RESUMEN
Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.
Asunto(s)
Dimetilsulfóxido , Ratones Endogámicos C57BL , Animales , Ratones , Dimetilsulfóxido/farmacología , Biomarcadores/metabolismo , Ratones Transgénicos , Tomografía de Coherencia Óptica , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Sensibilidad de Contraste/efectos de los fármacos , Sensibilidad de Contraste/fisiología , Modelos Animales de Enfermedad , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Visión Ocular/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
BACKGROUND: Iron-induced oxidative stress was thought to be the reason why the a-wave amplitude of the electroretinogram (ERG) dropped when iron ions were present. It is assumed that reactive oxygen species (ROS) are generated in the presence of iron ions, and this leads to a decrease in hyperpolarization of the photoreceptor. It is known that in age-related macular degeneration (AMD), sodium iodate can induce oxidative stress, apoptosis, and retinal damage, which mimic the effects of clinical AMD. Here, the reduction of the a-wave amplitude in mice with sodium iodate-induced age-related macular degeneration is explained. METHODS: The leading edge of the a-wave is divided into voltages developed by cones and rods. The same oxidative stress model is applied here since sodium iodate causes the creation of ROS in a manner similar to that caused by iron ions, with the exception that the retina is treated as a circuit of various resistances when computing the photoresponse. Moreover, sodium iodate also leads to apoptosis and, hence, may cause misalignment in cones (not in rods) during the initial stage of apoptosis in AMD. To include the effects of apoptosis and shortening in cones and rods, we have used a factor representing the fraction of total cones and rods that are alive. To include the effect of misalignment of cones on the reduction of the a-wave amplitude, we have used the Stiles-Crawford function to calculate the number of photoisomerizations occurring in a photoreceptor misaligned at an angle θ. The results are compared with experimental data. RESULTS: In sodium iodate-treated eyes, the ROS produced can attract calcium ions in the photoreceptor, which increases the calcium influx. In the case of the cones, the inclusion of the misalignment angle in the phototransduction process helps in determining the voltage and slope of the voltage vs. time graph.The smaller the fraction of active photoreceptors, the smaller the amplitude of the a-wave. The calcium influx, misaligned photoreceptors, and total photoreceptor loss all cause the amplitude of the a-wave to decrease, and at any time from the beginning of phototransduction cascade, the calcium influx causes the slope of the a-wave to increase. CONCLUSION: The reduction in the a-wave amplitude in the eyes of sodium iodate-treated mice is attributed to oxidative stress in both cones and rods and cone misalignment, which ultimately lead to apoptosis and vision loss in AMD.
Asunto(s)
Electrorretinografía , Yodatos , Degeneración Macular , Estrés Oxidativo , Especies Reactivas de Oxígeno , Células Fotorreceptoras Retinianas Conos , Animales , Degeneración Macular/patología , Degeneración Macular/fisiopatología , Degeneración Macular/inducido químicamente , Ratones , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Apoptosis/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Modelos Animales de Enfermedad , Modelos BiológicosRESUMEN
Retinitis pigmentosa (RP) is a form of retinal degeneration affecting a young population with an unmet medical need. Photoreceptor degeneration has been associated with increased guanosine 3',5'-cyclic monophosphate (cGMP), which reaches toxic levels for photoreceptors. Therefore, inhibitory cGMP analogues attract interest for RP treatments. Here we present the synthesis of dithio-CN03, a phosphorodithioate analogue of cGMP, prepared using the H-phosphonothioate route. Two crystal modifications were identified as a trihydrate and a tetrahydrofuran monosolvates. Dithio-CN03 featured a lower aqueous solubility than its RP-phosphorothioate counterpart CN03, a drug candidate, and this characteristic might be favorable for sustained-release formulations aimed at retinal delivery. Dithio-CN03 was tested in vitro for its neuroprotective effects in photoreceptor models of RP. The comparison of dithio-CN03 to CN03 and its diastereomer SP-CN03, and to their phosphate derivative oxo-CN03 identifies dithio-CN03 as the compound with the highest efficacy in neuroprotection and thus as a promising new candidate for the treatment of RP.
