RESUMO
Cell-penetrating peptides (CPPs) hold great promise for intracellular delivery of therapeutic proteins. However, endosomal entrapment of transduced cargo is a major bottleneck hampering their successful application. While developing a transducible zinc finger protein-based artificial transcription factor targeting the expression of endothelin receptor A, we identified interaction between the CPP and the endosomal membrane or endosomal entanglement as a main culprit for endosomal entrapment. To achieve endosomal disentanglement, we utilized endosome-resident proteases to sever the artificial transcription factor from its CPP upon arrival inside the endosome. Using this approach, we greatly enhanced the correct subcellular localization of the disentangled artificial transcription factor, significantly increasing its biological activity and distribution in vivo. With rational engineering of proteolytic sensitivity, we propose a new design principle for transducible therapeutic proteins, helping CPPs attain their full potential as delivery vectors for therapeutic proteins.
Assuntos
Peptídeos Penetradores de Células , Receptores de Endotelina , Peptídeos Penetradores de Células/metabolismo , Endossomos/metabolismo , Receptores de Endotelina/metabolismo , Fatores de Transcrição/metabolismoRESUMO
For the survival and maintenance of retinal ganglion cells (RGCs), axonal transportation is fundamental. Axonal transportation defects can cause severe neuropathies leading to neuronal loss. Axonal transport defects usually precede axonal degeneration and RGC loss in disease models. To date, the main causes of axonal transport defects have not been fully understood. Therefore, elucidation of the mechanisms that lead to transport defects will help us to develop novel therapeutic targets and early diagnostic tools. In this review, we provide an overview of optic neuropathies and axonal degeneration with a focus on axonal transport.
Assuntos
Doenças do Nervo Óptico , Células Ganglionares da Retina , Animais , Humanos , Células Ganglionares da Retina/fisiologia , Transporte Axonal/fisiologia , Modelos Animais de Doenças , Axônios/metabolismoRESUMO
Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Camundongos/genética , Células Ganglionares da Retina/metabolismo , Strongylocentrotus purpuratus/genética , Fator de Transcrição Brn-3B/genética , Animais , Embrião de Mamíferos/embriologia , Embrião não Mamífero/embriologia , Proteínas de Homeodomínio/metabolismo , Camundongos/crescimento & desenvolvimento , Camundongos/metabolismo , Células Ganglionares da Retina/citologia , Strongylocentrotus purpuratus/metabolismo , Fator de Transcrição Brn-3B/metabolismoRESUMO
BACKGROUND: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage. RESULTS: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells. CONCLUSIONS: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.
Assuntos
Células Ependimogliais/metabolismo , Fator Inibidor de Leucemia/metabolismo , Estabilidade de RNA/genética , Retina/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Camundongos , Oxirredução , Ratos , Degeneração Retiniana/metabolismo , Transdução de Sinais/genéticaRESUMO
Degeneration of cells in the retina is a hallmark of various inherited and acquired blinding diseases in humans. One of the most challenging problems to establish successful treatments for these diseases is to understand the molecular mechanisms that result in retinal degeneration and to identify endogenous rescue pathways which support cell survival. In many mouse models for retinal degeneration, expression of LIF in glial cells in response to a disease condition is crucial for the activation of an elaborate protective system. This mini review will summarize the findings that are related to LIF signaling and discuss the neuroprotective effects of LIF in different animal models.
Assuntos
Apoptose/fisiologia , Fator Inibidor de Leucemia/metabolismo , Degeneração Retiniana/metabolismo , Transdução de Sinais/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Degeneração Retiniana/patologiaRESUMO
Gene editing and gene regulatory fields are continuously developing new and safer tools that move beyond the initial CRISPR/Cas9 technology. As more advanced applications are emerging, it becomes crucial to understand and establish more complex gene regulatory and editing tools for efficient gene therapy applications. Ophthalmology is one of the leading fields in gene therapy applications with more than 90 clinical trials and numerous proof-of-concept studies. The majority of clinical trials are gene replacement therapies that are ideal for monogenic diseases. Despite Luxturna's clinical success, there are still several limitations to gene replacement therapies including the size of the target gene, the choice of the promoter as well as the pathogenic alleles. Therefore, further attempts to employ novel gene regulatory and gene editing applications are crucial to targeting retinal diseases that have not been possible with the existing approaches. CRISPR-Cas9 technology opened up the door for corrective gene therapies with its gene editing properties. Advancements in CRISPR-Cas9-associated tools including base modifiers and prime editing already improved the efficiency and safety profile of base editing approaches. While base editing is a highly promising effort, gene regulatory approaches that do not interfere with genomic changes are also becoming available as safer alternatives. Antisense oligonucleotides are one of the most commonly used approaches for correcting splicing defects or eliminating mutant mRNA. More complex gene regulatory methodologies like artificial transcription factors are also another developing field that allows targeting haploinsufficiency conditions, functionally equivalent genes, and multiplex gene regulation. In this review, we summarized the novel gene editing and gene regulatory technologies and highlighted recent translational progress, potential applications, and limitations with a focus on retinal diseases.
