Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Different inflammation responses modulate Müller glia proliferation in the acute or chronically damaged zebrafish retina.

Unlike mammals, zebrafish regenerate in response to retinal damage. Because microglia are activated by retinal damage, we investigated their role during regeneration following either acute or chronic damage. At three weeks post-fertilization (wpf), both wild-type fish exhibiting NMDA-induced acute ganglion and amacrine cell death and gold rush (gosh) mutant fish possessing chronic cone photoreceptor degeneration displayed reactive microglia/macrophages and Müller glia proliferation. Dexamethasone-treated retinas, to inhibit the immune response, lacked reactive microglia/macrophages and possessed fewer PCNA-positive cells, while LPS treatment increased microglia/macrophages and PCNA-labeled cells. NMDA-injured retinas upregulated expression of il-1ß and tnfα pro-inflammatory cytokine genes, followed by increased expression of il-10 and arg1 anti-inflammatory/remodeling cytokine genes. A transient early TNFα pro-inflammatory microglia/macrophage population was visualized in NMDA-damaged retinas. In contrast, gosh mutant retinas exhibited a slight increase of pro-inflammatory cytokine gene expression concurrently with a greater increased anti-inflammatory/remodeling cytokine gene expression. Few TNFα pro-inflammatory microglia/macrophages were observed in the gosh retina. Understanding why acute and chronic damage results in different inflammation profiles and their effects on regulating zebrafish retinal regeneration would provide important clues toward improving therapeutic strategies for repairing injured mammalian tissues.

Inflammation induces zebrafish regeneration.

Tissue or organ regeneration is a complex process with successful outcomes depending on the type of tissue and organism. Upon damage, mammals can only efficiently restore a few tissues including the liver, skin, epithelia of the lung, kidney, and gut. In contrast, lower vertebrates such as zebrafish possess an extraordinary regeneration ability, which restores the normal function of a broad spectrum of tissues including heart, fin, brain, spinal cord, and retina. This regeneration process is either mediated by the proliferation of resident stem cells, or cells that dedifferentiate into a stem cell-like. In recent years, evidence has suggested that the innate immune system can modulate stem cell activity to initiate the regenerative response to damage. This review will explore some of the newer concepts of inflammation in zebrafish regeneration in different tissues. Understanding how inflammation regulates regeneration in zebrafish would provide important clues to improve the therapeutic strategies for repairing injured mammalian tissues that do not have an inherent regenerative capacity.

TNFα Induces Müller Glia to Transition From Non-proliferative Gliosis to a Regenerative Response in Mutant Zebrafish Presenting Chronic Photoreceptor Degeneration.

Unlike mammals, zebrafish have the capacity to regenerate neurons in response to damage. Most zebrafish retinal injury models employ acute damage, which is unlike the chronic, gradual damage that occurs in human retinal diseases. Here, we studied the regenerative response in the zebrafish aipl1b mutant, gold rush (gosh). In gosh mutants, both cones and rods degenerate by 3 weeks post-fertilization (wpf). Müller glia do not exhibit a regenerative response by 3 wpf; however, they do present non-proliferative gliosis. Only at 5 wpf, is proliferation of Müller cells and rod precursor cells activated. Rods start to recover at 5 wpf and by 12 wpf they reach a level of recovery comparable to wild type, but cones remain absent in the adult stage. TNFα was detected in degenerating cones at 5-7 wpf and in Müller glia at 7 wpf in gosh mutants. At 5 wpf, proliferating Müller glia express Sox2, followed by Pax6 expression in neuronal progenitor cells (NPCs), confirming that the neuronal regeneration program is activated in gosh mutants after 5 wpf. Although acute light-induced damage did not activate proliferation of Müller glia, TNFα injection caused Müller glia to commence a proliferative response at 3 wpf in gosh mutants. These results suggest that Müller glia transition from non-proliferative gliosis to a regenerative state in gosh mutants, and that ectopic introduction of TNFα promotes this Müller cell transition even at 3 wpf. Thus, zebrafish gosh mutants provide a useful model to investigate mechanisms underlying retinal regeneration in a chronic photoreceptor degeneration model.

Do cGMP Levels Drive the Speed of Photoreceptor Degeneration?

Humans with mutations in the phototransduction pathway develop forms of retinal degeneration, such as retinitis pigmentosa, cone dystrophy, or Leber congenital amaurosis. Similarly, numerous phototransduction mutant animal models resemble retinal degeneration. In our lab, using a zebrafish model, we study cone-specific phototransduction mutants. cGMP is the second messenger in the phototransduction pathway, and abnormal cGMP levels are associated with photoreceptor death. Rd1, a rod-specific phosphodiesterase 6 (Pde6) subunit mutant in mice, is one of the most widely used animal models for retinal degeneration. Rd1 mutant mice accumulate cGMP, causing rapid photoreceptor degeneration. However, much less is known about photoreceptor mutants producing abnormally low levels of cGMP. Here, focusing on Pde6 mutants in zebrafish and mice, we propose a correlation between cGMP levels and speed of photoreceptor degeneration.

Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches.

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Aipl1 is required for cone photoreceptor function and survival through the stability of Pde6c and Gc3 in zebrafish.

Genetic mutations in aryl hydrocarbon receptor interacting protein-like 1 (AIPL1) cause photoreceptor degeneration associated with Leber congenital amaurosis 4 (LCA4) in human patients. Here we report retinal phenotypes of a zebrafish aipl1 mutant, gold rush (gosh). In zebrafish, there are two aipl1 genes, aipl1a and aipl1b, which are expressed mainly in rods and cones, respectively. The gosh mutant gene encodes cone-specific aipl1, aipl1b. Cone photoreceptors undergo progressive degeneration in the gosh mutant, indicating that aipl1b is required for cone survival. Furthermore, the cone-specific subunit of cGMP phosphodiesterase 6 (Pde6c) is markedly decreased in the gosh mutant, and the gosh mutation genetically interacts with zebrafish pde6c mutation eclipse (els). These data suggest that Aipl1 is required for Pde6c stability and function. In addition to Pde6c, we found that zebrafish cone-specific guanylate cyclase, zGc3, is also decreased in the gosh and els mutants. Furthermore, zGc3 knockdown embryos showed a marked reduction in Pde6c. These observations illustrate the interdependence of cGMP metabolism regulators between Aipl1, Pde6c, and Gc3 in photoreceptors.

Valoración del estado nutricional en pacientes pre-quirúrgicos en un hospital de agudos de la provincia de Buenos Aires / Assessment of nutritional status in pre-surgical patients in an acute care hospital in the province of Buenos Aires

Introducción: la desnutrición en pacientes pre-quirúrgicos incrementa el riesgo de padecer complicaciones, aumentando la estancia hospitalaria y los costos para el sistema de salud. Objetivo: Evaluar el estado nutricional de los pacientes admitidos en el servicio de “Clínica Quirúrgica”, antes de someterse a una cirugía programada, durante los meses de marzo a mayo de 2015. Materiales y método: Estudio observacional, descriptivo, de corte transversal. Se evaluaron pacientes ingresados en la institución por el servicio de “Clínica Quirúrgica”, con motivo de cirugía. Las variables estudiadas fueron edad en dos estratos, sexo, grupo de patologías y estado nutricional. Para el diagnóstico de esta última se utilizó la Valoración Global Subjetiva (VGS). El análisis estadístico se realizó a través de la prueba de chi cuadrado (X²) o Fisher. Resultados: Se evaluaron 106 pacientes, de los cuales el 70,7% se categorizaron como bien nutridos, el 25,5% con riesgo de desnutrición o desnutrición moderada y el 3,8% como desnutrición severa. Ser ≥60 años tuvo una diferencia estadísticamente significativa con el riesgo de desnutrición. La prevalencia de desnutrición fue mayor en el grupo de pacientes con patologías de hígado y vía biliar. Dentro de la categoría C de la VGS, el 75% presentaba neoplasias. Conclusión: En la población estudiada se identificó un porcentaje considerable de pacientes con desnutrición, por lo que es necesaria la implementación de una estrategia de tamizaje nutricional al momento de la programación de la cirugía para realizar una intervención oportuna que permita que los pacientes candidatos a cirugía lleguen con el mejor estado nutricional posible.

Localization of Usher 1 proteins to the photoreceptor calyceal processes, which are absent from mice.

The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.

Genetic changes including gene copy number alterations and their relation to prognosis in childhood acute myeloid leukemia.

We studied a series of 68 subjects diagnosed with childhood acute myeloid leukemia (AML) using conventional cytogenetics and fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) to analyze mutations in FLT3 and NPM1 genes, and/or array comparative genomic hybridization (CGH). Cytogenetic/FISH abnormalities were observed in 71% of subjects, FLT3-ITD mutations in 15%, and NPM1 mutations in 13%. The array CGH alterations (average 3.6 per case) were observed in 96% of the tested subjects. The most frequent alterations were gains of 8q24.3 and 11p15.5-p15.4 in 16% of the samples. Six genes (AKT1, RUNX1, LTB, SDC1, RUNX1T1, and JAK2) from the imbalanced regions have been reported to be involved in AML, whereas other 30 cancer genes, not previously reported in an AML context, were identified as imbalanced. They probably correspond to non passenger alterations that cooperate with the recurrent translocations. The clinical data and genetic changes were tested to find out the possible association with prognosis. Genomic instability (four or more genomic imbalances) was correlated with poor patient outcome (p = 0.029).

