Retinal Transcriptome Research Articles

Impacts of ciliary neurotrophic factor on the retinal transcriptome in a mouse model of photoreceptor degeneration

Ciliary neurotrophic factor (CNTF) has been tested in clinical trials for human retinal degeneration due to its potent neuroprotective effects in various animal models. To decipher CNTF-triggered molecular events in the degenerating retina, we performed high-throughput RNA sequencing analyses using the Rds/Prph2 (P216L) transgenic mouse as a preclinical model for retinitis pigmentosa. In the absence of CNTF treatment, transcriptome alterations were detected at the onset of rod degeneration compared with wild type mice, including reduction of key photoreceptor transcription factors Crx, Nrl, and rod phototransduction genes. Short-term CNTF treatments caused further declines of photoreceptor transcription factors accompanied by marked decreases of both rod- and cone-specific gene expression. In addition, CNTF triggered acute elevation of transcripts in the innate immune system and growth factor signaling. These immune responses were sustained after long-term CNTF exposures that also affected neuronal transmission and metabolism. Comparisons of transcriptomes also uncovered common pathways shared with other retinal degeneration models. Cross referencing bulk RNA-seq with single-cell RNA-seq data revealed the CNTF responsive cell types, including Müller glia, rod and cone photoreceptors, and bipolar cells. Together, these results demonstrate the influence of exogenous CNTF on the retinal transcriptome landscape and illuminate likely CNTF impacts in degenerating human retinas.

Circadian analysis of the mouse retinal pigment epithelium transcriptome

The presence of a phagocytic peak of photoreceptor outer segments by the retinal pigment epithelium (RPE) one or 2 h after the onset of light has been reported for several diurnal and nocturnal species. This peak in phagocytic activity also persists under constant lighting conditions (i.e., constant light or dark) thus demonstrating that the timing of this peak is driven by a circadian clock. The aim of this study was to investigate the change in RPE whole transcriptome at two different circadian times (CT; 1 h before (CT23) and 1 h after (CT1) subjective light onset). C57BL/6J male mice were maintained in constant dark conditions for three days and euthanized under red light (<1 lux) at CT23 and CT1. RPE was isolated from whole eyes for RNA library preparation and sequencing on an Illumina HiSeq4000 platform. 14,083 mouse RPE transcripts were detected in common between CT23 and CT1. 12,005 were protein coding transcripts and 2078 were non-protein coding transcripts. 2421 protein coding transcripts were significantly upregulated whereas only 3 transcripts were significantly downregulated and 12 non-protein coding transcripts were significantly upregulated and 31 non-protein coding transcripts were significantly downregulated at CT1 when compared to CT23 (p < 0.05, fold change ≥ ±2.0). Of the protein coding transcripts, most of them were characterized as: enzymes, kinases, and transcriptional regulators with a large majority of activity in the cytoplasm, nucleus, and plasma membrane. Non-protein coding transcripts included biotypes such as long-non coding RNAs and pseudogenes. Gene ontology analysis and ingenuity pathway analysis revealed that differentially expressed transcripts were associated with integrin signaling, oxidative phosphorylation, protein phosphorylation, and actin cytoskeleton remodeling suggesting that these previously identified phagocytic pathways are under circadian control. Our analysis identified new pathways (e.g., increased mitochondrial respiration via increased oxidative phosphorylation) that may be involved in the circadian control of phagocytic activity. In addition, our dataset suggests a possible regulatory role for the identified non-protein coding transcripts in mediating the complex function of RPE phagocytosis. Finally, our results also indicate, as seen in other tissues, about 20% of the whole RPE transcriptome may be under circadian clock regulation.

Identification of novel differentially expressed genes in retinas of STZ-induced long-term diabetic rats through RNA sequencing.

