Investigative Ophthalmology & Visual Science, Jun 11, 2015
The photoreceptor cell death associated with the various genetic forms of retinitis pigmentosa (R... more The photoreceptor cell death associated with the various genetic forms of retinitis pigmentosa (RP) is currently untreatable and leads to partial or complete vision loss. Carnosic acid (CA) upregulates endogenous antioxidant enzymes and has proven neuroprotective in studies of neurodegenerative models affecting the brain. In this study, we examined the potential effect of CA on photoreceptor death in the Pde6b rd10 mouse model of RP. Our data shows that CA provided morphological and functional preservation of photoreceptors. CA appears to exert its neuroprotective effects through inhibition of oxidative stress and endoplasmic reticulum stress. Retinitis pigmentosa (RP) is a class of inherited diseases which are characterized by the gradual degeneration of rod photoreceptors followed by cone photoreceptor cell dysfunction and death 1 . RP is a significant cause of vision loss, and affects approximately 1 in 3,700 people 2 . The initial symptoms of RP impact the peripheral retina. RP in late stages will involve central vision and may result in legal blindness 3 . Although effective treatments for RP should start early in life, there are currently no effective medications available for controlling the development of RP due to the limited therapeutic benefits or potential side effects of currently available treatment options . It is therefore important to search for novel therapeutics for RP treatment . Photoreceptors work in a very challenging environment characterized by high oxygen supply 7 , excessive light exposure 8 , dim ambient light and active metabolism 9 . These stressors induce oxidative damage of the biological macromolecules that comprise photoreceptors 10 . Increasing evidence obtained from animal models of RP suggests that oxidative stress , as well as endoplasmic reticulum (ER) stress , may be the critical mechanisms underlying photoreceptor damage and death . Consistent with this hypothesis, a number of studies have demonstrated that early administration of agents that inhibit oxidative stress could significantly decrease the rate of photoreceptor cell death in animal models of RP . Carnosic acid (CA) is a potent antioxidant isolated from Rosmarinus officinalis. CA can readily cross the blood-brain barrier 19 and exert its protective effects after conversion from its catechol form to an electrophilic quinone form. This conversion allows CA to bind to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and subsequently release protective transcription factors . Unlike other antioxidants, CA does not deplete the endogenous antioxidant glutathione . The Pde6b rd10 (rd10) mouse is a well-characterized model of RP . The rd10 mouse carries a missense mutation in exon 13 of the beta subunit of the rod phosphodiesterase gene (Pde6b) , mutations in which also cause human RP . In rd10 mice, rod cell death begins around postnatal day (P) 18 26 , and is near complete by P35 . In this study, we demonstrate that CA slows rod degeneration in the rd10 mouse, by reducing oxidative stress and ER stress. Electroretinography. Electroretinography (ERG) was used to compare outer retinal function of mice (Fig. ). Figure ,b compare representative ERG recordings made from wild-type (WT) and rd10 mice at post
Automatic discriminatory analysis of waveforms of visual evoked potential and its clinical application
Sheng wu hua xue yu sheng wu wu li jin zhan, 1998
The amplitudes and the phases of first to sixth harmonics of P VEP waveforms were calculated, thu... more The amplitudes and the phases of first to sixth harmonics of P VEP waveforms were calculated, thus 12 parameters of the P VEP waveform were obtained for the discriminatory analysis. The P VEP waveforms of thirty two normal eyes, thirty one amblyopic eyes and thirty eyes with retrobulbar neuritis (RBN) were used to establish the discriminatory system. In the test of the discriminatory system, thirty of the thirty two normal P VEP waveforms were classified into normal group, thirty four of the thirty five amblyopic P VEP waveforms were classified into amblyopic group, and twenty eight of thirty RBN P VEP waveforms were classified into RBN group.
Regulation of Nucleocytoplasmic Partitioning and Proteostasis of Substrates and Chemokine Signaling by Ran-binding protein 2 (Ranbp2) in Thy1+-Ganglionic Neurons
Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosi... more Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ranbinding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1 + -motoneurons develop cardinal ALS-like traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This understanding will facilitate to discern the role of nucleocytoplasmic transport in the differential vulnerability of neurons to ALS and to develop therapeutic approaches and biomarkers in ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1 + -RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified Cd11b + and CD45 + -microglia, increase of F4\80 +microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80 + -microglia intermingled between RGNs of naive mice. This immunogenic phenotype was accompanied by the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neural-type selective substrates. Metabolic and immune-modulators underpinning RGN-tomicroglial signaling are regulated by Ranbp2, and this neuroglial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of ALS.
