Wt Retinas Research Articles

The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1β and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1β in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1β. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases.

The Expression of Major Facilitator Superfamily Domain-Containing Protein2a (Mfsd2a) and Aquaporin 4 Is Altered in the Retinas of a 5xFAD Mouse Model of Alzheimer's Disease.

Cerebral amyloid angiopathy (CAA) is characterized by amyloid β (Aβ) accumulation in the blood vessels and is associated with cognitive impairment in Alzheimer's disease (AD). The increased accumulation of Aβ is also present in the retinal blood vessels and a significant correlation between retinal and brain amyloid deposition was demonstrated in living patients and animal AD models. The Aβ accumulation in the retinal blood vessels can be the result of impaired transcytosis and/or the dysfunctional ocular glymphatic system in AD and during aging. We analyzed the changes in the mRNA and protein expression of major facilitator superfamily domain-containing protein2a (Mfsd2a), the major regulator of transcytosis, and of Aquaporin4 (Aqp4), the key player implicated in the functioning of the glymphatic system, in the retinas of 4- and 12-month-old WT and 5xFAD female mice. A strong decrease in the Mfsd2a mRNA and protein expression was observed in the 4 M and 12 M 5xFAD and 12 M WT retinas. The increase in the expression of srebp1-c could be at least partially responsible for the Mfsd2a decrease in the 4 M 5xFAD retinas. The decrease in the pericyte (CD13+) coverage of retinal blood vessels in the 4 M and 12 M 5xFAD retinas and in the 12 M WT retinas suggests that pericyte loss could be associated with the Mfsd2a downregulation in these experimental groups. The observed increase in Aqp4 expression in 4 M and 12 M 5xFAD and 12 M WT retinas accompanied by the decreased perivascular Aqp4 expression is indicative of the impaired glymphatic system. The findings in this study reveal the impaired Mfsd2a and Aqp4 expression and Aqp4 perivascular mislocalization in retinal blood vessels during physiological (WT) and pathological (5xFAD) aging, indicating their importance as putative targets for the development of new treatments that can improve the regulation of transcytosis or the function of the glymphatic system.

Electrically-evoked responses for retinal prostheses are differentially altered depending on ganglion cell types in outer retinal neurodegeneration caused by Crb1 gene mutation.

Microelectronic prostheses for artificial vision stimulate neurons surviving outer retinal neurodegeneration such as retinitis pigmentosa (RP). Yet, the quality of prosthetic vision substantially varies across subjects, maybe due to different levels of retinal degeneration and/or distinct genotypes. Although the RP genotypes are remarkably diverse, prosthetic studies have primarily used retinal degeneration (rd) 1 and 10 mice, which both have Pde6b gene mutation. Here, we report the electric responses arising in retinal ganglion cells (RGCs) of the rd8 mouse model which has Crb1 mutation. We first investigated age-dependent histological changes of wild-type (wt), rd8, and rd10 mice retinas by H&E staining. Then, we used cell-attached patch clamping to record spiking responses of ON, OFF and direction selective (DS) types of RGCs to a 4-ms-long electric pulse. The electric responses of rd8 RGCs were analyzed in comparison with those of wt RGCs in terms of individual RGC spiking patterns, populational characteristics, and spiking consistency across trials. In the histological examination, the rd8 mice showed partial retinal foldings, but the outer nuclear layer thicknesses remained comparable to those of the wt mice, indicating the early-stage of RP. Although spiking patterns of each RGC type seemed similar to those of the wt retinas, correlation levels between electric vs. light response features were different across the two mouse models. For example, in comparisons between light vs. electric response magnitudes, ON/OFF RGCs of the rd8 mice showed the same/opposite correlation polarity with those of wt mice, respectively. Also, the electric response spike counts of DS RGCs in the rd8 retinas showed a positive correlation with their direction selectivity indices (r = 0.40), while those of the wt retinas were negatively correlated (r = -0.90). Lastly, the spiking timing consistencies of late responses were largely decreased in both ON and OFF RGCs in the rd8 than the wt retinas, whereas no significant difference was found across DS RGCs of the two models. Our results indicate the electric response features are altered depending on RGC types even from the early-stage RP caused by Crb1 mutation. Given the various degeneration patterns depending on mutation genes, our study suggests the importance of both genotype- and RGC type-dependent analyses for retinal prosthetic research.