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GMP Cíclico , Fármacos Neuroprotectores , Células Fotorreceptoras Retinianas Bastones , GMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/uso terapéutico , Degeneración Retiniana/tratamiento farmacológico , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/metabolismo , Relación Estructura-ActividadRESUMEN
To increase computational flexibility, the processing of sensory inputs changes with behavioural context. In the visual system, active behavioural states characterized by motor activity and pupil dilation1,2 enhance sensory responses, but typically leave the preferred stimuli of neurons unchanged2-9. Here we find that behavioural state also modulates stimulus selectivity in the mouse visual cortex in the context of coloured natural scenes. Using population imaging in behaving mice, pharmacology and deep neural network modelling, we identified a rapid shift in colour selectivity towards ultraviolet stimuli during an active behavioural state. This was exclusively caused by state-dependent pupil dilation, which resulted in a dynamic switch from rod to cone photoreceptors, thereby extending their role beyond night and day vision. The change in tuning facilitated the decoding of ethological stimuli, such as aerial predators against the twilight sky10. For decades, studies in neuroscience and cognitive science have used pupil dilation as an indirect measure of brain state. Our data suggest that, in addition, state-dependent pupil dilation itself tunes visual representations to behavioural demands by differentially recruiting rods and cones on fast timescales.
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Color , Pupila , Reflejo Pupilar , Visión Ocular , Corteza Visual , Animales , Oscuridad , Aprendizaje Profundo , Ratones , Estimulación Luminosa , Pupila/fisiología , Pupila/efectos de la radiación , Reflejo Pupilar/fisiología , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/fisiología , Factores de Tiempo , Rayos Ultravioleta , Visión Ocular/fisiología , Corteza Visual/fisiologíaRESUMEN
Hereditary degeneration of photoreceptors has been linked to over-activation of Ca2+-permeable channels, excessive Ca2+-influx, and downstream activation of Ca2+-dependent calpain-type proteases. Unfortunately, after more than 20 years of pertinent research, unequivocal evidence proving significant and reproducible photoreceptor protection with Ca2+-channel blockers is still lacking. Here, we show that both D- and L-cis enantiomers of the anti-hypertensive drug diltiazem were very effective at blocking photoreceptor Ca2+-influx, most probably by blocking the pore of Ca2+-permeable channels. Yet, unexpectedly, this block neither reduced the activity of calpain-type proteases, nor did it result in photoreceptor protection. Remarkably, application of the L-cis enantiomer of diltiazem even led to a strong increase in photoreceptor cell death. These findings shed doubt on the previously proposed links between Ca2+ and retinal degeneration and are highly relevant for future therapy development as they may serve to refocus research efforts towards alternative, Ca2+-independent degenerative mechanisms.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/metabolismo , Diltiazem/farmacología , Degeneración Retiniana/metabolismo , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , GMP Cíclico/metabolismo , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Diltiazem/química , Activación del Canal Iónico/efectos de los fármacos , Cinética , Ratones , Proteolisis , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patologíaRESUMEN
Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.
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Retinitis Pigmentosa/etiología , Retinitis Pigmentosa/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Biomarcadores , Línea Celular , Células Cultivadas , Clonación Molecular , Susceptibilidad a Enfermedades , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Ratones , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/etiología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/patologíaRESUMEN
Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the Pde6b gene, rd1 mice display elevated cGMP in conjunction with higher intracellular Ca 2+ in their rod photoreceptors, resulting in fast retinal degeneration. Ca 2+ has been linked to activation of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway integrates extracellular and intracellular signals to sense the supply of nutrients and plays a central role in regulating protein and lipid synthesis as well as apoptosis and autophagy. In the present study, we showed that mTOR and phosphorylated mTOR (p-mTOR, activated form of mTOR) are up-regulated in rd1 photoreceptors at postnatal day 10 (P10), a pre-degenerative stage. Moreover, the downstream effectors of mTOR, such as pS6K and S6K, are also increased, suggesting activation of the mTOR signaling pathway. Intravitreal administration of rapamycin, a negative regulator of mTOR, inhibits the mTOR pathway in rd1 photoreceptors. Consequently, the progression of retinal degeneration is slower and retinal function is enhanced, possibly mediated by activation of autophagy in the photoreceptors. Taken together, these results highlight rapamycin as a potential therapeutic avenue for retinal degeneration.
