ABSTRACT
The vasculature of the central nervous system is a 3D lattice composed of laminar vascular beds interconnected by penetrating vessels. The mechanisms controlling 3D lattice network formation remain largely unknown. Combining viral labeling, genetic marking, and single-cell profiling in the mouse retina, we discovered a perivascular neuronal subset, annotated as Fam19a4/Nts-positive retinal ganglion cells (Fam19a4/Nts-RGCs), directly contacting the vasculature with perisomatic endfeet. Developmental ablation of Fam19a4/Nts-RGCs led to disoriented growth of penetrating vessels near the ganglion cell layer (GCL), leading to a disorganized 3D vascular lattice. We identified enriched PIEZO2 expression in Fam19a4/Nts-RGCs. Piezo2 loss from all retinal neurons or Fam19a4/Nts-RGCs abolished the direct neurovascular contacts and phenocopied the Fam19a4/Nts-RGC ablation deficits. The defective vascular structure led to reduced capillary perfusion and sensitized the retina to ischemic insults. Furthermore, we uncovered a Piezo2-dependent perivascular granule cell subset for cerebellar vascular patterning, indicating neuronal Piezo2-dependent 3D vascular patterning in the brain.
Subject(s)
Cerebellum , Neurons , Retina , Animals , Female , Male , Mice , Cerebellum/metabolism , Cerebellum/blood supply , Cerebellum/cytology , Ion Channels/metabolism , Mice, Inbred C57BL , Neurons/metabolism , Retina/cytology , Retina/metabolism , Retinal Ganglion Cells/metabolism , Retinal Vessels/metabolismABSTRACT
Retinal regeneration has been mostly studied after widespread tissue injury, but it is not well understood how the retina regenerates at the cellular level following loss of specific cell types. In a new study, Jeff Mumm and colleagues selectively ablate retinal ganglion cells in zebrafish and find that the retina elicits different genetic responses in a context-dependent manner to replace lost cells. To find out more about the story behind the paper, we caught up with first author Kevin Emmerich and corresponding author Jeff Mumm, Associate Professor in Ophthalmology at Johns Hopkins University.
Subject(s)
Zebrafish , Animals , Humans , History, 21st Century , Retinal Ganglion Cells/physiology , Retinal Ganglion Cells/metabolism , Retina , History, 20th Century , Regeneration/physiology , Ophthalmology/historyABSTRACT
Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased 'fate bias'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.
Subject(s)
Animals, Genetically Modified , Retinal Ganglion Cells , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Nerve Regeneration/genetics , Nerve Regeneration/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , CRISPR-Cas Systems/genetics , Regeneration/genetics , Regeneration/physiology , Retina/metabolism , Retina/cytology , Stem Cells/metabolism , Stem Cells/cytology , Transcription FactorsABSTRACT
As essential components of gene expression networks, transcription factors regulate neural circuit assembly. The homeobox transcription factor encoding gene, gs homeobox 1 (gsx1), is expressed in the developing visual system; however, no studies have examined its role in visual system formation. In zebrafish, retinal ganglion cell (RGC) axons that transmit visual information to the brain terminate in ten arborization fields (AFs) in the optic tectum (TeO), pretectum (Pr), and thalamus. Pretectal AFs (AF1-AF9) mediate distinct visual behaviors, yet we understand less about their development compared to AF10 in the TeO. Using gsx1 zebrafish mutants, immunohistochemistry, and transgenic lines, we observed that gsx1 is required for vesicular glutamate transporter, Tg(slc17a6b:DsRed), expression in the Pr, but not overall neuron number. gsx1 mutants have normal eye morphology, yet they exhibit impaired visual ability during prey capture. RGC axon volume in the gsx1 mutant Pr and TeO is reduced, and AF7 that is active during feeding is missing which is consistent with reduced hunting performance. Timed laser ablation of Tg(slc17a6b:DsRed)-positive cells reveals that they are necessary for AF7 formation. This work is the first to implicate gsx1 in establishing cell identity and functional neural circuits in the visual system.