Photochemical Restoration of Light Sensitivity in the Degenerated Canine Retina.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 μL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway

BackgroundMicroglia, the innate immune cells in the central nervous system, play an essential role in brain homeostasis, neuroinflammation and brain infections. Dysregulated microglia, on the other hand, are associated with neurodegenerative diseases, yet the mechanisms underlying pro-inflammatory gene expression in microglia are incompletely understood.MethodsWe investigated the role of the actin-associated protein tropomyosin 1 (TPM1) in regulating pro-inflammatory phenotype of microglia in the retina by using a combination of cell culture, immunocytochemistry, Western blot, qPCR, TUNEL, RNA sequencing and electroretinogram analysis. TREM2−/− mice were used to investigate whether TPM1 regulated pro-inflammatory responses downstream of TREM2. To conditionally deplete microglia, we backcrossed CX3CR1CreER mice with Rosa26iDTR mice to generate CX3CR1CreER:Rosa26iDTR mice.ResultsWe revealed a vital role for TPM1 in regulating pro-inflammatory phenotype of microglia. We found that TPM1 drove LPS-induced inflammation and neuronal death in the retina via the PKA/CREB pathway. TPM1 knockdown ameliorated LPS-induced inflammation in WT retinas yet exaggerated the inflammation in TREM2−/− retinas. RNA sequencing revealed that genes associated with M1 microglia and A1 astrocytes were significantly downregulated in LPS-treated WT retinas but upregulated in LPS-treated TREM2−/− retinas after TPM1 knockdown. Mechanistically, we demonstrated that CREB activated by TPM1 knockdown mediated anti-inflammatory genes in LPS-treated WT retinas but pro-inflammatory genes in LPS-treated TREM2−/− retinas, suggesting a novel role for TREM2 as a brake on TPM1-mediated inflammation. Furthermore, we identified that TPM1 regulated inflammation downstream of TREM2 and in a microglia-dependent manner.ConclusionsWe demonstrate that TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway. Our findings suggest that TPM1 could be a potential target for therapeutic intervention in brain diseases.

Retinoid X receptor activation promotes photoreceptor survival and modulates the inflammatory response in a mouse model of retinitis pigmentosa

Photoreceptor cell (PHR) death is a hallmark of most retinal neurodegenerative diseases, in which inflammation plays a critical role. Activation of retinoid X receptors (RXR) modulates and integrates multiple cell functions, and has beneficial effects in animal models of chronic inflammatory diseases. Nonetheless, the mechanisms involved and their role in retina neuroprotection are poorly understood. In this work we assessed whether RXR activation prevents inflammation and/or PHR death in retinitis pigmentosa, an inherited retina neurodegeneration, using as an ex vivo model, retinas from the rd1 mice, a murine model of this disease. We demonstrated that rd1 retinas had lower levels of RXR alpha isoform than their wt counterparts at early developmental times, whereas its distribution pattern remained similar. In mixed neuro-glial cultures obtained from either rd1 or wt retinas, both PHR and Müller glial cells (MGC) expressed RXRalpha, and RXR activation by its synthetic pan-agonist PA024 selectively increased mRNA levels of RXRgamma isoform. PA024 decreased PHR death in rd1 mixed cultures; it reduced the amount of non-viable neurons, delayed the onset of PHR apoptosis, and decreased Bax mRNA levels. PA024 also reduced MGC reactivity in vitro before and at the onset of degeneration, decreasing GFAP expression, increasing glutamine synthetase mRNA levels, and promoting the transcription of the anti-inflammatory cytokine, Il-10. These results suggest that RXR activation rescues rd1 PHR and decreases MGC reactivity, promoting an anti-inflammatory environment in the rd1 retina, thus supporting the potential of RXR agonists as pharmacological tools for treating retina degenerative diseases.