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Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/prevención & control , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/patología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Ratones , Degeneración Retiniana/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Sirolimus/uso terapéuticoRESUMEN
Retinitis pigmentosa (RP) is a major cause of inherited blindness, and there is presently no cure for RP. Rd1 mouse is the most commonly used RP animal model. Re-expression of cell cycle proteins in post-mitotic neurons is considered an important mechanism of neurodegenerative diseases, including RP. The retinoblastoma tumor suppressor (Rb) is a major regulator of cell cycle progression, yet its role in rd1 mouse retina and related signaling pathways have never been analyzed. By crossing α-Cre, Rbf/f mice with rd1 mice, p21cip1-/- mice, Cdk1f/f mice and Cdk2f/f mice, we established multiple rd1 mouse models with deletions of Rb gene, Cdkn1a (p21cip1) gene, Cdk1 and Cdk2 gene in the retina. Cdk inhibitor CR8 was injected into the vitreous of rd1 mouse to investigate its effects on photoreceptor survival. Rb gene knockout (KO) induces cell death in excitatory retinal neurons (rods, rod bipolar and ganglions) and ectopic proliferation of retinal cells; but it paradoxically delays the rod death of rd1 mice, which is primarily mediated by the Cdk inhibitor Cdkn1a (p21cip1). Interestingly, p21cip1 protects the ectopic dividing rd1 rod cells by inhibiting Cdk1 and Cdk2. However, inhibiting Cdk1 and Cdk2 in rd1 mice with non-dividing rods only has limited and transient protective effects. Our data suggest that there is no ectopic division of rd1 rod cells, and RbKO induces ectopic division but delays the death of rd1 rod cells. This reveals the important protective role of Rb-p21cip1-Cdk axis in rd1 rod cells. P21cip1 is a potential target for future therapy of RP.
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Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Degeneración Retiniana/prevención & control , Proteína de Retinoblastoma/deficiencia , Animales , Apoptosis , Proteína Quinasa CDC2/metabolismo , Supervivencia Celular/fisiología , Quinasa 2 Dependiente de la Ciclina/metabolismo , Electrorretinografía , Inhibidores Enzimáticos/farmacología , Etiquetado Corte-Fin in Situ , Inyecciones Intravítreas , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Microscopía Fluorescente , Purinas/farmacología , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patologíaRESUMEN
Epigenetic modifiers are increasingly being investigated as potential therapeutics to modify and overcome disease phenotypes. Diseases of the nervous system present a particular problem as neurons are postmitotic and demonstrate relatively stable gene expression patterns and chromatin organization. We have explored the ability of epigenetic modifiers to prevent degeneration of rod photoreceptors in a mouse model of retinitis pigmentosa (RP), using rd10 mice of both sexes. The histone modification eraser enzymes lysine demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) are known to have dramatic effects on the development of rod photoreceptors. In the RP mouse model, inhibitors of these enzymes blocked rod degeneration, preserved vision, and affected the expression of multiple genes including maintenance of rod-specific transcripts and downregulation of those involved in inflammation, gliosis, and cell death. The neuroprotective activity of LSD1 inhibitors includes two pathways. First, through targeting histone modifications, they increase accessibility of chromatin and upregulate neuroprotective genes, such as from the Wnt pathway. We propose that this process is going in rod photoreceptors. Second, through nonhistone targets, they inhibit transcription of inflammatory genes and inflammation. This process is going in microglia, and lack of inflammation keeps rod photoreceptors alive.SIGNIFICANCE STATEMENT Retinal degenerations are a leading cause of vision loss. RP is genetically very heterogeneous, and the multiple pathways leading to cell death are one reason for the slow progress in identifying suitable treatments for patients. Here we demonstrate that inhibition of LSD1and HDAC1 in a mouse model of RP leads to preservation of rod photoreceptors and visual function, retaining of expression of rod-specific genes, and with decreased inflammation, cell death, and Müller cell gliosis. We propose that these epigenetic inhibitors cause more open and accessible chromatin, allowing expression of neuroprotective genes. A second mechanism that allows rod photoreceptor survival is suppression of inflammation by epigenetic inhibitors in microglia. Manipulation of epigenetic modifiers is a new strategy to fight neurodegeneration in RP.