BackgroundThe aim of this research was to investigate the retinal transcriptome changes in long‐term streptozotocin (STZ)‐induced rats' retinas using RNA sequencing (RNA‐seq), to explore the molecular mechanisms of diabetic retinopathy (DR), and to identify novel targets for the treatment of DR by comparing the gene expression profile we obtained.MethodsIn this study, 6 healthy male SD rats were randomly divided into wild‐type (WT) group and streptozotocin (STZ)‐induced group, 3 rats each group. After 6 months, 3 normal retina samples and 3 DM retina samples (2 retinas from the same rat were considered as 1 sample) were tested and differentially expressed genes (DEGs) were measured by RNA‐seq technology. Then, we did Gene Ontology (GO) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and validated the results of RNA‐seq through qRT–PCR.ResultsA total of 118 DEGs were identified, of which 72 were up‐regulated and 46 were down‐regulated. The enriched GO terms showed that 3 most significant enrichment terms were binding (molecular function), cell part (cellular component), and biological regulation (biological process). The results of the KEGG pathway analysis revealed a significant enrichment in cell adhesion molecules, PI3K‐Akt signaling pathway, and allograft rejection, etc.ConclusionOur research has identified specific DEGs and also speculated their potential functions, which will provide novel targets to explore the molecular mechanisms of DR.

PRPF31 reduction causes mis-splicing of the phototransduction genes in human organotypic retinal culture.

PRPF31 is ubiquitously expressed splicing factor and has an essential role in the pre-mRNA splicing in all tissues. However, it is not clear how reduced expression of this general splicing factor leads to retinal restricted disease, retinitis pigmentosa (RP). In this study, we used RNA interference and RNA-sequencing to mimic the PRPF31 haploinsufficiency in human organotypic retinal cultures (HORCs). We examined the effects of PRPF31 deficiency on splicing by analyzing the differential exon usages (DEUs) and intron retentions of the retinal transcriptome. Our results revealed that the PRPF31 deficiency causes mis-splicing of genes involved in RNA processing (PRPF3, PRPF8, PRPF4, and PRPF19) and phototransduction (RHO, ROM1, FSCN2, GNAT2, and GNAT1) in the retina in the PRPF31 reduced samples. Mis-splicing of genes implicated in phototransduction was associated with photoreceptor degeneration observed in RP patients. Our data revealed that PRPF31 deficiency leads to the mis-splicing of a distinct subset of pre-mRNAs with a widespread effect on phototransduction and RNA processing.

Acupuncture Treatment Reverses Retinal Gene Expression Induced by Optic Nerve Injury via RNA Sequencing Analysis.

Glaucoma and traumatic optic nerve crush (ONC) injury result in progressive loss of retinal ganglion cells (RGCs) and defects in visual function. In clinical trials of Traditional Chinese Medicine, acupuncture has been widely used for the treatment of ocular diseases. However, the molecular mechanisms of acupuncture treatment are still unclear. In this study, we used technique of RNA sequencing (RNA-seq) to study the effects of acupuncture treatment on retinal transcriptome after axotomy injury. RNA-seq results revealed that 436 genes including 31 transcription factors (TFs) were changed after injury, among them were many well-known neural degeneration related TFs such as Jun, Ddit3, Atf3, and Atf4. Interestingly, acupuncture treatment at acupoint GB20 (Fengchi) significantly reversed a series of differential expressed genes (DEGs) induced by optic nerve injury. While treatments at BL1 (Jingming) or GB20 sham control acupoint-GV16 (Fengfu), led to limited DEG reversal. In contrast, treatments at these two sites further enhanced the trend of DEG expression induced by axotomy injury. At last, retina immunostaining results revealed that only GB20 acupoint treatment increased RGC survival, in consistent with RNA-seq results. Therefore, our study first reported that acupuncture treatment regulated retinal transcriptome and reversed the gene expression induced by axotomy injury, and GB20 acupoint treatment increased RGC survival, which will provide novel therapeutic targets for treatment of ocular diseases.

Disruptions of Autophagy in the Rat Retina with Age During the Development of Age-Related-Macular-Degeneration-like Retinopathy.

