Stimulation of α7 nAChR leads to regeneration of damaged neurons in adult mammalian retinal disease models.

The adult mammal lacks the ability to regenerate neurons lost to retinal damage or disease in a meaningful capacity. However, previous studies from this laboratory have demonstrated that PNU-282987, an α7 nicotinic acetylcholine receptor agonist, elicits a robust neurogenic response in the adult murine retina. With eye drop application of PNU-282987, Müller glia cells re-enter the cell cycle and produce progenitor-like cells that can differentiate into various types of retinal neurons. In this study, we analyzed the regenerative capability of PNU-282987 in two retinal disease models and identified the source of newly regenerated neurons. Wild-type mice and mice with a transgenic Müller-glia lineage tracer were manipulated to mimic loss of retinal cells associated with glaucoma or photoreceptor degeneration. Following treatment with PNU-282987, the regenerative response of retinal neurons was quantified and characterized. After onset of photoreceptor degeneration, PNU-282987 was able to successfully regenerate both rod and cone photoreceptors. Quantification of this response demonstrated significant regeneration, restoring photoreceptors to near wild-type density. In mice that had glaucoma-like conditions induced, PNU-282987 treatment led to a significant increase in retinal ganglion cells. Retrograde labeling of optic nerve axon fibers demonstrated that newly regenerated axons projected into the optic nerve. Lineage tracing analysis demonstrated that these new neurons were derived from Müller glia. These results demonstrate that PNU-282987 can induce retinal regeneration in adult mice following onset of retinal damage. The ability of PNU-282987 to regenerate retinal neurons in a robust manner offers a new direction for developing novel and potentially transformative treatments to combat neurodegenerative disease.

Stimulation of Retinal Pigment Epithelium With an α7 nAChR Agonist Leads to Müller Glia Dependent Neurogenesis in the Adult Mammalian Retina.

Purpose: The adult mammalian retina is typically incapable of regeneration when damaged by disease or trauma. Restoration of function would require generation of new adult neurons, something that until recently, mammals were thought to be incapable of doing. However, previous studies from this laboratory have shown that the α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, PNU-282987, induces cell cycle reentry of Müller glia and generation of mature retinal neurons in adult rats, in the absence of detectible injury. This study analyzes how PNU-282987 treatment in RPE leads to robust BrdU incorporation in Müller glia in adult mice and leads to generation of Müller-derived retinal progenitors and neuronal differentiation. Methods: Retinal BrdU incorporation was examined after eye drop application of PNU-282987 in adult wild-type and transgenic mice that contain tamoxifen-inducible tdTomato Müller glia, or after intraocular injection of conditioned medium from PNU-282987-treated cultured RPE cells. Results: PNU-282987 induced robust incorporation of BrdU in all layers of the adult mouse retina. The α7 nAChR agonist was found to stimulate cell cycle reentry of Müller glia and their generation of new retinal progenitors indirectly, via the RPE, in an α7 nAChR-dependent fashion. Conclusions: The results from this study point to RPE as a contributor to Müller glial neurogenic responses. The manipulation of the RPE to stimulate retinal neurogenesis offers a new direction for developing novel and potentially transformative treatments to reverse the loss of neurons associated with neurodegenerative disease, traumatic injury, or aging.

Class I histone deacetylases in retinal progenitors and differentiating ganglion cells.

BACKGROUND: The acetylation state of histones has been used as an indicator of the developmental state of progenitor and differentiating cells. The goal of this study was to determine the nuclear localization patterns of Class I histone deacetylases (HDACs) in retinal progenitor cells (RPCs) and retinal ganglion cells (RGCs), as the first step in understanding their potential importance in cell fate determination within the murine retina. RESULTS: The only HDAC to label RPC nuclei at E16 and P5 was HDAC1. In contrast, there was generally increased nuclear localization of all Class I HDACs in differentiating RGCs. Between P5 and P30, SOX2 expression becomes restricted to Müller glial, cholinergic amacrine cells, and retinal astrocytes. Cholinergic amacrine showed a combination of changes in nuclear localization of Class I HDACs. Strikingly, although Müller glia and retinal astrocytes express many of the same genes, P30 Müller glial cells showed nuclear localization only of HDAC1, while retinal astrocytes were positive for HDACs 1, 2, and 3. CONCLUSION: These results indicate there may be a role for one or more of the Class I HDACs in retinal cell type-specific differentiation.