Subject(s)
Animals, Genetically Modified , Gene Expression Regulation, Developmental , Homeodomain Proteins , Retinal Ganglion Cells , Zebrafish Proteins , Zebrafish , Animals , Axons/metabolism , Axons/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mutation , Retinal Ganglion Cells/metabolism , Superior Colliculi/metabolism , Superior Colliculi/growth & development , Transcription Factors/genetics , Transcription Factors/metabolism , Visual Pathways/growth & development , Visual Pathways/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.
Subject(s)
Mice, Inbred C57BL , Retina , Retinal Ganglion Cells , Synapses , Animals , Mice , Retina/metabolism , Retina/physiology , Male , Synapses/physiology , Synapses/metabolism , Female , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/physiology , Amacrine Cells/physiology , Amacrine Cells/metabolism , Motion Perception/physiology , Nerve Net/physiology , Nerve Net/metabolism , Visual Pathways/physiology , Visual Pathways/metabolismABSTRACT
Glaucoma is characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, and its risk increases with aging. Yet comprehensive insights into the complex mechanisms are largely unknown. Here, we found that anti-aging molecule Sirt6 was highly expressed in RGCs. Deleting Sirt6 globally or specifically in RGCs led to progressive RGC loss and optic nerve degeneration during aging, despite normal intraocular pressure (IOP), resembling a phenotype of normal-tension glaucoma. These detrimental effects were potentially mediated by accelerated RGC senescence through Caveolin-1 upregulation and by the induction of mitochondrial dysfunction. In mouse models of high-tension glaucoma, Sirt6 level was decreased after IOP elevation. Genetic overexpression of Sirt6 globally or specifically in RGCs significantly attenuated high tension-induced degeneration of RGCs and their axons, whereas partial or RGC-specific Sirt6 deletion accelerated RGC loss. Importantly, therapeutically targeting Sirt6 with pharmacological activator or AAV2-mediated gene delivery ameliorated high IOP-induced RGC degeneration. Together, our studies reveal a critical role of Sirt6 in preventing RGC and optic nerve degeneration during aging and glaucoma, setting the stage for further exploration of Sirt6 activation as a potential therapy for glaucoma.
Subject(s)
Aging , Disease Models, Animal , Glaucoma , Optic Nerve , Retinal Ganglion Cells , Sirtuins , Animals , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Mice , Sirtuins/metabolism , Sirtuins/genetics , Glaucoma/metabolism , Glaucoma/genetics , Glaucoma/pathology , Glaucoma/etiology , Optic Nerve/metabolism , Optic Nerve/pathology , Aging/metabolism , Aging/genetics , Intraocular Pressure , Humans , Axons/metabolism , Axons/pathology , Mice, Knockout , Nerve Degeneration/metabolismABSTRACT
The death of retinal ganglion cells (RGCs) can cause irreversible injury in visual function. Clarifying the mechanism of RGC degeneration is critical for the development of therapeutic strategies. Circular RNAs (circRNAs) are important regulators in many biological and pathological processes. Herein, we performed circRNA microarrays to identify dysregulated circRNAs following optic nerve crush (ONC). The results showed that 221 circRNAs were differentially expressed between ONC retinas and normal retinas. Notably, the levels of circular RNA-Dcaf6 (cDcaf6) expression in aqueous humor of glaucoma patients were higher than that in cataract patients. cDcaf6 silencing could reduce oxidative stress-induced RGC apoptosis in vitro and alleviate retinal neurodegeneration in vivo as shown by increased neuronal nuclei antigen (NeuN, neuronal bodies) and beta-III-tubulin (TUBB3, neuronal filaments) staining and reduced glial fibrillary acidic protein (GFAP, activated glial cells) and vimentin (activated glial cells) staining. Collectively, this study identifies a promising target for treating retinal neurodegeneration.