Age-related macular degeneration (AMD) is one of the main causes of vision impairment in the elderly. Autophagy is the process of delivery of cytoplasmic components into lysosomes for cleavage; its age-related malfunction may contribute to AMD. Here we showed that the development of AMD-like retinopathy in OXYS rats is accompanied by retinal transcriptome changes affecting genes involved in autophagy. These genes are associated with kinase activity, immune processes, and FoxO, mTOR, PI3K-AKT, MAPK, AMPK, and neurotrophin pathways at preclinical and manifestation stages, as well as vesicle transport and processes in lysosomes at the progression stage. We demonstrated a reduced response to autophagy modulation (inhibition or induction) in the OXYS retina at age 16 months: expression of genes Atg5, Atg7, Becn1, Nbr1, Map1lc3b, p62, and Gabarapl1 differed between OXYS and Wistar (control) rats. The impaired reactivity of autophagy was confirmed by a decreased number of autophagosomes under the conditions of blocked autophagosome–lysosomal fusion according to immunohistochemical analysis and transmission electron microscopy. Thus, the development of AMD signs occurs against the background of changes in the expression of autophagy-related genes and a decrease in autophagy reactivity: the ability to enhance autophagic flux in response to stress.

Cardinalfishes (Apogonidae) show visual system adaptations typical of nocturnally and diurnally active fish.

Animal visual systems adapt to environmental light on various timescales. In scotopic conditions, evolutionary time-scale adaptations include spectral tuning to a narrower light spectrum, loss (or inactivation) of visual genes, and pure-rod or rod-dominated retinas. Some fishes inhabiting shallow coral reefs may show activity during the day and at night. It is unclear whether these fishes show adaptations typical of exclusively nocturnal or deep-sea fishes, or of diurnally active shallow-water species. Here, we investigated visual pigment diversity in cardinalfishes (Apogonidae). Most cardinalfishes are nocturnal foragers, yet they aggregate in multispecies groups in and around coral heads during the day, engaging in social and predator avoidance behaviours. We sequenced retinal transcriptomes of 28 species found on the Great Barrier Reef, assessed the diversity of expressed opsin genes and predicted the spectral sensitivities of resulting photopigments using sequence information. Predictions were combined with microspectrophotometry (MSP) measurements in seven cardinalfish species. Retinal opsin expression was rod opsin (RH1) dominated (>87%), suggesting the importance of scotopic vision. However, all species retained expression of multiple cone opsins also, presumably for colour vision. We found five distinct quantitative expression patterns among cardinalfishes, ranging from short-wavelength-shifted to long-wavelength-shifted. These results indicate that cardinalfishes are both well adapted to dim-light conditions and have retained a sophisticated colour vision sense. Other reef fish families also show both nocturnal and diurnal activity while most are strictly one or the other. It will be interesting to compare these behavioural differences across different phylogenetic groups using the criteria and methods developed here.

Retinal Transcriptome Analysis in the Treatment of Endotoxin-Induced Uveitis with Tetramethylpyrazine Eye Drops.

Purpose: To investigate retinal gene expression of tetramethylpyrazine (TMP) eye drop-treated endotoxin-induced uveitis (EIU) in mice and to explore the mechanisms. Methods: The inflammatory signs of the anterior segment were evaluated, and clinical scores were graded. The retinal transcriptome from the TMP eye drop-treated and the untreated mice was identified by RNA sequencing (RNA-seq) strategy. Differentially expressed genes (DEGs) were validated by real-time PCR. The protein-protein interaction was analyzed using the STRING software. Results: Compared with the TMP-treated group, the inflammatory responses of the untreated control group were much severe and clinical score was remarkably higher (P < 0.001) at 24 h after lipopolysaccharide administration. RNA-seq assay identified 407 DEGs, among which 356 were upregulated and 51 were downregulated. There were 12 upregulated gene ontology terms enriched and 27 upregulated pathways. Seven DEGs, including inflammation-related, complement system-related, and interferon-related genes, were validated using quantitative PCR. Conclusions: TMP exerted anti-inflammatory effect in EIU. Local application of TMP inhibited retinal inflammatory response by regulating the inflammation-related genes, suggesting that TMP may be a potential novel therapeutic drug for ocular inflammation.

Deletion of miR-182 Leads to Retinal Dysfunction in Mice.