Expression of Antimicrobial Peptides by Uveal and Cutaneous Melanoma Cells and Investigation of Their Role in Tumor Cell Migration and Vasculogenic Mimicry.

AIMS: Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy. METHODS: mRNA expression of the AMPs α defensin (HNP-1); cathelicidin (LL-37); and ß defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy. RESULTS: Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process. CONCLUSIONS: Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.

Regulation of Stem Cell Properties of Müller Glia by JAK/STAT and MAPK Signaling in the Mammalian Retina.

In humans and other mammals, the neural retina does not spontaneously regenerate, and damage to the retina that kills retinal neurons results in permanent blindness. In contrast to embryonic stem cells, induced pluripotent stem cells, and embryonic/fetal retinal stem cells, Müller glia offer an intrinsic cellular source for regenerative strategies in the retina. Müller glia are radial glial cells within the retina that maintain retinal homeostasis, buffer ion flux associated with phototransduction, and form the blood/retinal barrier within the retina proper. In injured or degenerating retinas, Müller glia contribute to gliotic responses and scar formation but also show regenerative capabilities that vary across species. In the mammalian retina, regenerative responses achieved to date remain insufficient for potential clinical applications. Activation of JAK/STAT and MAPK signaling by CNTF, EGF, and FGFs can promote proliferation and modulate the glial/neurogenic switch. However, to achieve clinical relevance, additional intrinsic and extrinsic factors that restrict or promote regenerative responses of Müller glia in the mammalian retina must be identified. This review focuses on Müller glia and Müller glial-derived stem cells in the retina and phylogenetic differences among model vertebrate species and highlights some of the current progress towards understanding the cellular mechanisms regulating their regenerative response.

Talkin' about my (re)generation: The who of intrinsic retinal stem cells.

World-wide, two degenerative retinal diseases, glaucoma and age-related macular degeneration, are estimated to affect more than 12% of individuals over the age of 40 (Tham et al., 2014; Wong et al., 2014). Current therapies can slow progression, but cannot restore lost neurons or vision. Thus, there is increasing interest in developing strategies for therapeutic retinal regeneration. Nearly 50years of research on retinal neurogenesis and regeneration has identified Müller glia as intrinsic retinal stem cells in teleost fish. In the mammalian retina, there is no de novo neurogenesis in adults and only very limited injury-induced regeneration has been induced using exogenous growth factors. The study by (Webster et al., 2017) (Evidence of BrdU Positive Retinal Neurons after Application of an Alpha7 Nicotinic Acetylcholine Receptor Agonist, this issue) is the first to show robust, retinal neurogenesis in an adult, mammalian retina in the absence of overt injury and provides evidence that the source of the new neurons is likely to be the Müller glia. This exciting finding has the potential to be a game-changer in the field of retinal regeneration.

Novel Roles and Mechanism for Krüppel-like Factor 16 (KLF16) Regulation of Neurite Outgrowth and Ephrin Receptor A5 (EphA5) Expression in Retinal Ganglion Cells.

Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies.

Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells.

In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1×10(12) to 1×10(14) ions/cm(2)), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation.

Histone deacetylase expression patterns in developing murine optic nerve.