Subject(s)
Optic Nerve Injuries , RNA, Circular , Animals , Humans , Disease Models, Animal , Optic Nerve/metabolism , Optic Nerve/pathology , Optic Nerve Injuries/genetics , Optic Nerve Injuries/drug therapy , Optic Nerve Injuries/metabolism , Retina , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , RNA, Circular/genetics , RNA, Circular/metabolismABSTRACT
BACKGROUND: During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood. RESULTS: Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected. CONCLUSIONS: Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching.
Subject(s)
Axons , Neurogenesis , Retinal Ganglion Cells , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/genetics , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/physiology , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Axons/metabolism , Axons/physiology , Neurogenesis/genetics , Neurogenesis/physiology , Motor Neurons/metabolism , Motor Neurons/physiology , Gene Expression Regulation, Developmental , Embryo, Nonmammalian/metabolism , Retina/metabolism , Retina/embryologyABSTRACT
Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for ß-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.
Subject(s)
Armadillo Domain Proteins , Retinal Ganglion Cells , Animals , Mice , Armadillo Domain Proteins/genetics , Armadillo Domain Proteins/metabolism , Mice, Knockout , Optic Nerve/metabolism , Retina/metabolism , Retinal Ganglion Cells/metabolismABSTRACT
AIMS/HYPOTHESIS: The aim of this study was to explore whether diabetic retinopathy is associated with alterations of the circadian system, and to examine the role of reduced intrinsically photosensitive retinal ganglion cell (ipRGC) function. METHODS: Participants with type 2 diabetes, with diabetic retinopathy (n=14) and without diabetic retinopathy (n=9) underwent 24 h blood sampling for melatonin and cortisol under controlled laboratory conditions. ipRGC function was inferred from the post-illumination pupil response (PIPR). Habitual sleep duration, efficiency and variability were assessed by actigraphy. RESULTS: Participants with diabetic retinopathy compared to participants without diabetic retinopathy had smaller PIPR (p=0.007), lower 24 h serum melatonin output (p=0.042) and greater day-to-day sleep variability (p=0.012). By contrast, 24 h cortisol profiles, sleep duration and efficiency were similar in both groups. Six individuals with diabetic retinopathy had no detectable dim-light melatonin onset. PIPR correlated with 24 h mean melatonin levels (r=0.555, p=0.007). CONCLUSIONS/INTERPRETATION: ipRCG dysfunction in diabetic retinopathy is associated with disruptions of the 24 h melatonin rhythm, suggesting circadian dysregulation in diabetic retinopathy.
Subject(s)
Circadian Rhythm , Diabetes Mellitus, Type 2 , Diabetic Retinopathy , Melatonin , Retinal Ganglion Cells , Humans , Melatonin/blood , Melatonin/metabolism , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/blood , Diabetic Retinopathy/physiopathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Male , Female , Cross-Sectional Studies , Middle Aged , Circadian Rhythm/physiology , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/physiopathology , Aged , Hydrocortisone/blood , Hydrocortisone/metabolism , Sleep/physiology , AdultABSTRACT
Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken ß-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.
Subject(s)
Cytomegalovirus Infections , Parvovirinae , Humans , Retinal Ganglion Cells/metabolism , Genetic Therapy , Transgenes , Optic Nerve , Dependovirus/genetics , Parvovirinae/genetics , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/metabolism , Genetic Vectors/geneticsABSTRACT
Neuromodulation in the retina is crucial for effective processing of retinal signal at different levels of illuminance. Intrinsically photosensitive retinal ganglion cells (ipRGCs), the neurons that drive nonimage-forming visual functions, express a variety of neuromodulatory receptors that tune intrinsic excitability as well as synaptic inputs. Past research has examined actions of neuromodulators on light responsiveness of ipRGCs, but less is known about how neuromodulation affects synaptic currents in ipRGCs. To better understand how neuromodulators affect synaptic processing in ipRGC, we examine actions of opioid and dopamine agonists have on inhibitory synaptic currents in ipRGCs. Although µ-opioid receptor (MOR) activation had no effect on γ-aminobutyric acid (GABA) currents, dopamine [via the D1-type dopamine receptor (D1R)]) amplified GABAergic currents in a subset of ipRGCs. Furthermore, this D1R-mediated facilitation of the GABA conductance in ipRGCs was mediated by a cAMP/PKA-dependent mechanism. Taken together, these findings reinforce the idea that dopamine's modulatory role in retinal adaptation affects both nonimage-forming and image-forming visual functions.NEW & NOTEWORTHY Neuromodulators such as dopamine are important regulators of retinal function. Here, we demonstrate that dopamine increases inhibitory inputs to intrinsically photosensitive retinal ganglion cells (ipRGCs), in addition to its previously established effect on intrinsic light responsiveness. This indicates that dopamine, in addition to its ability to intrinsically modulate ipRGC activity, can also affect synaptic inputs to ipRGCs, thereby tuning retina circuits involved in nonimage-forming visual functions.