Advances in Experimental Medicine and Biology, 2018
The monocarboxylate transporter 1 (MCT1) is highly expressed in the outer retina, suggesting that... more The monocarboxylate transporter 1 (MCT1) is highly expressed in the outer retina, suggesting that it plays a critical role in photoreceptors. We examined MCT1 +/-heterozygotes, which express half of the normal complement of MCT1. The MCT1 +/-retina developed normally and retained normal function, indicating that MCT1 is expressed at sufficient levels to support outer retinal metabolism.
Background: Cyclophilins harbor ill-defined chaperone and prolyl isomerase activities toward phys... more Background: Cyclophilins harbor ill-defined chaperone and prolyl isomerase activities toward physiological substrates. Results: Nonoverlapping chaperone or prolyl isomerase activity loss of Ran-binding protein 2 (Ranbp2) cyclophilin domain triggers unique impairments of proteostasis in distinct cell types and ubiquitin-proteasome system. Conclusion: Ranbp2 cyclophilin subdomains present discriminating physiological activities toward substrates or regulation of ubiquitin-proteasome system. Significance: Ranbp2-mediated mechanistic links in proteostasis with physiological and therapeutic relevance are uncovered. The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2 WT-HA ) or without PPIase activities (Tg-Ranbp2 R2944A-HA ). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Tg-Ranbp2 R2944A-HA ::Ranbp2 ؊/؊ . Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2 R2944A-HA ::Ranbp2 ؊/؊ . This is accompanied by the age-and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2 CLDm-HA ::Ranbp2 ؊/؊ , harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil distinct mechanistic and biological links between PPIase and chaperone activities of Ranbp2 cyclophilin toward proteostasis of selective substrates and with novel therapeutic potential. Peptidyl cis-trans-prolyl isomerization is a rate-limiting step in protein folding (1-3). The catalysis of the cis-trans interconversion of the peptidyl-prolyl isomers is catalyzed by peptidylprolyl cis-trans isomerases (PPIase) 5 (4 -6). PPIases compose three families of structurally unrelated proteins, the cyclophilins (CyP), FK506-binding proteins (FKBP), and parvulins (7).
Purpose-Retinitis pigmentosa (RP), a group of inherited diseases characterized by the death of ro... more Purpose-Retinitis pigmentosa (RP), a group of inherited diseases characterized by the death of rod photoreceptors, followed by the death of cone photoreceptors, progressively leading to partial or complete blindness. Currently no specific treatment is available for RP patients. Sulforaphane (SFN) has been confirmed to be an effective antioxidant in the treatment of many diseases. In this study, we tested the therapeutic effects of SFN against photoreceptor degeneration in Pde6 rd10 (rd10) mice. Methods-rd10 mice and C57BL/6J wild-type (WT) mice were treated with SFN and saline, respectively, from P6 to P20. Electroretinography (ERG), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and western blot were tested, respectively, at P21 for the analysis of retinal function, retinal cell apoptosis or death and the protein express of GRP78/BiP (TUNEL) as a marker of endoplasmic reticulum stress. Results-Compared to the saline group, the SFN-treated group showed significantly higher ERG a-wave and b-wave amplitudes, less photoreceptor death, and downregulation of GRP78/BiP. Conclusions-Our data showed that SFN ameliorated the retinal degeneration of rd10 mice, which is possibly related to the downregulation of GRP78 expression.
Investigative Ophthalmology & Visual Science, Nov 19, 2012
PURPOSE. The association of single nucleotide polymorphisms of components of the complement alter... more PURPOSE. The association of single nucleotide polymorphisms of components of the complement alternative pathway with the risk of age-related macular degeneration (AMD) indicates that complement signaling plays an important role in retinal physiology. How genetic variation leads to retinal degeneration is unknown. It has been assumed that complement activation augments immune responses, which in turn initiate AMD pathogenesis. To better understand the relationship between complement and the outer retina, we examined mice lacking the main complement component C3 and the receptors for complement activation fragments C3a (C3aR) and/or C5a (C5aR). METHODS. Complement mutant mice were studied along with wild-type (WT) littermates from 6 weeks to 14 months of age. Strobe flash electroretinography (ERG) was used to examine outer retinal function and a dc-ERG technique was used to measure ERG components generated by the retinal pigment epithelium. Retinas were examined by histology, immunohistochemistry, and biochemistry. Mice lacking C3aR and/or C5aR developed early onset and progressive retinal degeneration, accompanied by cleaved caspase-3 upregulation. Genetic deletion of C3aR and/or C5aR led to cell-specific defects that matched the cellular localization of these receptors in the WT retina. Compared to WT, C3aR À/À and C3aR À/À C5aR À/À mice showed increased retinal dysfunction upon light exposure. C3aR À/À C5aR À/À mice immunized with 4hydroxynonenal-adducted protein developed severe retinal impairment unrelated to immune response. CONCLUSIONS. C3aR-and C5aR-mediated signaling was necessary to maintain normal retinal function and structure. These receptors may be important biomarkers for predicting retinal degeneration including AMD. (Invest
Background: Ranbp2 and its Ran-GTP-binding domains' roles in RPE survival/function, a multidiseas... more Background: Ranbp2 and its Ran-GTP-binding domains' roles in RPE survival/function, a multidisease target, are elusive. Results: RPE undergoes degeneration, disruptions of proteostasis of Ranbp2 partners, and blood-retinal barrier upon Ranbp2 ablation. Impairment of selective Ran-GTP-binding domains of Ranbp2 suffices to promote RPE degeneration. GTPase regulation by Ranbp2 is vital to RPE. Significance: Ranbp2-dependent targets/mechanisms with therapeutic potential in RPE degeneration are uncovered. Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2 ؊/؊ , with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2 ؊/؊ background. Among the transgenic lines produced, only Tg RBD2/3*-HA ::RPE-cre::Ranbp2 ؊/؊ -expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2 ؊/؊ . By contrast, Tg RBD2/3*-HA expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2 ؊/؊ and Tg RBD2/3*-HA ::RPE-cre::Ranbp2 ؊/؊ share proteostatic deregulation of Ran GTPase, serotransferrin, and ␥-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.