MicroRNA-182 (miR-182) is abundantly expressed in mammalian retinas; however, the association between miR-182 and retinal function remains unclear. In this study, we explored whether miR-182 contributes to functional decline in retinas using a miR-182 depleted mouse. Electroretinogram (ERG) amplitudes at different ages were measured in miR-182 knockout (KO) mice. The thickness and lamination of retinas were assessed using a color fundus camera and high-resolution optical coherence tomography. Expression levels of key photoreceptor-specific genes and the miR-183/96/182 cluster (miR-183C) were quantified using quantitative real-time PCR. RNA sequencing and light-induced damage were carried out to observe the changes in the retinal transcriptome and sensitivity to light damage in the miR-182 KO mice. The ERG recording reveals that the ERG response amplitude decreased both at early and later ages when compared with control littermates. The expression of some key photoreceptor-specific genes was down-regulated with deletion of miR-182 in retina. RNA sequencing indicated that some biological processes of visual system were affected, and the numbers of potential target genes of miR-182 were presented in the mouse retina using bioinformatics analysis. The miR-182 KO mice were characterized by progressively losing the outer segment after being treated with light-damage exposure. The thickness and lamination of retina as well as compensatory expression of miR-183C showed no apparent changes in retina of miR-182 KO mice under normal laboratory lighting condition. Our findings provided new insights into the relationship between the miR-182 and retinal development and revealed that miR-182 may play a critical role in maintaining retinal function.

Owls lack UV-sensitive cone opsin and red oil droplets, but see UV light at night: Retinal transcriptomes and ocular media transmittance

Most diurnal birds have cone-dominated retinae and tetrachromatic colour vision based on ultra-violet/violet-sensitive UV/V cones expressing short wavelength-sensitive opsin 1 (SWS1), S cones expressing short wavelength-sensitive opsin 2 (SWS2), M cones expressing medium wavelength-sensitive opsin (RH2) and L cones expressing long wavelength-sensitive opsin (LWS). Double cones (D) express LWS but do not contribute to colour vision. Each cone is equipped with an oil droplet, transparent in UV/V cones, but pigmented by carotenoids: galloxanthin in S, zeaxanthin in M, astaxanthin in L and a mixture in D cones. Owls (Strigiformes) are crepuscular or nocturnal birds with rod-dominated retinae and optical adaptations for high sensitivity. For eight species, the absence of functional SWS1 opsin has recently been documented, functional RH2 opsin was absent in three of these. Here we confirm the absence of SWS1 transcripts for the Long-eared owl (Asio otus) and demonstrate its absence for the Short-eared owl (Asio flammeus), Tawny owl (Strix aluco) and Boreal owl (Aegolius funereus). All four species had transcripts of RH2, albeit with low expression. All four species express all enzymes needed to produce galloxanthin, but lack CYP2J19 expression required to produce astaxanthin from dietary precursors. We also present ocular media transmittance of the Eurasian eagle owl (Bubo bubo) and Short-eared owl and predict spectral sensitivities of all photoreceptors of the Tawny owl. We conclude that owls, despite lacking UV/V cones, can detect UV light. This increases the sensitivity of their rod vision allowing them, for instance, to see UV-reflecting feathers as brighter signals at night.

Retinal transcriptome and eQTL analyses identify genes associated with age-related macular degeneration.

Genome-wide association studies (GWAS) have identified genetic variants at 34 loci contributing to age-related macular degeneration (AMD)1–3. We generated transcriptional profiles of postmortem retina from 453 controls and cases at distinct stages of AMD and integrated retinal transcriptomes, covering 13,662 protein-coding and 1,462 non-coding genes, with genotypes at over 9 million common single nucleotide polymorphisms (SNPs) for expression quantitative trait loci (eQTL) analysis of a tissue not included in Genotype-Tissue Expression (GTEx) and other large datasets4, 5. Cis-eQTL analysis identified 10,474 genes under genetic regulation, including 4,541 eQTLs detected only in the retina. Integrated analysis of AMD-GWAS with eQTLs ascertained likely target genes at six reported loci. Using transcriptome-wide association analysis (TWAS), we identified three additional genes, RLBP1, HIC1 and PARP12, after Bonferroni correction. Our studies expand the genetic landscape of AMD and establish the Eye Genotype Expression (EyeGEx) database as a resource for post-GWAS interpretation of multifactorial ocular traits.

Variable vision in variable environments: the visual system of an invasive cichlid (Cichla monoculus) in Lake Gatun, Panama.