Asunto(s)
Histona Desacetilasa 1/antagonistas & inhibidores , Histona Demetilasas/antagonistas & inhibidores , Degeneración Nerviosa/patología , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinitis Pigmentosa/patología , Tranilcipromina/farmacologíaRESUMEN
In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.
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Retina/fisiopatología , Epitelio Pigmentado de la Retina/fisiopatología , Retinitis Pigmentosa/fisiopatología , Animales , Dexametasona/farmacología , Modelos Animales de Enfermedad , Estudios de Evaluación como Asunto , Inflamación/metabolismo , Inflamación/fisiopatología , Ratones , Ratones Endogámicos C57BL , Retina/efectos de los fármacos , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismo , Vasos Retinianos/efectos de los fármacos , Vasos Retinianos/metabolismo , Retinitis Pigmentosa/metabolismo , Rodopsina/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.
Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.
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Fármacos Neuroprotectores/farmacología , Retinitis Pigmentosa/tratamiento farmacológico , Animales , Animales Modificados Genéticamente , Células Cultivadas/efectos de los fármacos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Mutación , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Pez CebraRESUMEN
Fatty acids, and especially docosahexaenoic acid (DHA), are essential for photoreceptor cell integrity and are involved in the phototransduction cascade. In this study, we analyzed the changes in the fatty acid profile in the retina of the rd10 mouse, model of retinitis pigmentosa, in order to identify potential risk factors for retinal degeneration and possible therapeutic approaches. Fatty acids from C57BL/6J and rd10 mouse retinas were extracted with Folch's method and analyzed by gas chromatography/mass spectrometry. Changes in retinal morphology were evaluated by immunohistochemistry. The rd10 mouse retina showed a decreased number of photoreceptor rows and alterations in photoreceptor morphology compared to C57BL/6J mice. The total amount of fatty acids dropped by 29.4% in the dystrophic retinas compared to C57BL/6J retinas. A positive correlation was found between the retinal content of specific fatty acids and the number of photoreceptor rows. We found that the amount of several short-chain and long-chain saturated fatty acids, as well as monounsaturated fatty acids, decreased in the retina of rd10 mice. Moreover, the content of the n-6 polyunsaturated fatty acid arachidonic acid and the n-3 polyunsaturated DHA decreased markedly in the dystrophic retina. The fall of DHA was more pronounced, hence the n-6/n-3 ratio was significantly increased in the diseased retina. The content of specific fatty acids in the retina decreased with photoreceptor degeneration in retinitis pigmentosa mice, with a remarkable reduction in DHA and other saturated and unsaturated fatty acids. These fatty acids could be essential for photoreceptor cell viability, and they should be evaluated for the design of therapeutical strategies and nutritional supplements.
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Ácidos Docosahexaenoicos/farmacología , Ácidos Grasos/farmacología , Lipidómica/métodos , Células Fotorreceptoras Retinianas Bastones/patología , Retinitis Pigmentosa/tratamiento farmacológico , Animales , Muerte Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Retinitis Pigmentosa/diagnósticoRESUMEN
Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.