Metabolic Features of Mouse and Human Retinas: Rods versus Cones, Macula versus Periphery, Retina versus RPE.

SummaryPhotoreceptors, especially cones, which are enriched in the human macula, have high energy demands, making them vulnerable to metabolic stress. Metabolic dysfunction of photoreceptors and their supporting retinal pigment epithelium (RPE) is an important underlying cause of degenerative retinal diseases. However, how cones and the macula support their exorbitant metabolic demand and communicate with RPE is unclear. By profiling metabolite uptake and release and analyzing metabolic genes, we have found cone-rich retinas and human macula share specific metabolic features with upregulated pathways in pyruvate metabolism, mitochondrial TCA cycle, and lipid synthesis. Human neural retina and RPE have distinct but complementary metabolic features. Retinal metabolism centers on NADH production and neurotransmitter biosynthesis. The retina needs aspartate to sustain its aerobic glycolysis and mitochondrial metabolism. RPE metabolism is directed toward NADPH production and biosynthesis of acetyl-rich metabolites, serine, and others. RPE consumes multiple nutrients, including proline, to produce metabolites for the retina.

Targeting connexin37 alters angiogenesis and arteriovenous differentiation in the developing mouse retina.

Connexin37 (Cx37) forms intercellular channels between endothelial cells (EC), and contributes to coordinate the motor tone of vessels. We investigated the contribution of this protein during physiological angiogenesis. We show that, compared to WT littermates, mice lacking Cx37 (Cx37-/- ) featured (i) a decreased extension of the superficial vascular plexus during the first 4days after birth; (ii) an increased vascular density at the angiogenic front at P6, due to an increase in the proliferative rate of EC and in the sprouting of the venous compartment, as well as to a somewhat displaced position of tip cells; (iii) a decreased coverage of newly formed arteries and veins by mural cells; (iv) altered ERK-dependent endothelial cells proliferation through the EphB4 signaling pathway, which is involved in the specification of veins and arteries. In vitro studies documented that, in the absence of Cx37, human venous EC (HUVEC) released less platelet-derived growth factor (PDGF) and more Angiopoietin-2, two molecules involved in the recruitment of mural cells. Treatment of mice with DAPT, an inhibitor of the Notch pathway, decreased the expression of Cx37, and partially mimicked in WT retinas, the alterations observed in Cx37-/- mice. Thus, Cx37 contributes to (i) the early angiogenesis of retina, by interacting with the Notch pathway; (ii) the growth and maturation of neo-vessels, by modulating tip, stalk, and mural cells; (iii) the regulation of arteriovenous specification, thus, representing a novel target for treatments of retina diseases.

Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution

The mammalian CNS is capable of tolerating chronic hypoxia, but cell type-specific responses to this stress have not been systematically characterized. In the Norrin KO (NdpKO ) mouse, a model of familial exudative vitreoretinopathy (FEVR), developmental hypovascularization of the retina produces chronic hypoxia of inner nuclear-layer (INL) neurons and Muller glia. We used single-cell RNA sequencing, untargeted metabolomics, and metabolite labeling from 13C-glucose to compare WT and NdpKO retinas. In NdpKO retinas, we observe gene expression responses consistent with hypoxia in Muller glia and retinal neurons, and we find a metabolic shift that combines reduced flux through the TCA cycle with increased synthesis of serine, glycine, and glutathione. We also used single-cell RNA sequencing to compare the responses of individual cell types in NdpKO retinas with those in the hypoxic cerebral cortex of mice that were housed for 1 week in a reduced oxygen environment (7.5% oxygen). In the hypoxic cerebral cortex, glial transcriptome responses most closely resemble the response of Muller glia in the NdpKO retina. In both retina and brain, vascular endothelial cells activate a previously dormant tip cell gene expression program, which likely underlies the adaptive neoangiogenic response to chronic hypoxia. These analyses of retina and brain transcriptomes at single-cell resolution reveal both shared and cell type-specific changes in gene expression in response to chronic hypoxia, implying both shared and distinct cell type-specific physiologic responses.