An adaptive visual system is essential for organisms inhabiting new or changing light environments. The Panama Canal exhibits such variable environments owing to its anthropogenic origin and current human activities. Within the Panama Canal, Lake Gatun harbors several exotic fish species including the invasive peacock bass (Cichla monoculus), a predatory Amazonian cichlid. In this research, through spectral measurements and molecular and physiological experiments, we studied the visual system of C. monoculus and its adaptive capabilities. Our results suggest that (1) Lake Gatun is a highly variable environment, where light transmission changes throughout the canal waterway, and that (2) C. monoculus has several visual adaptations suited for this red-shifted light environment. Cichla monoculus filters short wavelengths (∼400 nm) from the environment through its ocular media and tunes its visual sensitivities to the available light through opsin gene expression. More importantly, based on shifts in spectral sensitivities of photoreceptors alone, and on transcriptome analysis, C. monoculus exhibits extreme intraspecific variation in the use of vitamin A1/A2 chromophore in their photoreceptors. Fish living in turbid water had higher proportions of vitamin A2, shifting sensitivities to longer wavelengths, than fish living in clear water. Furthermore, we also found variation in retinal transcriptomes, where fish from turbid and clear waters exhibited differentially expressed genes that vary greatly in their function. We suggest that this phenotypic plasticity has been key in the invasion success of C. monoculus.

Evolution of the visual sensory system in the deep-sea fishes from the Sargasso Sea

Evolution of the visual sensory system in the deep-sea fishes from the Sargasso Sea

Gene expression in response to optical defocus of opposite signs reveals bidirectional mechanism of visually guided eye growth.

Myopia (nearsightedness) is the most common eye disorder, which is rapidly becoming one of the leading causes of vision loss in several parts of the world because of a recent sharp increase in prevalence. Nearwork, which produces hyperopic optical defocus on the retina, has been implicated as one of the environmental risk factors causing myopia in humans. Experimental studies have shown that hyperopic defocus imposed by negative power lenses placed in front of the eye accelerates eye growth and causes myopia, whereas myopic defocus imposed by positive lenses slows eye growth and produces a compensatory hyperopic shift in refractive state. The balance between these two optical signals is thought to regulate refractive eye development; however, the ability of the retina to recognize the sign of optical defocus and the composition of molecular signaling pathways guiding emmetropization are the subjects of intense investigation and debate. We found that the retina can readily distinguish between imposed myopic and hyperopic defocus, and identified key signaling pathways underlying retinal response to the defocus of different signs. Comparison of retinal transcriptomes in common marmosets exposed to either myopic or hyperopic defocus for 10 days or 5 weeks revealed that the primate retina responds to defocus of different signs by activation or suppression of largely distinct pathways. We also found that 29 genes differentially expressed in the marmoset retina in response to imposed defocus are localized within human myopia quantitative trait loci (QTLs), suggesting functional overlap between genes differentially expressed in the marmoset retina upon exposure to optical defocus and genes causing myopia in humans. These findings identify retinal pathways involved in the development of myopia, as well as potential new strategies for its treatment.

Ndr kinases regulate retinal interneuron proliferation and homeostasis

Ndr2/Stk38l encodes a protein kinase associated with the Hippo tumor suppressor pathway and is mutated in a naturally-occurring canine early retinal degeneration (erd). To elucidate the retinal functions of Ndr2 and its paralog Ndr1/Stk38, we generated Ndr1 and Ndr2 single knockout mice. Although retinal lamination appeared normal in these mice, Ndr deletion caused a subset of Pax6-positive amacrine cells to proliferate in differentiated retinas, while concurrently decreasing the number of GABAergic, HuD and Pax6-positive amacrine cells. Retinal transcriptome analyses revealed that Ndr2 deletion increased expression of neuronal stress genes and decreased expression of synaptic organization genes. Consistent with the latter, Ndr deletion dramatically reduced levels of Aak1, an Ndr substrate that regulates vesicle trafficking. Our findings indicate that Ndr kinases are important regulators of amacrine and photoreceptor cells and suggest that Ndr kinases inhibit the proliferation of a subset of terminally differentiated cells and modulate interneuron synapse function via Aak1.