Cannabinoid receptors in conjunctival epithelium: identification and functional properties.

PURPOSE: Preservation of the ocular surface barrier requires complex control of epithelial cell proliferation and inflammation mechanisms. The endocannabinoid system may be regulating these processes. Therefore, the authors explored the presence and properties of cannabinoid receptors (CB1 and CB2) in conjunctival epithelial cells. METHODS: The authors used immunohistochemistry to detect CB1 and CB2 in normal mouse conjunctiva, human conjunctival cryosections and impression samples, and IOBA-NHC cells, a human conjunctiva-derived cell line. The presence of CB1 and CB2 proteins and transcripts was studied in IOBA-NHC cells by Western blot and RT-PCR, respectively. The authors also used this cell line to assay cannabinoid ligand-induced changes in cAMP levels, cell growth, and tumor necrosis factor-alpha (TNF-alpha)-induced activation of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB (NF-kappaB). RESULTS: Mouse and human conjunctival epithelial cells displayed CB1 and CB2 proteins and transcripts. Cannabinoid receptor activation decreased cAMP levels in IOBA-NHC cells, and specific CB1 and CB2 antagonists canceled this effect. Cannabinoid ligands also increased cell growth and blocked stress pathways activated by TNF-alpha in vitro. CONCLUSIONS: Cannabinoid receptors are present in mouse and human conjunctival cells. Functional responses, such as decreased cAMP levels, proliferation, and modulation of stress signaling pathways, were mediated by CB1 and CB2 stimulation. Thus, these receptors might be involved in the regulation of epithelial renewal and inflammatory processes at the ocular surface.

Blockade of endothelinergic receptors prevents development of proliferative vitreoretinopathy in mice.

Proliferative vitreoretinopathy (PVR) is characterized by severe glial remodeling. Glial activation and proliferation that occur in brain diseases are modulated by endothelin-1 (ET-1) and its receptor B (ETR-B). Because retinal astrocytes contain ET-1 and express ETR-B, we studied the changes of these molecules in an experimental mouse model of PVR and in human PVR. Both ET-1 and ETR-B immunoreactivities increased in mouse retina after induction of PVR with dispase. Epi- and subretinal outgrowths also displayed these immunoreactivities in both human and experimental PVR. Additionally, myofibroblasts and other membranous cell types showed both ET-1 and ETR-B immunoreactivities. In early stages of experimentally induced PVR, prepro-ET-1 and ETR-B mRNA levels increased in the retina. These mRNA levels also increased after retinal detachment (RD) produced by subretinal injection. Treatment of mice with tezosentan, an antagonist of endothelinergic receptors, reduced the histopathological hallmarks of dispase-induced PVR: retinal folding, epiretinal outgrowth, and gliosis. Our findings in human and in dispase-induced PVR support the involvement of endothelinergic pathways in retinal glial activation and the phenotypic transformations that underlie the growth of membranes in this pathology. Elucidating these pathways further will help to develop pharmacological treatments to prevent PVR. In addition, the presence of ET-1 and ETR-B in human fibrous membranes suggests that similar treatments could be helpful after PVR has been established.

Controlling retinal pigment epithelium injury after experimental detachment of the retina.

PURPOSE: Damage induced by detachment of the neural retina and the retinal pigment epithelium (RPE) can be reduced by dark adaptation. The authors evaluated the influence of the duration of dark adaptation, time of day, and modification of the melatonin-dopamine pathway on acute RPE lesions induced by mechanical detachment. METHODS: BALB/c mice were studied at different times of day and different periods of dark adaptation. Some mice were treated with melatonin or sulpiride, a D2 dopamine receptor antagonist. Enucleated eyes and different saline solutions were used in experiments ex vivo. Retinal detachments in vivo were made by subretinal injections of hyaluronic acid. RPE cell damage was quantitatively evaluated with a dye exclusion procedure, and their viability was tested by preservation of tight junctions in culture. Lectin histochemistry was used to examine the interphotoreceptor matrix (IPM). RESULTS: Significant propidium iodide (PI) incorporation in RPE cells was detected after ex vivo separation during daytime, but it was very low when detachment took place at night after 24 to 48 hours of dark adaptation. PI exclusion was achieved during daytime after a single hour of dark adaptation when mice were pretreated with melatonin or sulpiride. Reduction of RPE cell damage was accompanied by decreased lectin binding to cone sheaths. CONCLUSIONS: A combination of time of day and length of dark adaptation decreased damage induced by detachment of the retina ex vivo and in vivo. Melatonin or sulpiride could replace these environmental factors. Therefore, melatonin and dopamine pathways might be involved in the control of IPM properties and retina/RPE interactions.

Inhibition of salivary secretion by activation of cannabinoid receptors.