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Adenosina Trifosfato/toxicidad , Células Ependimogliales/fisiología , Regeneración Nerviosa/fisiología , Neuroglía/fisiología , Degeneración Retiniana/inducido químicamente , Células Fotorreceptoras Retinianas Bastones/fisiología , Pez Cebra/fisiología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Etiquetado Corte-Fin in Situ , Inyecciones Intravítreas , Masculino , Degeneración Retiniana/fisiopatología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/fisiología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/patologíaRESUMEN
Retinal detachment (RD) causes damage, including disjunction, of the rod photoreceptor-bipolar synapse, which disrupts vision and may contribute to the poor visual recovery observed after retinal reattachment surgery. We created a model of iatrogenic RD in adult female pigs to study damage to the rod-bipolar synapse after injury and the ability of a highly specific Rho-kinase (ROCK) inhibitor to preserve synaptic structure and function. This model mimics procedures used in humans when viral vectors or cells are injected subretinally for treatment of retinal disease. Synaptic disjunction by retraction of rod spherules, quantified by image analysis of confocal sections, was present 2 h after detachment and remained 2 days later even though the retina had spontaneously reattached by then. Moreover, spherule retraction occurred in attached retina 1-2 cms from detached retina. Synaptic damage was significantly reduced by ROCK inhibition in detached retina whether injected subretinally or intravitreally. Dark-adapted full-field electroretinograms were recorded in reattached retinas to assess rod-specific function. Reduction in synaptic injury correlated with increases in rod-driven responses in drug-treated eyes. Thus, ROCK inhibition helps prevent synaptic damage and improves functional outcomes after retinal injury and may be a useful adjunctive treatment in iatrogenic RD and other retinal degenerative diseases.
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Modelos Animales de Enfermedad , Lesiones Oculares/complicaciones , Inhibidores de Proteínas Quinasas/farmacología , Desprendimiento de Retina/prevención & control , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Sinapsis/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidores , Animales , Femenino , Desprendimiento de Retina/etiología , Desprendimiento de Retina/patología , PorcinosRESUMEN
Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH+) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH+ ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH+ ACs. Optogenetic stimulation of local CRH+ ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH+ ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.
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Inhibición Neural/fisiología , Células Fotorreceptoras de Vertebrados/fisiología , Retina/fisiología , Células Ganglionares de la Retina/fisiología , Células Amacrinas/fisiología , Animales , Hormona Liberadora de Corticotropina/fisiología , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Uniones Comunicantes/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Optogenética , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Opsinas de Bastones/metabolismo , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (RN) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, RN of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, RN did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.
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Membrana Celular/metabolismo , Uniones Comunicantes/metabolismo , Células Bipolares de la Retina/metabolismo , Células Amacrinas/efectos de los fármacos , Células Amacrinas/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Fenómenos Electrofisiológicos/efectos de los fármacos , Femenino , Uniones Comunicantes/efectos de los fármacos , Masculino , Ácido Meclofenámico/farmacología , Ratas , Células Bipolares de la Retina/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/metabolismoRESUMEN
All neurodegenerative diseases involve a relatively long period of timeframe from the onset of the disease to complete loss of functions. Extending this timeframe, even at a reduced level of function, would improve the quality of life of patients with these devastating diseases. The retina, as the part of the central nervous system and a frequent site of many distressing neurodegenerative disease, provides an ideal model to investigate the feasibility of extending the functional timeframe through pharmacologic intervention. Retinitis Pigmentosa (RP) is a group of blinding diseases. Although the rate of progression and degree of visual loss varies, there is usually a prolonged time before patients totally lose their photoreceptors and vision. It is believed that inhibitory mechanisms are still intact and may become relatively strong after the gradual loss of photoreceptors in RP patients. Therefore, it is possible that light-evoked responses of retinal ganglion cells and visual information processes in retinal circuits could be "unmasked" by blocking these inhibitory mechanisms restoring some level of visual function. Our results indicate that if the inhibition in the inner retina was unmasked in the retina of the rd10 mouse (the well-characterized RP mimicking, clinically relevant mouse model), the light-evoked responses of many retinal ganglion cells can be induced and restore their normal light sensitivity. GABA A receptor plays a major role in this masking inhibition. ERG b-wave and behavioral tests of spatial vision partly recovered after the application of PTX. Hence, removing retinal inhibition unmasks signalling mediated by surviving cones, thereby restoring some degree of visual function. These results may offer a novel strategy to restore the visual function with the surviving cones in RP patients and other gradual and progressive neurodegenerative diseases.