Subject(s)
Dopamine , Receptors, GABA-A , Retinal Ganglion Cells , Animals , Retinal Ganglion Cells/physiology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Dopamine/metabolism , Dopamine/pharmacology , Receptors, GABA-A/metabolism , Mice , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D1/antagonists & inhibitors , Mice, Inbred C57BL , Receptors, Opioid, mu/metabolism , Male , Inhibitory Postsynaptic Potentials/physiology , Inhibitory Postsynaptic Potentials/drug effects , Female , Dopamine Agonists/pharmacologyABSTRACT
BACKGROUND: Vascular dysregulation is one of the major risk factors of glaucoma, and endothelin-1 (ET-1) may have a role in the pathogenesis of vascular-related glaucoma. Fruit extract from Lycium Barbarum (LB) exhibits anti-ageing and multitarget mechanisms in protecting retinal ganglion cells (RGC) in various animal models. To investigate the therapeutic efficacy of LB glycoproteins (LbGP) in ET-1 induced RGC degeneration, LbGP was applied under pre- and posttreatment conditions to an ET-1 mouse model. Retina structural and functional outcomes were characterised using clinical-based techniques. METHODS: Adult C57BL/6 mice were randomly allocated into four experimental groups, namely vehicle control (n = 9), LbGP-Pretreatment (n = 8), LbGP-Posttreatment (day 1) (n = 8) and LbGP-Posttreatment (day 5) (n = 7). Oral administration of LbGP 1 mg/Kg or PBS for vehicle control was given once daily. Pre- and posttreatment (day 1 or 5) were commenced at 1 week before and 1 or 5 days after intravitreal injections, respectively, and were continued until postinjection day 28. Effects of treatment on retinal structure and functions were evaluated using optical coherence tomography (OCT), doppler OCT and electroretinogram measurements at baseline, post-injection days 10 and 28. RGC survival was evaluated by using RBPMS immunostaining on retinal wholemounts. RESULTS: ET-1 injection in vehicle control induced transient reductions in arterial flow and retinal functions, leading to significant RNFL thinning and RGC loss at day 28. Although ET-1 induced a transient loss in blood flow or retinal functions in all LbGP groups, LbGP treatments facilitated better restoration of retinal flow and retinal functions as compared with the vehicle control. Also, all three LbGP treatment groups (i.e. pre- and posttreatments from days 1 or 5) significantly preserved thRNFL thickness and RGC densities. No significant difference in protective effects was observed among the three LbGP treatment groups. CONCLUSION: LbGP demonstrated neuroprotective effects in a mouse model of ET-1 induced RGC degeneration, with treatment applied either as a pretreatment, immediate or delayed posttreatment. LbGP treatment promoted a better restoration of retinal blood flow, and protected the RNFL, RGC density and retinal functions. This study showed the translational potential of LB as complementary treatment for glaucoma management.