Deletion of Arginase 2 reduces neurodegeneration in a model of Multiple Sclerosis

PurposeVisual impairment due to optic neuritis is a major clinical feature of Multiple Sclerosis (MS), and can lead to temporary or permanent vision loss. Previous studies from our lab have demonstrated the critical involvement of arginase 2 (A2) in retinal neurodegeneration. The current study was undertaken to investigate the role of A2 signaling in MS mediated retinal neuronal damage.MethodExperimental Autoimmune Encephalomyelitis (EAE) was induced in WT mice and A2 knock out (A2 KO) mice (both on C57Bl6 background) according to established procedures. Briefly, WT and A2 KO mice were immunized by subcutaneous injections of an emulsion containing MOG35‐55 peptide (300 μg/mouse) along with Complete Freund's adjuvant (CFA, killed Mycobacterium tuberculosis H37Ra, 400 μg/ul). Each mouse additionally received 300 ng of pertussis toxin (PT, Sigma, St. Louis, MO) by i.p. injection in 200 μl of PBS on day 0 and day 2 post‐immunization. Control group will be immunized with CFA without antigen (MOG), and two doses of PT will be given on day 0 and 2. Clinical disease scores were monitored daily in all groups in a blinded fashion by measuring paralysis according to the conventional grading system. Neurodegeneration studies were performed using retinal flatmount analysis. Expression of arginase and the downstream signaling molecules were examined by Western blotting, and inflammatory molecules were analyzed by qPCR.ResultsIncreased expression A2 was observed in WT retinas in response to EAE induction. EAE‐induced motor deficits and body weight loss were markedly reduced in A2 KO mice compared to WT controls. Retinal flat mount studies demonstrated a significant reduction in the number of Brn3a positive and NeuN positive neurons in WT EAE retina (p<0.01; N=6–10) in comparison to normal control mice. A significant increase in the number of surviving neurons (NeuN‐positive and Brn3a‐positive) was evident in retinas of EAE‐induced A2KO mice compared to WT (p<0.05; N=6–10). RNA levels of proinflammatory molecules such as CCL2 ((C‐C motif) ligand 2), COX2 (Cyclooxygenase‐2), IL1a (Interleukin 1a), IL12a, and IL‐18 were significantly reduced in the A2KO EAE retinas compared to WT EAE (p<0.05, N=5–6). Signaling studies showed increased levels of phospho‐ERK1/2 and reduced levels phospho‐BAD in the WT EAE retina, while these changes were normalized in EAE‐induced A2 retinas.ConclusionOur studies establish EAE as an excellent model to study MS‐mediated retinal neuronal damage and suggest the potential value of targeting arginase 2 signaling as a therapy to prevent MS‐mediated vision loss.Support or Funding InformationThese studies supported by the National Multiple Sclerosis Society (PP‐1606‐08778 to S.P.N) and Augusta University Culver Vision Discovery Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Opposing Effects of Valproic Acid Treatment Mediated by Histone Deacetylase Inhibitor Activity in Four Transgenic X. laevis Models of Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in Rhodopsin; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA on Xenopus laevis models of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases.SIGNIFICANCE STATEMENT Retinitis pigmentosa (RP) is an inherited, degenerative retinal disease that leads to blindness for which no therapy is available. We determined that valproic acid (VPA), currently undergoing a phase II trial for RP, has both beneficial and detrimental effects in animal models of RP depending on the underlying disease mechanism and that both effects are due to histone deacetylase (HDAC) inhibition possibly linked to autophagy regulation. Off-label use of VPA and other HDAC inhibitors for the treatment of RP should be limited to the research setting until this effect is understood and can be predicted. Our study suggests that, unless genotype is accounted for, clinical trials for RP treatments may give negative results due to multiple disease mechanisms with differential responses to therapeutic interventions.

Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition.AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies.

Blue light-induced retinal lesions, intraretinal vascular leakage and edema formation in the all-cone mouse retina

Little is known about the mechanisms underlying macular degenerations, mainly for the scarcity of adequate experimental models to investigate cone cell death. Recently, we generated R91W;Nrl−/− double-mutant mice, which display a well-ordered all-cone retina with normal retinal vasculature and a strong photopic function that generates useful vision. Here we exposed R91W;Nrl−/− and wild-type (wt) mice to toxic levels of blue light and analyzed their retinas at different time points post illumination (up to 10 days). While exposure of wt mice resulted in massive pyknosis in a focal region of the outer nuclear layer (ONL), the exposure of R91W;Nrl−/− mice led to additional cell death detected within the inner nuclear layer. Microglia/macrophage infiltration at the site of injury was more pronounced in the all-cone retina of R91W;Nrl−/− than in wt mice. Similarly, vascular leakage was abundant in the inner and outer retina in R91W;Nrl−/− mice, whereas it was mild and restricted to the subretinal space in wt mice. This was accompanied by retinal swelling and the appearance of cystoid spaces in both inner and ONLs of R91W;Nrl−/− mice indicating edema in affected areas. In addition, basal expression levels of tight junction protein-1 encoding ZO1 were lower in R91W;Nrl−/− than in wt retinas. Collectively, our data suggest that exposure of R91W;Nrl−/− mice to blue light not only induces cone cell death but also disrupts the inner blood–retinal barrier. Macular edema in humans is a result of diffuse capillary leakage and microaneurysms in the macular region. Blue light exposure of the R91W;Nrl−/− mouse could therefore be used to study molecular events preceding edema formation in a cone-rich environment, and thus potentially help to develop treatment strategies for edema-based complications in macular degenerations.

CX3CL1 (fractalkine) protein expression in normal and degenerating mouse retina: in vivo studies.

We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1.

Inhibition of dopamine signaling suppresses cGMP accumulation in rd1 retinal organ cultures

The rd1 mouse is a model of retinitis pigmentosa, an inherited photoreceptor neurodegenerative disease. In rd1 retina, early onset rod degeneration is caused by a Pde6b mutation that leads to high levels of intracellular cyclic guanosine monophosphate (cGMP). Cyclic nucleotide-gated ion channels (CNGCs), necessary for phototransduction, are regulated by cGMP. We have previously demonstrated that inhibition of dopamine signaling blocks rd1 photoreceptor degeneration in retinal organ cultures. The mechanism underlying this protection remains unknown. The aim of this study was to determine whether inhibition of dopamine signaling alters cGMP accumulation or CNGC expression. Dopamine depletion from rd1 retinal organ cultures resulted in a significant decrease in cGMP compared with untreated rd1 organ cultures. However, cGMP levels in both treated and untreated rd1 organ cultures significantly exceeded cGMP levels in wild-type (wt) retinal organ cultures. The CNGC expression profile was first determined in vivo. Both channel subunits, Cnga1 and Cngb1, are expressed at low levels by postnatal day 2 (P2), increasing sharply by P6 with a modest increase after P12 in wt retina. A similar pattern is seen in rd1 retina until P12 when expression levels decrease, leading to cell death. No significant difference was observed in the expression of either Cnga1 or Cngb1 in organ cultures from wt, rd1, and dopamine-depleted rd1 retinas. Our results show that dopamine depletion significantly decreases cGMP levels in rd1 retinal organ cultures, but that cGMP accumulation remains high, requiring additional mechanisms for photoreceptor protection. These mechanisms may include activation of protein kinase G-signaling pathways and/or crosstalk with dopamine signaling through cyclic adenosine monophosphate pathways.