Cone-rod homeobox CRX controls presynaptic active zone formation in photoreceptors of mammalian retina.

In the mammalian retina, rod and cone photoreceptors transmit the visual information to bipolar neurons through highly specialized ribbon synapses. We have limited understanding of regulatory pathways that guide morphogenesis and organization of photoreceptor presynaptic architecture in the developing retina. While neural retina leucine zipper (NRL) transcription factor determines rod cell fate and function, cone-rod homeobox (CRX) controls the expression of both rod- and cone-specific genes and is critical for terminal differentiation of photoreceptors. A comprehensive immunohistochemical evaluation of Crx-/- (null), CrxRip/+ and CrxRip/Rip (models of dominant congenital blindness) mouse retinas revealed abnormal photoreceptor synapses, with atypical ribbon shape, number and length. Integrated analysis of retinal transcriptomes of Crx-mutants with CRX- and NRL-ChIP-Seq data identified a subset of differentially expressed CRX target genes that encode presynaptic proteins associated with the cytomatrix active zone (CAZ) and synaptic vesicles. Immunohistochemistry of Crx-mutant retina validated aberrant expression of REEP6, PSD95, MPP4, UNC119, UNC13, RGS7 and RGS11, with some reduction in Ribeye and no significant change in immunostaining of RIMS1, RIMS2, Bassoon and Pikachurin. Our studies demonstrate that CRX controls the establishment of CAZ and anchoring of ribbons, but not the formation of ribbon itself, in photoreceptor presynaptic terminals.

Convergent phenotypic evolution of the visual system via different molecular routes: How Neotropical cichlid fishes adapt to novel light environments.

How predictable is evolution? This remains a fundamental but contested issue in evolutionary biology. When independent lineages colonize the same environment, we are presented with a natural experiment that allows us to ask if genetic and ecological differences promote species‐specific evolutionary outcomes or whether species phenotypically evolve in a convergent manner in response to shared selection pressures. If so, are the molecular mechanisms underlying phenotypic convergence the same? In Nicaragua, seven species of cichlid fishes concurrently colonized two novel photic environments. Hence, their visual system represents a compelling model to address these questions, particularly since the adaptive value of phenotypic changes is well‐understood. By analyzing retinal transcriptomes, we found that differential expression of genes responsible for color vision (cone opsins and cyp27c1) produced rapid and mostly convergent changes of predicted visual sensitivities. Notably, these changes occurred in the same direction in all species although there were differences in underlying gene expression patterns illustrating nonconvergence at the molecular level. Adaptive phenotypes evolved deterministically, even when species differ substantially in ecology and genetic variation. This provides strong evidence that phenotypic evolution of the visual system occurred in response to similar selective forces of the photic environment.

Retinal transcriptome profile in mice following dexamethasone treatment for endotoxin-induced uveitis

To investigate the changes in retinal transcriptome profile of mice with endotoxin-induced uveitis (EIU)following dexamethasone (DEX) treatment and explore the mechanisms underlying the therapeutic effect of DEX. EIU was induced in BALB/c mice by intravitreal injection of 125 ng lipopolysaccharide (LPS), followed by topical applicationof DEX (0.1%) eye drops every 4 h for 24 h. The anterior chamber inflammation was examined with a slit lamp and the clinicalscores were assessed. The morphological changes in the eyes were assessed at 24 h after LPS injection. The retinas wereharvested for analysis of transcriptome profile using the next-generation sequencing (NGS)-based RNA sequencing (RNA-seq), and the expressions of the inflammatory cytokines and the differentially expressed genes (DEGs) were verified using real-timePCR. DEX alleviated the inflammatory response and reduced the mRNA expressions of IL-6, TNF- a, MCP-1 andICAM-1 at 24 h after LPS injection. A total of 52 DEGs were identified by RNA-seq. Within these DEGs, 37 genes were upregulated and 15 genes were down-regulated in LPS group as compared with DEX+LPS group. No significantly enriched GeneOntology (GO) terms was noted. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed 6up-regulated and 2 down-regulated KEGG pathways. RIG-I-like receptor signaling pathway and several immune- andinflammation-related genes including Ifit1, H2-T24, Mx2 and Eif2ak2 were significantly down regulated by DEX. Verificationwith RT-PCR yielded results consistent with these findings. DEX alleviates LPS-induced inflammatory response inthe retina of mice, and such protective effect is probably mediated by RIG-I like receptor signal pathway and the immune-andinflammation-related genes.