It is known that marijuana use decreases saliva secretion. Therefore, we hypothesized that cannabinoid receptors (CBs) are located in salivary glands to mediate that effect. In these experiments, we used the submandibular gland (SMG) of male rats, which is one of the major salivary glands. Mammalian tissues contain at least two types of CBs, CB1 and CB2, mainly located in the nervous system and peripheral tissues, respectively. Both receptors are coupled to Gi protein and respond by inhibiting the activity of adenylyl cyclase. We demonstrated that both CB1 and CB2 are present in the SMG, each showing specific localizations. The best-known endocannabinoid is anandamide (AEA), which binds with high affinity to CB1 and CB2. We showed that AEA markedly reduced forskolin-induced increase of cAMP content in vitro. This effect was blocked by AM251 and AM630 (CB1 and CB2 antagonists, respectively), indicating that both receptors are implicated in SMG physiology. In addition, we showed that AEA injected intraglandularly to anesthetized rats inhibited norepinephrine (NE)- and methacholine (MC)-stimulated saliva secretion in vivo and that both AM251 or AM630 prevented the inhibitory action of AEA. Also, the intraglandular injection of AM251 increased saliva secretion induced by lower doses of NE or MC. This increase was synergized after coinjection with AM630. Therefore, we concluded that AEA decreases saliva secretion in the SMG acting through CB1 and CB2 receptors.

Endothelin receptors in light-induced retinal degeneration.

Excessive light exposure leads to retinal degeneration in albino animals and exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In previous studies we have described the presence of endothelin-1 (ET-1) and its receptors (ET-A and ET-B) in different sites of the mouse retina, including the retinal pigment epithelium, the outer plexiform layer (OPL), astrocytes, the ganglion cell layer (GCL), and vascular endothelia. After light-induced degeneration of photoreceptors, endothelinergic structures disappear from the OPL, but ET-1 and ET-B immunoreactivities increase in astrocytes. Here, we present novel observations about the course of light-induced retinal degeneration in BALB-c mice exposed to 1500 lux during 4 days with or without treatment with tezosentan, a mixed endothelinergic antagonist. Retinal whole mounts were immunostained with anticleaved caspase-3 (CC-3) serum to identify apoptotic photoreceptor cells within the outer nuclear layer (ONL). Glial activation was measured as glial fibrillary acidic protein (GFAP) immunoreactivity in retinal whole mounts and in Western blots from retinal extracts. Tezosentan treatment significantly reduced both the number of CC3-immunoreactive cells and GFAP levels, suggesting that inhibition of endothelinergic receptors could play a role in photoreceptor survival. Using confocal double immunofluorescence, we have observed that ET-A seems to be localized in bipolar cell dendrites, whereas ET-B is localized in horizontal cells. Our observations suggest the existence of an endothelinergic mechanism modulating synaptic transmission in the OPL. This mechanism could perhaps explain the effects of tezosentan treatment on photoreceptor survival.

Endothelin-1 and endothelin receptors in light-induced retinal degeneration.

We have studied the distribution of endothelinergic molecules: prepro-endothelin-1 (PPET-1), endothelin-1 (ET-1), and receptors A and B (ET-A) and (ET-B) in the retina of mice. The localization of these molecules in normal mice was compared to their localization in retinas from animals submitted to continuous illumination during 1, 6, 9 or 18 days. We also evaluated the distribution of smooth muscle actin (SMA) and glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthase (GS). PPET-1 immunoreactivity mainly appeared in retinal pigment epithelium (RPE) and cells of the ganglion cell layer (GCL), whereas ET-1 immunoreactivity was present in the RPE, outer plexiform layer (OPL) and astrocytes. Astrocytes exhibited the strongest immunostaining in the retina. ET-A immunoreactivity was observed in endothelium, RPE, OPL and cells of the GCL. By contrast, ET-B immunoreactivity could be detected in endothelial cells, horizontal cells and astrocytes. Astrocytes of the optic nerve also exhibited ET-1, ET-A, and ET-B immunoreactivities. After light-induced degeneration, there was an increase of RPE immunostaining. Degeneration of photoreceptors was accompanied by disappearance of immunoreactivity in the OPL. However, ET-A immunoreactivity appeared in the amacrine sublayer of the INL. There was an enormous increase in astrocytes and its cell processes. The increase of astrocytic immunoreactivities for ET-1 and ET-B was confirmed by quantitative image analysis. Growth of astrocytic cell processes was most marked around retinal blood vessels. Our findings indicate that there are at least three endothelinergic pathways in the normal retina: (1) between the RPE and choriocapillaris, (2) at the OPL, and (3) between blood vessels, astrocytes and cells of the GCL. After light-induced degeneration of photoreceptors, endothelinergic molecules were overexpressed at the RPE and astrocytes, but mostly disappeared from the OPL.