SLITRK6 mutations cause myopia and deafness in humans and mice

Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness.

Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell–cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl −/− model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl −/− compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function.

A role for prenylated rab acceptor 1 in vertebrate photoreceptor development

BackgroundThe rd1 mouse retina is a well-studied model of retinal degeneration where rod photoreceptors undergo cell death beginning at postnatal day (P) 10 until P21. This period coincides with photoreceptor terminal differentiation in a normal retina. We have used the rd1 retina as a model to investigate early molecular defects in developing rod photoreceptors prior to the onset of degeneration.ResultsUsing a microarray approach, we performed gene profiling comparing rd1 and wild type (wt) retinas at four time points starting at P2, prior to any obvious biochemical or morphological differences, and concluding at P8, prior to the initiation of cell death. Of the 143 identified differentially expressed genes, we focused on Rab acceptor 1 (Rabac1), which codes for the protein Prenylated rab acceptor 1 (PRA1) and plays an important role in vesicular trafficking. Quantitative RT-PCR analysis confirmed reduced expression of PRA1 in rd1 retina at all time points examined. Immunohistochemical observation showed that PRA1-like immunoreactivity (LIR) co-localized with the cis-Golgi marker GM-130 in the photoreceptor as the Golgi translocated from the perikarya to the inner segment during photoreceptor differentiation in wt retinas. Diffuse PRA1-LIR, distinct from the Golgi marker, was seen in the distal inner segment of wt photoreceptors starting at P8. Both plexiform layers contained PRA1 positive punctae independent of GM-130 staining during postnatal development. In the inner retina, PRA1-LIR also colocalized with the Golgi marker in the perinuclear region of most cells. A similar pattern was seen in the rd1 mouse inner retina. However, punctate and significantly reduced PRA1-LIR was present throughout the developing rd1 inner segment, consistent with delayed photoreceptor development and abnormalities in Golgi sorting and vesicular trafficking.ConclusionsWe have identified genes that are differentially regulated in the rd1 retina at early time points, which may give insights into developmental defects that precede photoreceptor cell death. This is the first report of PRA1 expression in the retina. Our data support the hypothesis that PRA1 plays an important role in vesicular trafficking between the Golgi and cilia in differentiating and mature rod photoreceptors.

Retinal Expression of Wnt-Pathway Mediated Genes in Low-Density Lipoprotein Receptor-Related Protein 5 (Lrp5) Knockout Mice

Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5−/−) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5−/− mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5−/− and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5−/− retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5−/− retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5−/− retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.

LiGluR Restores Visual Responses in Rodent Models of Inherited Blindness

Inherited retinal degeneration results from many different mutations in either photoreceptor-specific or nonphotoreceptor-specific genes. However, nearly all mutations lead to a common blinding phenotype that initiates with rod cell death, followed by loss of cones. In most retinal degenerations, other retinal neuron cell types survive for long periods after blindness from photoreceptor loss. One strategy to restore light responsiveness to a retina rendered blind by photoreceptor degeneration is to express light-regulated ion channels or transporters in surviving retinal neurons. Recent experiments in rodents have restored light-sensitivity by expressing melanopsin or microbial opsins either broadly throughout the retina or selectively in the inner segments of surviving cones or in bipolar cells. Here, we present an approach whereby a genetically and chemically engineered light-gated ionotropic glutamate receptor (LiGluR) is expressed selectively in retinal ganglion cells (RGCs), the longest-surviving cells in retinal blinding diseases. When expressed in the RGCs of a well-established model of retinal degeneration, the rd1 mouse, LiGluR restores light sensitivity to the RGCs, reinstates light responsiveness to the primary visual cortex, and restores both the pupillary reflex and a natural light-avoidance behavior.