Subject(s)
Endothelin-1 , Mice, Inbred C57BL , Neuroprotection , Retinal Ganglion Cells , Animals , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Endothelin-1/metabolism , Neuroprotection/drug effects , Electroretinography , Lycium/chemistry , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Retinal Degeneration/drug therapy , Retinal Degeneration/pathology , Tomography, Optical Coherence , Male , Mice , Nerve Degeneration/pathology , Nerve Degeneration/drug therapyABSTRACT
BACKGROUND: Retinal ischemia/reperfusion (RIR) is implicated in various forms of optic neuropathies, yet effective treatments are lacking. RIR leads to the death of retinal ganglion cells (RGCs) and subsequent vision loss, posing detrimental effects on both physical and mental health. Apigenin (API), derived from a wide range of sources, has been reported to exert protective effects against ischemia/reperfusion injuries in various organs, such as the brain, kidney, myocardium, and liver. In this study, we investigated the protective effect of API and its underlying mechanisms on RGC degeneration induced by retinal ischemia/reperfusion (RIR). METHODS: An in vivo model was induced by anterior chamber perfusion following intravitreal injection of API one day prior to the procedure. Meanwhile, an in vitro model was established through 1% oxygen and glucose deprivation. The neuroprotective effects of API were evaluated using H&E staining, spectral-domain optical coherence tomography (SD-OCT), Fluoro-Gold retrograde labeling, and Photopic negative response (PhNR). Furthermore, transmission electron microscopy (TEM) was employed to observe mitochondrial crista morphology and integrity. To elucidate the underlying mechanisms of API, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry assay, western blot, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, JC-1 kit assay, dichlorofluorescein-diacetate (DCFH-DA) assay, as well as TMRE and Mito-tracker staining were conducted. RESULTS: API treatment protected retinal inner plexiform layer (IPL) and ganglion cell complex (GCC), and improved the function of retinal ganglion cells (RGCs). Additionally, API reduced RGC apoptosis and decreased lactate dehydrogenase (LDH) release by upregulating Bcl-2 and Bcl-xL expression, while downregulating Bax and cleaved caspase-3 expression. Furthermore, API increased mitochondrial membrane potential (MMP) and decreased extracellular reactive oxygen species (ROS) production. These effects were achieved by enhancing mitochondrial function, restoring mitochondrial cristae morphology and integrity, and regulating the expression of OPA1, MFN2, and DRP1, thereby regulating mitochondrial dynamics involving fusion and fission. CONCLUSION: API protects RGCs against RIR injury by modulating mitochondrial dynamics, promoting mitochondrial fusion and fission.
Subject(s)
Apigenin , Mitochondrial Dynamics , Neuroprotective Agents , Reperfusion Injury , Retinal Ganglion Cells , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Apigenin/pharmacology , Apigenin/therapeutic use , Animals , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Mitochondrial Dynamics/drug effects , Male , Apoptosis/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Models, Biological , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Blast Injuries , Retinal Ganglion Cells , Mice , Animals , Retinal Ganglion Cells/metabolism , Blast Injuries/metabolism , Retina , Electroretinography , Cell Death , Disease Models, Animal , Mice, Inbred C57BLABSTRACT
The retinal ganglion cells (RGCs) serve as the critical pathway for transmitting visual information from the retina to the brain, yet they can be dramatically impacted by diseases such as glaucoma. When investigating disease processes affecting RGCs in mouse models, accurately quantifying affected cells becomes essential. However, the use of pan RGC markers like RBPMS or THY1 presents challenges in accurate total cell counting. While Brn3a serves as a reliable RGC nuclear marker for automated counting, it fails to encompass all RGC subtypes in mice. To address this limitation and enable precise automated counting, our research endeavors to develop a method for labeling nuclei in all RGC subtypes. Investigating RGC subtypes labeled with the nuclear marker POU6F2 revealed that numerous RGCs unlabeled by Brn3a were, in fact, labeled with POU6F2. We hypothesize that using antibodies against both Brn3a and POU6F2 would label virtually all RGC nuclei in the mouse retina. Our experiments confirmed that staining retinas with both markers resulted in the labeling of all RGCs. Additionally, when using the cell body marker RBPMS known to label all mouse RGCs, all RBPMS-labeled cells also exhibited Brn3a or POU6F2 labeling. This combination of Brn3a and POU6F2 antibodies provides a pan-RGC nuclear stain, facilitating accurate automated counting by labeling cell nuclei in the retina.