BACKGROUND: Histone deacetylases (HDACs) play important roles in glial cell development and in disease states within multiple regions of the central nervous system. However, little is known about HDAC expression or function within the optic nerve. As a first step in understanding the role of HDACs in optic nerve, this study examines the spatio-temporal expression patterns of methylated histone 3 (K9), acetylated histone 3 (K18), and HDACs 1-6 and 8-11 in the developing murine optic nerve head. RESULTS: Using RT-qPCR, western blot and immunofluorescence, three stages were analyzed: embryonic day 16 (E16), when astrocyte precursors are found in the optic stalk, postnatal day 5 (P5), when immature astrocytes and oligodendrocytes are found throughout the optic nerve, and P30, when optic nerve astrocytes and oligodendrocytes are mature. Acetylated and methylated histone H3 immunoreactivity was co-localized in the nuclei of most SOX2 positive glia within the optic nerve head and adjacent optic nerve at all developmental stages. HDACs 1-11 were expressed in the optic nerve glial cells at all three stages of optic nerve development in the mouse, but showed temporal differences in overall levels and subcellular localization. HDACs 1 and 2 were predominantly nuclear throughout optic nerve development and glial cell maturation. HDACs 3, 5, 6, 8, and 11 were predominantly cytoplasmic, but showed nuclear localization in at least one stage of optic nerve development. HDACs 4, 9 and10 were predominantly cytoplasmic, with little to no nuclear expression at any time during the developmental stages examined. CONCLUSIONS: Our results showing that HDACs 1, 2, 3, 5, 6, 8, and 11 were each localized to the nuclei of SOX2 positive glia at some stages of optic nerve development and maturation and extend previous reports of HDAC expression in the aging optic nerve. These HDACs are candidates for further research to understand how chromatin remodeling through acetylation, deacetylation and methylation contributes to glial development as well as their injury response.

Distinct neurogenic potential in the retinal margin and the pars plana of mammalian eye.

Unlike many other vertebrates, a healthy mammalian retina does not grow throughout life and lacks a ciliary margin zone capable of actively generating new neurons. The isolation of stem-like cells from the ciliary epithelium has led to speculation that the mammalian retina and/or surrounding tissues may retain neurogenic potential capable of responding to retinal damage. Using genetically altered mouse lines with varying degrees of retinal ganglion cell loss, we show that the retinal margin responds to ganglion cell loss by prolonging specific neurogenic activity, as characterized by increased numbers of Atoh7(LacZ)-expressing cells. The extent of neurogenic activity correlated with the degree of ganglion cell deficiency. In the pars plana, but not the retinal margin, cells remain proliferative into adulthood, marking the junction of pars plana and retinal margin as a niche capable of producing proliferative cells in the mammalian retina and a potential cellular source for retinal regeneration.

Muller cell expression of genes implicated in proliferative vitreoretinopathy is influenced by substrate elastic modulus.

PURPOSE: Matrix stiffness is recognized increasingly as a significant factor in cell and tissue function. To understand better the mechanosensitivity of Müller cells and its association with vitreoretinal disorders, we examined morphology, propagation, and expression of genes in Müller cells that were cultured on substrates of varying elastic moduli. METHODS: A conditionally immortalized mouse Müller cell line was cultured on laminin-coated polyacrylamide substrates with calibrated Young's moduli. Glass was used as a control. Phase contrast, fluorescence, and atomic force microscopy were used to study cell morphology and propagation. Expression of extracellular matrix (ECM) genes was analyzed using quantitative reverse-transcription PCR. RESULTS: The adherent area, stiffness, and propagation of Müller cells all are affected by matrix stiffness, but to different extents and with different ranges of sensitivity. Of 85 ECM genes tested 11 showed a continuous >4-fold increase or decrease in mRNA expression as a function of the substrate elastic modulus. The changes were statistically significant in four genes: connective tissue growth factor (Ctgf, P = 0.04), tenascin C (Tnc, P = 0.035), Collagen Iα1 (Col1a1, P = 0.0001), and Collagen IVα3 (Col4a3, P = 0.05), with all showing increased expression on softer substrates. CONCLUSIONS: There are significant changes in morphology, cytoskeletal integrity, and gene regulation in Müller cells as a function of the stiffness of the substrate. Changes in local tissue elastic modulus may have a role in vitreoretinal disorders. These findings also may have implications for strategies for improved integration of retinal prosthetics, and for stem cell therapies, particularly targeting the transcriptional regulators YAP and TAZ.

A role for DNA methylation in regulation of EphA5 receptor expression in the mouse retina.

Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cells but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up-regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.

Differential expression of neuronal genes in Müller glia in two- and three-dimensional cultures.

