose: The development and progression of myopia are influenced by the Wnt7b/β-catenin signaling pathway. This study investigated the specific impacts of this pathway on the biomechanical properties of the sclera by altering the expression of matrix metalloproteinase-2 (MMP-2) to regulate type I collagen (collagen I) levels. Methods: We examined the effects of the Wnt7b/β-catenin signaling pathway and MMP-2 on human fetal scleral fibroblasts (HFSFs) and the sclera of guinea pigs with form-deprivation myopia (FDM). To explore the effects of the Wnt7b/β-catenin pathway and the role of MMP-2 in this context, we treated HFSFs and guinea pig sclera with specific agonists and inhibitors targeting Wnt7b/β-catenin and MMP-2. The expression levels of Wnt7b, MMP-2, and collagen I were subsequently analyzed quantitatively via western blot (WB) analysis, immunofluorescence, and quantitative real-time PCR (qRT-PCR) to assess protein and mRNA changes in response to pathway manipulation. Atomic force microscopy (AFM) was used to measure the elastic modulus of the treated HFSFs and guinea pig sclera to directly evaluate changes in cell and tissue stiffness. In the FDM model, essential ocular parameters such as refractive error and axial length (AL) were also assessed. Results: In vivo and in vitro activation of the Wnt7b/β-catenin signaling pathway significantly upregulated MMP-2 expression, which was accompanied by a notable decrease in collagen I levels. This change led to a reduction in the elastic modulus of both HFSFs and the sclera of guinea pigs with FDM. These significant biomechanical changes in the scleral tissue were indicated by a reduction in stiffness. Alterations in scleral biomechanics were associated with changes in ocular parameters, including an increase in AL and a myopic shift in refraction. Conclusions: The Wnt7b/β-catenin pathway regulates scleral biomechanics by upregulating MMP-2 expression, which leads to increased collagen I degradation and, consequently, an increase in axial elongation and a myopic shift in refractive error.

Purpose: Retinal vein occlusion (RVO) is a common retinal vascular disease that severely threatens visual function. This study aims to elucidate the role of the complement C3/C3aR signaling pathway in a laser-induced RVO mouse model and to explore its potential as a therapeutic target. Methods: RVO was induced in C57BL/6J mice using laser photocoagulation combined with photosensitizer dye administration. Two days later, retinal tissues were collected for bulk RNA sequencing. The activation of the C3/C3aR signaling pathway was validated through RT-qPCR and Western blot. The C3aR antagonist SB290157 (C3aRA) was administered intravitreally and retinal morphological and functional changes were examined 1, 2, and 8 days later by optical coherence tomography (OCT), fundus photography (FP), and fluorescein angiography (FA), optomotor response (OKR) test, and electroretinogram (ERG). Results: RVO mice exhibited marked increases in retinal thickness (P < 0.001) and fluorescence leakage (P < 0.01) compared to the sham-laser group. Bulk RNA-seq revealed significant upregulation of the complement pathway. Elevated expression of C3 and C3aR (P < 0.05) was confirmed by RT-qPCR and Western blot. Blocking C3aR with SB290157 significantly alleviated RVO-induced retinal edema, vascular leakage, and structural damage. Functional assessment showed that SB290157 treatment significantly improved contrast sensitivity (P < 0.05), increased b-wave (P < 0.001), and oscillatory potentials (Ops) amplitudes (P < 0.05) in RVO mice. RNA-seq analysis demonstrated that SB290157 significantly reduced the inflammatory mediator-related pathways and upregulated visual perception pathways (P < 0.05). Conclusions: The complement C3/C3aR signaling pathway is critically involved in RVO-induced retinal damage and targeting this pathway may be a promising approach for RVO treatment.

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.

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.