Characteristics of Visual Electrophysiology in Albinism
Springer eBooks, 2019
This chapter summarizes the application of visual evoked potential (VEP) and electroretinogram in... more This chapter summarizes the application of visual evoked potential (VEP) and electroretinogram in patients with albinism that has partial misrouted ganglion cell axons of temporal retina to the contralateral side of visual cortex. The VEP amplitude and polarity recorded from scalp overlying the right and left visual cortex shows characteristic change due to the change of the contribution from the temporal side and nasal side of the retina, providing useful information for diagnosis.
Characteristics of Visual Electrophysiology in the Diseases of Optic Nerve or Visual Pathway
This chapter summarizes the application of electrophysiologic tests in diseases associated with o... more This chapter summarizes the application of electrophysiologic tests in diseases associated with optic nerve or higher visual pathway dysfunction. Electrophysiologic test results are shown in amblyopia, Leber’s hereditary optic neuropathy, multiple sclerosis, ischemic optic neuropathy, optic neuritis, optic nerve hypoplasia, traumatic optic neuropathy, neurofibromatosis, and optic nerve toxicities that can provide clinicians information for differential diagnoses. In addition, this chapter will help clinicians choose suitable electrophysiologic tests in specific patients.
Expression of the constitutively active serine/threonine kinase Akt in oligodendrocytes results i... more Expression of the constitutively active serine/threonine kinase Akt in oligodendrocytes results in enhanced myelination in the CNS. Here, we have examined the effects of this Akt overexpression on optic nerve structure and on optic nerve function, assessed using the visual evoked potential (VEP). Transgenic mice have been generated with the Plp promoter driving expression of a modified form of Akt, in which aspartic acids are substituted for Thr308 and Ser473. These Plp-Akt-DD (Akt-DD) mice, and littermate controls, were studied at different ages. Optic nerves were examined anatomically at 2 and 6 months of age. At 2 months of age, optic nerves were substantially thicker in Akt-DD mice, reflecting an increase in myelination of optic nerve axons. By electron microscopy, myelin thickness was increased in Akt-DD optic nerve, with extended paranodal domains having excess paranodal loops, and the density of nodes of Ranvier was reduced, relative to control mice. We recorded VEPs in response to strobe flash ganzfeld stimuli presented after overnight dark-and light-adapted conditions at ages ranging from 1 to 10 months. It was possible to record a clear VEP from Akt-DD mice at all ages examined. At 1 month of age, VEP implicit times were somewhat shorter in Akt-DD transgenic mice than in control animals. Beyond 6 months of age, VEP latencies were consistently delayed in Akt-DD transgenic mice. These abnormalities did not reflect an alteration in retinal function as there were no significant differences between ERGs obtained from control or Akt-DD transgenic mice. In young mice, the somewhat faster responses may reflect improved transmission due to increased myelination of
bioRxiv (Cold Spring Harbor Laboratory), Mar 2, 2022
Precise and reliable cell-specific gene delivery remains technically challenging. Here we report ... more Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of cellspecific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, sequence conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED vectors can leverage the strong expression of constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. We generated SLED vectors to selectively target all neurons, photoreceptors, or excitatory neurons, and demonstrated that specificity was retained in vivo when delivered using AAVs. We further demonstrated the utility of SLED by creating what would otherwise be unobtainable research tools, specifically a GluA2 flip/flop reporter and a dual excitatory/inhibitory neuronal calcium indicator. Finally, we show the translational potential of SLED by rescuing photoreceptor degeneration in Prph2 rds/rds mice and by developing an oncolytic vector that can selectively induce apoptosis in SF3B1 mutant cancer cells. The flexibility of SLED technology enables new avenues for basic and translational research.
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