PURPOSE: Müller glia in the mammalian retina have some stem cell-like characteristics, although their capacity for neurogenesis remains limited both in vivo and in vitro. In vitro studies to date have used traditional two-dimensional (2D) cell culture to assess neuronal differentiation of Müller glia. The purpose of this study was to compare the effects of 2D and three-dimensional (3D) environments on Müller glial gene expression after growth factor stimulation. METHODS: Conditionally immortalized mouse Müller glia cells (ImM10) were cultured under nonimmortalizing conditions with EGF/FGF2 to generate spheres that were differentiated in vitro on uncoated culture dishes (2D) or encapsulated in self-assembling, RADA-16 peptide hydrogels (3D) under identical media and growth factor supplementation conditions. Gene expression was analyzed using quantitative RT-PCR and immunocytochemistry. Cellular morphology was analyzed with light and confocal microscopy; sphere ultrastructure was analyzed with transmission electron microscopy. RESULTS: ImM10 Müller cells express numerous genes associated with neural stem cells and retinal progenitors in both normal growth conditions and sphere-forming conditions. When encapsulated in the 3D hydrogel, cells can migrate and send processes into the hydrogel. Many genes associated with neurogenesis, as well as retinal neuron-specific genes, are differentially expressed in 2D and 3D differentiation conditions. CONCLUSIONS: ImM10 Müller glia upregulate genes characteristic of retinal neurons after growth factor stimulation in vitro, and gene expression patterns are altered in 3D hydrogel cultures.

Eyes on DNA methylation: current evidence for DNA methylation in ocular development and disease.

Epigenetic modulation of chromatin states constitutes a vital component of the cellular repertoire of transcriptional regulatory mechanisms. The development of new technologies capable of generating genome-wide maps of chromatin modifications has re-energized the field. We are now poised to determine how species- and tissue-specific patterns of DNA methylation, in concert with other chromatin modifications, function to establish and maintain cell- and tissue-specific patterns of gene expression during normal development, cellular differentiation, and disease. This review addresses our current understanding of the major mechanisms and function of DNA methylation in vertebrates with a historical perspective and an emphasis on what is known about DNA methylation in eye development and disease.

A conditional immortalized mouse muller glial cell line expressing glial and retinal stem cell genes.

PURPOSE: Müller glia have multiple functions in the retina, including synthesis of neurotrophic factors, uptake and metabolism of neurotransmitters, spatial buffering of ions, maintenance of the blood-retinal barrier, and response to injury. A population of Müller glia has some stem cell-like characteristics both in vivo and in vitro. The purpose of this study was to generate and characterize novel Müller glial cell lines from the postnatal mouse retina. METHODS: Cells were cultured from postnatal day (P) 10 double heterozygous transgenic (H-2K(b)-tsA58/+; HRhoGFP/+) or C57BL/6 mice after papain dissociation. Interferon gamma (IFNγ) induction of the SV40 T-antigen (TAg) was assayed by immunohistochemistry and Western blot analysis. Proliferation was assayed by BrdU uptake and cell counts of calcein AM/ethidium bromide-stained cells. Gene expression was analyzed by RT-PCR and immunohistochemistry. RESULTS: Conditionally immortalized (ImM10 [Immortmouse Müller P10]) and spontaneously immortalized (C57M10 [C57BL/6 Müller P10]) Müller glial cell lines were selected by differential adherence to laminin; both consisted of adherent flat cells with large, diffusely staining nuclei and an epithelial morphology. TAg induction stimulated BrdU uptake by Müller glia in mixed retinal cultures from H-2K(b)-tsA58/+; HRhoGFP/+ mice and increased the proliferation of ImM10 cells. ImM10 and C57M10 cells expressed genes characteristic of Müller glia but not genes characteristic of differentiated retinal neurons. ImM10 cells also expressed retinal stem cell genes. CONCLUSIONS: The ImM10 cell line is a novel, conditionally immortalized Müller glial cell line isolated from the P10 mouse retina that expresses genes characteristic of Müller glial and retinal stem cells.

Progression of neuronal and synaptic remodeling in the rd10 mouse model of retinitis pigmentosa.