Downregulation of splicing regulator RBFOX1 compromises visual depth perception.

Rbfox1 is a splicing regulator that has been associated with various neurological conditions such as autism spectrum disorder, mental retardation, epilepsy, attention-deficit/hyperactivity disorder and schizophrenia. We show that in adult rodent retinas, Rbfox1 is expressed in all types of retinal ganglion cells (RGCs) and in certain subsets of amacrine cells (ACs), within the inner nuclear (INL) and ganglion cell (GCL) layers. In the INL, all Rbfox1-positive cells were colocalized with GABAergic ACs, however not all GABAergic ACs were immunostained for Rbfox1. In the GCL, a vast majority of GABAergic dACs were Rbfox1-immunopositive. Furthermore, all cholinergic starburst ACs (SACs) in the INL (type a) and in the GCL (type b) were Rbfox1 positive. The expression of Rbfox1 in the retina significantly overlapped with expression of Rbfox2, another member of Rbfox family of proteins. Rbfox2, in addition to RGCs and ACs, was also expressed in horizontal cells. In developing retinas at E12 and E15, Rbfox1 is localized to the cytoplasm of differentiating RGCs and ACs. Between P0 and P5, Rbfox1 subcellular localization switched from cytoplasmic to predominantly nuclear. Downregulation of Rbfox1 in adult Rbfox1loxP/loxP mice had no detectable effect on retinal gross morphology. However, the visual cliff test revealed marked abnormalities of depth perception of these animals. RNA sequencing of retinal transcriptomes of control and Rbfox1 knockout animals identified a number of Rbfox1-regulated genes that are involved in establishing neuronal circuits and synaptic transmission, including Vamp1, Vamp2, Snap25, Trak2, and Slc1A7, suggesting the role of Rbfox1 in facilitating synaptic communications between ACs and RGCs.

TLR4 modulates inflammatory gene targets in the retina during Bacillus cereus endophthalmitis

BackgroundEndophthalmitis is a serious intraocular infection that frequently results in significant inflammation and vision loss. Because current therapeutics are often unsuccessful in mitigating damaging inflammation during endophthalmitis, more rational targets are needed. Toll-like receptors (TLRs) recognize specific motifs on invading pathogens and initiate the innate inflammatory response. We reported that TLR4 contributes to the robust inflammation which is a hallmark of Bacillus cereus endophthalmitis. To identify novel, targetable host inflammatory factors in this disease, we performed microarray analysis to detect TLR4-dependent changes to the retinal transcriptome during B. cereus endophthalmitis.ResultsC57BL/6 J and TLR4−/− mouse eyes were infected with B. cereus and retinas were harvested at 4 h postinfection, a time representing the earliest onset of neutrophil infiltration. Genes related to acute inflammation and inflammatory cell recruitment including CXCL1 (KC), CXCL2 (MIP2-α), CXCL10 (IP-10), CCL2 (MCP1), and CCL3 (MIP1-α)) were significantly upregulated 5-fold or greater in C57BL/6 J retinas. The immune modulator IL-6, intercellular adhesion molecule ICAM1, and the inhibitor of cytokine signal transduction SOCS3 were upregulated 25-, 11-, and 10-fold, respectively, in these retinas. LIF, which is crucial for photoreceptor cell survival, was increased 6-fold. PTGS2/COX-2, which converts arachidonic acid to prostaglandin endoperoxide H2, was upregulated 9-fold. PTX3, typically produced in response to TLR engagement, was induced 15-fold. None of the aforementioned genes were upregulated in TLR4−/− retinas following B. cereus infection.ConclusionsOur results have identified a cohort of mediators driven by TLR4 that may be important in regulating pro-inflammatory and protective pathways in the retina in response to B. cereus intraocular infection. This supports the prospect that blocking the activation of TLR-based pathways might serve as alternative targets for Gram-positive and Gram-negative endophthalmitis therapies in general.