RESUMO
Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.
Assuntos
Dependovirus , Vetores Genéticos , Animais , Dependovirus/metabolismo , Vetores Genéticos/genética , Camundongos , Reação em Cadeia da Polimerase , Retina/metabolismo , Transdução GenéticaRESUMO
Dominant optic atrophy (DOA) is an inherited mitochondrial disease leading to specific degeneration of retinal ganglion cells (RGCs), thus compromising transmission of visual information from the retina to the brain. Usually, DOA starts during childhood and evolves to poor vision or legal blindness, affecting the central vision, whilst sparing the peripheral visual field. In 20% of cases, DOA presents as syndromic disorder, with secondary symptoms affecting neuronal and muscular functions. Twenty years ago, we demonstrated that heterozygous mutations in OPA1 are the most frequent molecular cause of DOA. Since then, variants in additional genes, whose functions in many instances converge with those of OPA1, have been identified by next generation sequencing. OPA1 encodes a dynamin-related GTPase imported into mitochondria and located to the inner membrane and intermembrane space. The many OPA1 isoforms, resulting from alternative splicing of three exons, form complex homopolymers that structure mitochondrial cristae, and contribute to fusion of the outer membrane, thus shaping the whole mitochondrial network. Moreover, OPA1 is required for oxidative phosphorylation, maintenance of mitochondrial genome, calcium homeostasis and regulation of apoptosis, thus making OPA1 the Swiss army-knife of mitochondria. Understanding DOA pathophysiology requires the understanding of RGC peculiarities with respect to OPA1 functions. Besides the tremendous energy requirements of RGCs to relay visual information from the eye to the brain, these neurons present unique features related to their differential environments in the retina, and to the anatomical transition occurring at the lamina cribrosa, which parallel major adaptations of mitochondrial physiology and shape, in the pre- and post-laminar segments of the optic nerve. Three DOA mouse models, with different Opa1 mutations, have been generated to study intrinsic mechanisms responsible for RGC degeneration, and these have further revealed secondary symptoms related to mitochondrial dysfunctions, mirroring the more severe syndromic phenotypes seen in a subgroup of patients. Metabolomics analyses of cells, mouse organs and patient plasma mutated for OPA1 revealed new unexpected pathophysiological mechanisms related to mitochondrial dysfunction, and biomarkers correlated quantitatively to the severity of the disease. Here, we review and synthesize these data, and propose different approaches for embracing possible therapies to fulfil the unmet clinical needs of this disease, and provide hope to affected DOA patients.
Assuntos
Atrofia Óptica Autossômica Dominante , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Camundongos , Mitocôndrias , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Bacterial artificial chromosomes (BACs) are genomic tools that can carry several hundred kilobases of exogenous genomic material. This allows to incorporate sufficiently large DNA stretches to include most if not all upstream and downstream cis-regulatory elements of a gene in order to mimic and analyze its endogenous regulation of expression using a reporter protein in vivo. Here, we illustrate the generation of a BAC:LIF-EGFP transgenic mouse line to describe a simplified version of BAC transgenesis using galK-based recombineering.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Camundongos Transgênicos/genética , Retina/fisiologia , Transgenes/genética , Animais , Técnicas de Transferência de Genes , Genes Reporter/genética , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética/genética , Elementos Reguladores de Transcrição/genéticaRESUMO
Stress adaptation is essential for neuronal health. While the fundamental role of mitochondria in neuronal development has been demonstrated, it is still not clear how adult neurons respond to alterations in mitochondrial function and how neurons sense, signal, and respond to dysfunction of mitochondria and their interacting organelles. Here, we show that neuron-specific, inducible in vivo ablation of the mitochondrial fission protein Drp1 causes ER stress, resulting in activation of the integrated stress response to culminate in neuronal expression of the cytokine Fgf21. Neuron-derived Fgf21 induction occurs also in murine models of tauopathy and prion disease, highlighting the potential of this cytokine as an early biomarker for latent neurodegenerative conditions.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Animais , CamundongosRESUMO
Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.
Assuntos
Retina/patologia , Oclusão da Artéria Retiniana/patologia , Oclusão da Veia Retiniana/patologia , Tomografia de Coerência Óptica/métodos , Animais , Modelos Animais de Doenças , Angiofluoresceinografia , Fotocoagulação a Laser , Masculino , CamundongosRESUMO
PURPOSE: Due to the low abundance of cone photoreceptors in the mouse retina and the scarcity of alternative animal models, little is known about mechanisms of cone degeneration. Nrl knockout mice develop exclusively the cone-type of photoreceptors. However, the cone photoreceptor layer in Nrl(-/-) mice displays an irregular morphology with severe rosette formation. Retinas of Rpe65(-/-);Nrl(-/-) mice have no rosettes due to the lack of 11-cis-retinal, but also are not functional. To develop a model with a functional all-cone retina that is morphologically well structured, we generated R91W;Nrl(-/-) double-mutant mice, which express a hypomorphic Rpe65 allele (R91W). METHODS: The following analyses were used to characterize the R91W;Nrl(-/-)mice: morphology by light and electron microscopy, protein distribution by immunofluorescence, cone function by electroretinography and optomotor response, RNA levels by RT-PCR, and chromophore levels by HPLC. Cone degeneration was assessed in R91W;Nrl(-/-) mice treated with MNU, and in triple R91W;Nrl(-/-);Cpfl1 and quadruple R91W;Nrl(-/-);Cpfl1;rd10 mutant mice. RESULTS: The all-cone retina of R91W;Nrl(-/-) mice is functional and relatively stable with only very slow age-related degeneration. Using triple and quadruple mutant mice, or a chemical treatment, we demonstrated that cone degeneration could be induced and analyzed in these mice. CONCLUSIONS: The reduced levels of visual chromophore prevented rosette formation and sustained function in the R91W;Nrl(-/-) retina. Thus, the R91W;Nrl(-/-) mouse allows study of the etiology of diseases related to cone degeneration in a "morphologically intact" and functional all-cone photoreceptor retina.
Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Retiniana/patologia , Animais , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Eletrorretinografia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Retina/metabolismo , Degeneração Retiniana/fisiopatologia , Retinaldeído/deficiência , cis-trans-Isomerases/metabolismoRESUMO
Several behavioral and electrophysiological studies indicate that all classes of echinoderms, including Echinoidia, the class to which sea urchins belong, are photosensitive and exhibit complex behavioral responses to light or changes in light intensity. However, no discrete photosensitive structure has been identified in sea urchins. The purpose of this study was to provide new insights into eye evolution by determining whether distinct photosensory structures are present in adult sea urchins. Recently, we showed that the Strongylocentrotus purpuratus genome contains orthologs of many mammalian retinal genes and that these genes are expressed in tube feet, suggesting the presence of photoreceptor neurons. To determine whether this is so, we identified several features of tube feet that relate to a possible invertebrate phototransduction system. We show that rhabdomeric opsin is expressed severalfold higher within the disk region of the tube feet and is the most abundant opsin. Immunostaining identified ßIII-tubulin-expressing cells at the periphery of disk in the vicinity of the synaptotagmin-expressing nerve fibers. We also showed that Pax6 expression in the disk was restricted to the periphery, where small clusters of putative sensory neurons reside. Our results reveal neuromuscular organization of the tube foot neuromuscular system. They further support earlier studies suggesting the presence of a photosensory system in tube feet.
Assuntos
Junção Neuromuscular/crescimento & desenvolvimento , Células Fotorreceptoras de Invertebrados/fisiologia , Células Receptoras Sensoriais/fisiologia , Strongylocentrotus purpuratus/crescimento & desenvolvimento , Animais , Junção Neuromuscular/citologia , Ouriços-do-Mar , Strongylocentrotus purpuratus/citologiaRESUMO
The sea urchin oral-aboral (OA) axis is established in part by Nodal signaling. The OA axis is also influenced by treatments affecting respiration and Nodal transcription is influenced by redox-dependent transcription factors. This suggests that intracellular redox state plays a role in OA axis specification. Since cellular redox state can be altered by the formation of excess reactive oxygen species (ROS), and hypoxia and paraquat generate ROS in cells, we asked whether these treatments affected specification of the OA axis and Nodal expression. Embryos cultured under conditions that elevate ROS, demonstrate perturbed ectoderm specification, but other territories are not affected. Immunohistochemical and Q-RT-PCR analyses revealed that both oral and aboral ectoderm genes are downregulated. Our results argue that elevating ROS in sea urchin embryos by these treatments blocks early steps in ectoderm differentiation preceding the polarization of the ectoderm into oral and aboral territories.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Ectoderma/efeitos dos fármacos , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Ouriços-do-Mar/embriologia , Animais , Biomarcadores/análise , Células Cultivadas , Ectoderma/embriologia , Técnicas de Cultura Embrionária , Embrião não Mamífero , Desenvolvimento Embrionário/efeitos dos fármacos , Herbicidas/farmacologia , Níquel/farmacologia , Paraquat/farmacologia , Ouriços-do-Mar/efeitos dos fármacosRESUMO
In the sea urchin embryo, Nodal is the earliest known signal to play a role in the specification of the oral ectodermal territory. Nodal, a TGF-beta ligand, is first expressed in the presumptive oral ectoderm at approximately 7 H of development. Nodal overexpression produces a distinctive bell-shaped phenotype with expanded oral ectoderm, which resembles the oralized phenotype obtained as a result of nickel (Ni) treatment. To date, a detailed analysis of gene expression in Ni-treated embryos has not been undertaken. Because treatment with cobalt (Co) produces similar results to those seen with Ni treatment in other systems, we were interested in determining how Co influences sea urchin embryonic development. Here we report that Co also induces oralization of the ectoderm, and the effects of Ni and Co depend on functional Nodal signaling. Although both metals upregulate nodal gene expression, they do not initiate nodal transcription precociously. Analysis of the perturbation of Nodal receptor function suggests that Ni and Co contribute to nodal upregulation in the absence of nodal autoregulation, but cannot fully oralize the ectoderm in the absence of Nodal signaling.