Delivering drugs to the posterior eye segment is a complex task, particularly for treating retinal diseases. Neuroprotective approaches to maintain neuronal integrity have garnered significant attention in recent research. Here, we developed a mucoadhesive nanoparticulate system based on thiolated hyaluronic acid-modified cationic liposomes (HA-SH@liposomes) for topical administration. To fabricate these liposomes, we utilized microfluidic technology with a toroidal mixer to ensure consistent size and stability. Cationic liposomes were prepared by using the microfluidic method, and Epoetin-β (EPOβ), a neuroprotective agent, was loaded into the liposomes. Following this, HA-SH was conjugated to the EPOβ/HA-SH@liposomes using a postmicrofluidics conjugation method, wherein HA-SH was added dropwise to facilitate electrostatic interactions between the cationic liposomes and the anionic polymer. The resulting liposomes exhibited a mean size of 144 ± 1.3 nm and a polydispersity index (PDI) of 0.09 ± 0.01, indicating their uniformity. We evaluated the biocompatibility of the EPOβ/HA-SH@liposomes in vitro using live/dead and MTS assays on the RGC-5 cell line, demonstrating no notable cytotoxicity compared to the controls. To assess the in vivo performance, we conducted extensive ophthalmological examinations in C57/BL6 mice, including immunofluorescence staining to track the distribution of EPOβ and EPOβ/HA-SH@liposomes within the eyeball. Additionally, we quantified EPOβ levels in the retina using an enzyme-linked immunosorbent assay (ELISA) kit after the topical application of free EPOβ and the EPOβ/HA-SH@liposome formulation. The immunofluorescence staining revealed efficient delivery of EPOβ into the retina and choroid via the transcorneal route when administered as EPOβ/HA-SH@liposomes. ELISA results showed that the liposomal formulation achieved approximately 1.9× greater penetration efficiency than free EPOβ. Furthermore, optokinetic response (OKR) assays indicated that animals treated with EPOβ/HA-SH@liposomes exhibited slightly improved visual acuity compared with those treated with free EPOβ, though the difference was not statistically significant. In conclusion, the topical ocular administration of EPOβ/HA-SH@liposomes facilitated the efficient delivery of EPOβ to the retina, promoting retinal recovery and confirming its neuroprotective properties. This preclinical study provides a foundation for innovative strategies in the topical delivery of neuroprotective agents in ocular therapy.

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.

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.

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.

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.

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.

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

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.

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

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

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.

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.

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

As part of the central nervous system, the optic nerve, composed of axons from retinal ganglion cells (RGCs), generally fails to regenerate on its own when injured in adult mammals. An innovative approach to promoting optic nerve regeneration involves manipulating the interactions between amacrine cells (ACs) and RGCs. Here, we identified a unique AC subtype, dopaminergic ACs (DACs), that responded early after optic nerve crush by down-regulating neuronal activity and reducing retinal dopamine (DA) release. Activating DACs or augmenting DA release with levodopa demonstrated neuroprotective effects and modestly enhanced axon regeneration. Within this context, we pinpointed the DA receptor D1 (DRD1) as a critical mediator of DAC-derived DA and showed that RGC-specific Drd1 overexpression effectively overcame subtype-specific barriers to regeneration. This strategy markedly boosted RGC survival and axon regeneration after crush and preserved vision in a glaucoma model. This study unveils the crucial role of DAC-derived DA signaling in optic nerve regeneration, holding promise for therapeutic insights into neural repair.

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

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

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

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

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.

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.

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.  

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.

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

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.

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: 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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.

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.

Collectively, retinal neurodegenerative diseases are comprised of numerous subtypes of disorders which result in loss of a varying cell types in the retina. These diseases can range from glaucoma, which results in retinal ganglion cell death, to age-related macular degeneration and retinitis pigmentosa, which result in cell death of the retinal pigment epithelium, photoreceptors, or both. Regardless of the disease, it's been recently found that increased release of proinflammatory cytokines and proliferation of active microglia result in a remarkably proinflammatory microenvironment that assists in the pathogenesis of the disease; however, many of the details of these inflammatory events have yet to be elucidated. In an ongoing study, we have used systems genetics to identify possible models of spontaneous polygenic age-related macular degeneration by mining the BXD family of mice using single nucleotide polymorphism analyses of known genes associated with the human retinal disease. One BXD strain (BXD32) was removed from the study as the rate of degeneration observed in these animals was markedly increased with a resultant loss of most all photoreceptors by 6 months of age. Using functional and anatomical exams including optokinetic nystamography, funduscopy, fluorescein angiography, and optical coherence tomography, along with immunohistochemical analyses, we show that the BXD32 mouse strain exhibits a severe neurodegenerative phenotype accompanied by adverse effects on the retinal vasculature. We also expose the concurrent establishment of a chronic proinflammatory microenvironment including the TNFα secretion and activation of the NF-κB and JAK/STAT pathways with an associated increase in activated macrophages and phagoptosis. We conclude that the induced neuronal death and proinflammatory pathways work synergistically in the disease pathogenesis to enhance the rate of degeneration in this spontaneous polygenic model of inherited retinal dystrophy.

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.

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.

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.

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