Subject(s)
Cell Nucleus , Mice, Inbred C57BL , Retinal Ganglion Cells , Transcription Factor Brn-3A , Animals , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Mice , Cell Count , Cell Nucleus/metabolism , Transcription Factor Brn-3A/metabolism , Staining and Labeling/methods , Biomarkers/metabolismABSTRACT
Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.
Subject(s)
Dependovirus , Genetic Therapy , Genetic Vectors , Mice, Inbred C57BL , Retinal Ganglion Cells , Transduction, Genetic , Animals , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Mice , Dependovirus/genetics , Genetic Therapy/methods , Optic Nerve Injuries/therapy , Optic Nerve Injuries/metabolism , Disease Models, Animal , Cell Survival , Orbit/blood supplyABSTRACT
Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.
Subject(s)
Cytomegalovirus Infections , Retinal Ganglion Cells , Rats , Humans , Animals , Retinal Ganglion Cells/metabolism , Genetic Vectors , Retina/metabolism , Transgenes , Intravitreal Injections , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/metabolism , Dependovirus/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Transduction, GeneticABSTRACT
Sleep loss is common in modern society and is increasingly associated with eye diseases. However, the precise effects of sleep loss on retinal structure and function, particularly on the retinal circadian system, remain largely unexplored. This study investigates these effects using a chronic sleep deprivation (CSD) model in mice. Our investigation reveals that CSD significantly alters the retinal circadian transcriptome, leading to remarkable changes in the temporal patterns of enriched pathways. This perturbation extends to metabolic and immune-related transcriptomes, coupled with an accumulation of reactive oxygen species in the retina. Notably, CSD rhythmically affects the thickness of the ganglion cell complex, along with diurnal shifts in microglial migration and morphology within the retina. Most critically, we observe a marked decrease in both scotopic and photopic retinal function under CSD conditions. These findings underscore the broad impact of sleep deprivation on retinal health, highlighting its role in altering circadian gene expression, metabolism, immune response, and structural integrity. Our study provides new insights into the broader impact of sleep loss on retinal health.
Subject(s)
Circadian Rhythm , Mice, Inbred C57BL , Retina , Sleep Deprivation , Transcriptome , Animals , Sleep Deprivation/physiopathology , Sleep Deprivation/metabolism , Sleep Deprivation/genetics , Mice , Circadian Rhythm/physiology , Male , Retina/metabolism , Retina/physiopathology , Disease Models, Animal , Retinal Ganglion Cells/pathology , Retinal Ganglion Cells/metabolism , Electroretinography , Gene Expression Regulation , Chronic DiseaseABSTRACT
Autism spectrum disorder (ASD) is a group of neurodevelopment disorders characterized by deficits in social interaction and communication, and repetitive or stereotyped behavior. Autistic children are more likely to have vision problems, and ASD is unusually common among blind people. However, the mechanisms behind the vision disorders in autism are unclear. Stabilizing WNT-targeted scaffold protein Axin2 by XAV939 during embryonic development causes overproduction of cortical neurons and leads to autistic-like behaviors in mice. In this study, we investigated the relationship between vision abnormality and autism using an XAV939-induced mouse model of autism. We found that the mice receiving XAV939 had decreased amplitude of bright light-adaptive ERG. The amplitudes and latency of flash visual evoked potential recorded from XAV939-treated mice were lower and longer, respectively than in the control mice, suggesting that XAV939 inhibits visual signal processing and conductance. Anatomically, the diameters of RGC axons were reduced when Axin2 was stabilized during the development, and the optic fibers had defective myelin sheaths and reduced oligodendrocytes. The results suggest that the WNT signaling pathway is crucial for optic nerve development. This study provides experimental evidence that conditions interfering with brain development may also lead to visual problems, which in turn might exaggerate the autistic features in humans.