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: 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.

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.

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.

The optomotor reflex (OMR) provides a behavioral assessment of an animal's contrast sensitivity and visual acuity. Mice or rats are typically placed directly onto a small circular platform by hand; however, handling animals like this can stimulate stress and anxiety, which introduce confounding factors when interpreting data. It has been shown that non-aversive handling methods, such as picking up mice or rats in a familiar tunnel/tube, can reduce anxiety. This is of particular interest in studies where animals display heightened stress, overactivity, or motor dysfunction, resulting in an inability to stay on the platform. A team led by Drs. Kiyoharu J. Miyagishima and Francisco M. Nadal-Nicolás have redesigned the conventional OMR platform to provide semi-closed containment. This makes it possible for the first time to record the optomotor reflex in the 13-lined ground squirrel, which is one of the few mammals that can see color. It has a visual streak with a high density of cones similar to the human macula providing an attractive model for studying effects on the cone visual system.

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.

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: 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.

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.

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.

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

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

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.

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.

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

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.

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

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

Purpose: 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.

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.

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

Background. The neuronal ceroid lipofuscinoses (CLN diseases) are fatal lysosomal storage diseases causing neurodegeneration in the CNS. We have previously shown that neuroinflammation comprising innate and adaptive immune reactions drives axonal damage and neuron loss in the CNS of palmitoyl protein thioesterase 1-deficient (Ppt1−/−) mice, a model of the infantile form of the diseases (CLN1). Therefore, we here explore whether pharmacological targeting of innate immune cells modifies disease outcome in CLN1 mice. Methods. We applied treatment with PLX3397 (150 ppm in the chow), a potent inhibitor of the colony stimulating factor-1 receptor (CSF-1R) to target innate immune cells in CLN1 mice. Experimental long-term treatment was non-invasively monitored by longitudinal optical coherence tomography and rotarod analysis, as well as analysis of visual acuity, myoclonic jerks, and survival. Treatment effects regarding neuroinflammation, neural damage, and neurodegeneration were subsequently analyzed by histology and immunohistochemistry. Results. We show that PLX3397 treatment attenuates neuroinflammation in CLN1 mice by depleting pro-inflammatory microglia/macrophages. This leads to a reduction of T lymphocyte recruitment, an amelioration of axon damage and neuron loss in the retinotectal system, as well as reduced thinning of the inner retina and total brain atrophy. Accordingly, long-term treatment with the inhibitor also ameliorates clinical outcomes in CLN1 mice, such as impaired motor coordination, visual acuity, and myoclonic jerks. However, we detected a sex- and region-biased efficacy of CSF-1R inhibition, with male microglia/macrophages showing higher responsiveness toward depletion, especially in the gray matter of the CNS. This results in a better treatment outcome in male Ppt1−/− mice regarding some histopathological and clinical readouts and reflects heterogeneity of innate immune reactions in the diseased CNS. Conclusions. Our results demonstrate a detrimental impact of innate immune reactions in the CNS of CLN1 mice. These findings provide insights into CLN pathogenesis and may guide in the design of immunomodulatory treatment strategies.

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

N-Methyl-D-aspartate (NMDA)-induced neuronal cell death is involved in a large spectrum of diseases affecting the brain and the retina such as Alzheimer’s disease and diabetic retinopathy. Associated neurological impairments may result from the inhibition of neuronal plasticity by Nogo-A. The objective of the current study was to determine the contribution of Nogo-A to NMDA excitotoxicity in the mouse retina. We observed that Nogo-A is upregulated in the mouse vitreous during NMDA-induced inflammation. Intraocular injection of a function-blocking antibody specific to Nogo-A (11C7) was carried out 2 days after NMDA-induced injury. This treatment significantly enhanced visual function recovery in injured animals. Strikingly, the expression of potent pro-inflammatory molecules was downregulated by 11C7, among which TNFα was the most durably decreased cytokine in microglia/macrophages. Additional analyses suggest that TNFα downregulation may stem from cofilin inactivation in microglia/macrophages. 11C7 also limited gliosis presumably via P.Stat3 downregulation. Diabetic retinopathy was associated with increased levels of Nogo-A in the eyes of donors. In summary, our results reveal that Nogo-A-targeting antibody can stimulate visual recovery after retinal injury and that Nogo-A is a potent modulator of excitotoxicity-induced neuroinflammation. These data may be used to design treatments against inflammatory eye diseases.

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and the underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture at the expense of image detail. Here, we show that retinal optical quality improves 2-fold during terminal development, and that this enhancement is caused by nuclear inversion. We further demonstrate that improved retinal contrast transmission, rather than photon-budget or resolution, enhances scotopic contrast sensitivity by 18–27%, and improves motion detection capabilities up to 10-fold in dim environments. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast transmission as a decisive determinant of mammalian visual perception.

Spatial resolution is a key property of eyes when it comes to understanding how animals' visual signals are perceived. This property can be robustly estimated by measuring the contrast sensitivity as a function of different spatial frequencies, defined as the number of achromatic vertical bright and dark stripe pairs within one degree of visual angle. This contrast sensitivity function (CSF) has been estimated for different animal groups, but data on fish are limited to two free-swimming, freshwater species (i.e., goldfish and bluegill sunfish). In this study, we describe the CSF of a small marine cryptobenthic fish (Tripterygion delaisi) using an optokinetic reflex approach. Tripterygion delaisi features a contrast sensitivity that is as excellent as other fish species, up to 125 (reciprocal of Michelson contrast) at the optimal spatial frequency of 0.375 c/°. The maximum spatial resolution is instead relatively coarse, around 2.125 c/°. By comparing our results with acuity values derived from anatomical estimates of ganglion cells' density, we conclude that the optokinetic reflex seems to be adapted to process low spatial frequency information from stimuli in the peripheral visual field and show that small marine fish can feature excellent contrast sensitivity at optimal spatial frequency.

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

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

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