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Neuronas/fisiología , Picrotoxina/farmacología , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Células Fotorreceptoras Retinianas Bastones/fisiología , Visión Ocular/efectos de los fármacos , Animales , Conducta Animal , Modelos Animales de Enfermedad , Luz , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacosRESUMEN
Photoreceptor death is the ultimate cause of vision loss in many retinal degenerative conditions. Identifying novel therapeutic avenues for prolonging photoreceptor health and function has the potential to improve vision and quality of life for patients suffering from degenerative retinal disorders. Photoreceptors are metabolically unique among other neurons in that they process the majority of their glucose via aerobic glycolysis. One of the main regulators of aerobic glycolysis is hexokinase 2 (HK2). Beyond its enzymatic function of phosphorylating glucose to glucose-6-phosphate, HK2 has additional non-enzymatic roles, including the regulation of apoptotic signaling via AKT signaling. Determining the role of HK2 in photoreceptor homeostasis may identify novel signaling pathways that can be targeted with neuroprotective agents to boost photoreceptor survival during metabolic stress. Here we show that following experimental retinal detachment, p-AKT is upregulated and HK2 translocates to mitochondria. Inhibition of AKT phosphorylation in 661W photoreceptor-like cells results in translocation of mitochondrial HK2 to the cytoplasm, increased caspase activity, and decreased cell viability. Rod-photoreceptors lacking HK2 upregulate HK1 and appear to develop normally. Interestingly, we found that HK2-deficient photoreceptors are more susceptible to acute nutrient deprivation in the experimental retinal detachment model. Additionally, HK2 appears to be important for preserving photoreceptors during aging. We show that retinal glucose metabolism is largely unchanged after HK2 deletion, suggesting that the non-enzymatic role of HK2 is important for maintaining photoreceptor health. These results suggest that HK2 expression is critical for preserving photoreceptors during acute nutrient stress and aging. More specifically, p-AKT mediated translocation of HK2 to the mitochondrial surface may be critical for protecting photoreceptors from acute and chronic stress.
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Envejecimiento/patología , Hexoquinasa/metabolismo , Células Fotorreceptoras Retinianas Bastones/enzimología , Estrés Fisiológico , Animales , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Modelos Biológicos , Morfolinas/farmacología , Transporte de Proteínas/efectos de los fármacos , Desprendimiento de Retina/enzimología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Photoreceptor loss is the final common endpoint in most retinopathies that lead to irreversible blindness, and there are no effective treatments to restore vision1,2. Chemical reprogramming of fibroblasts offers an opportunity to reverse vision loss; however, the generation of sensory neuronal subtypes such as photoreceptors remains a challenge. Here we report that the administration of a set of five small molecules can chemically induce the transformation of fibroblasts into rod photoreceptor-like cells. The transplantation of these chemically induced photoreceptor-like cells (CiPCs) into the subretinal space of rod degeneration mice (homozygous for rd1, also known as Pde6b) leads to partial restoration of the pupil reflex and visual function. We show that mitonuclear communication is a key determining factor for the reprogramming of fibroblasts into CiPCs. Specifically, treatment with these five compounds leads to the translocation of AXIN2 to the mitochondria, which results in the production of reactive oxygen species, the activation of NF-κB and the upregulation of Ascl1. We anticipate that CiPCs could have therapeutic potential for restoring vision.
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Reprogramación Celular/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Degeneración Retiniana/terapia , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/trasplante , Visión Ocular/efectos de los fármacos , Animales , Proteína Axina/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Visión Ocular/fisiologíaRESUMEN
Most vertebrates express four arrestin subtypes: two visual ones in photoreceptor cells and two non-visuals expressed ubiquitously. The latter two interact with hundreds of G protein-coupled receptors, certain receptors of other types, and numerous non-receptor partners. Arrestins have no enzymatic activity and work by interacting with other proteins, often assembling multi-protein signaling complexes. Arrestin binding to every partner affects cell signaling, including pathways regulating cell survival, proliferation, and death. Thus, targeting individual arrestin interactions has therapeutic potential. This requires precise identification of protein-protein interaction sites of both participants and the choice of the side of each interaction which would be most advantageous to target. The interfaces involved in each interaction can be disrupted by small molecule therapeutics, as well as by carefully selected peptides of the other partner that do not participate in the interactions that should not be targeted.