The Pde6b(rd10) (rd10) mouse has a moderate rate of photoreceptor degeneration and serves as a valuable model for human autosomal recessive retinitis pigmentosa (RP). We evaluated the progression of neuronal remodeling of second- and third-order retinal cells and their synaptic terminals in retinas from Pde6b(rd10) (rd10) mice at varying stages of degeneration ranging from postnatal day 30 (P30) to postnatal month 9.5 (PNM9.5) using immunolabeling for well-known cell- and synapse-specific markers. Following photoreceptor loss, changes occurred progressively from outer to inner retina. Horizontal cells and rod and cone bipolar cells underwent morphological remodeling that included loss of dendrites, cell body migration, and the sprouting of ectopic processes. Gliosis, characterized by translocation of Müller cell bodies to the outer retina and thickening of their processes, was evident by P30 and became more pronounced as degeneration progressed. Following rod degeneration, continued expression of VGluT1 in the outer retina was associated with survival and expression of synaptic proteins by nearby second-order neurons. Rod bipolar cell terminals showed a progressive reduction in size and ectopic bipolar cell processes extended into the inner nuclear layer and ganglion cell layer by PNM3.5. Putative ectopic conventional synapses, likely arising from amacrine cells, were present in the inner nuclear layer by PNM9.5. Despite these changes, the laminar organization of bipolar and amacrine cells and the ON-OFF organization in the inner plexiform layer was largely preserved. Surviving cone and bipolar cell terminals continued to express the appropriate cell-specific presynaptic proteins needed for synaptic function up to PNM9.5.

Multimodal regulation of E2F1 gene expression by progestins.

An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.

Intracellular delivery of proteins into mouse Müller glia cells in vitro and in vivo using Pep-1 transfection reagent.

Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3-conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells.

Voltage-gated sodium channel alpha-subunits Na(v)1.1, Na(v)1.2, and Na(v)1.6 in the distal mammalian retina.

PURPOSE: Recent studies indicate the presence of functional voltage-gated sodium channels (Na(v) channels) in the distal retina in several species. This study examined the distribution of Na(v) channels in the outer plexiform layer (OPL) of rat, mouse, and rabbit retinas. METHODS: Immunohistochemical and electroretinographic approaches were used. RESULTS: Antibodies specific for Na(v)1 alpha-subunits appropriately labeled retinal ganglion cells, their axons, and amacrine cells that are known to have tetrodotoxin (TTX)-sensitive Na(v) channels. Pan-Na(v), Na(v)1.2, and Na(v)1.6 labeling was found in horizontal cells and processes in all three species. Weaker Na(v)1.1 labeling was observed in rodent horizontal cells, but some rabbit horizontal cells and processes were prominently labeled. Additional labeling for Na(v)1.1, Na(v)1.2, and Na(v)1.6 that was not attributable to horizontal cells was also present in the OPL. Much of this labeling was diffusely distributed. Some of the additional Na(v)1.1 labeling was associated with photoreceptor terminals. By exclusion using photoreceptor and horizontal cell markers, some of this labeling could have been associated with bipolar cell dendrites, although colocalization was not directly established due to the diffuse nature of the labeling and limits on anatomical resolution. No Na(v)1 alpha-subunit labeling was observed in bipolar cell bodies. Testing for functional Na(v) channels was performed by recording full field flash electroretinograms from dark-adapted rats before and after intravitreal injections of TTX, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), or TTX+CNQX. TTX and CNQX+TTX, but not CNQX alone, greatly attenuated the dark-adapted cone-driven b-waves. CONCLUSIONS: Horizontal cells from three different mammalian retinas showed prominent labeling for Na(v)1 alpha-subunits. Some additional diffuse Na(v)1 alpha-subunit labeling in the OPL was associated with photoreceptor terminals. Na(v)1 alpha-subunit labeling also may have been present on bipolar cell dendrites, although it was not possible to establish this localization unequivocally by immunostaining. However, cone-driven b-waves in rats were reduced in maximum amplitude by TTX in the presence of CNQX which blocks synaptic input to horizontal, amacrine, and ganglion cells. This finding is consistent with TTX effects on the b-wave being due to blockade of Na(v) channels in cone bipolar cell dendrites in the OPL. The role of Na(v) channels in horizontal cells remains to be determined.