RESUMO
Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson's disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.
Assuntos
Degeneração Retiniana , Proteínas de Transporte Vesicular , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Camundongos , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Endossomos/metabolismo , Microglia/metabolismo , Microglia/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Retina/metabolismo , Retina/patologia , Camundongos Knockout , Modelos Animais de Doenças , Humanos , Sinapses/metabolismo , Sinapses/patologia , MasculinoRESUMO
Recessive Stargardt disease (STGD1) is an inherited juvenile maculopathy caused by mutations in the ABCA4 gene, for which there is no suitable treatment. Loss of functional ABCA4 in the retinal pigment epithelium (RPE) alone, without contribution from photoreceptor cells, was shown to induce STGD1 pathology. Here, we identified cathepsin D (CatD), the primary RPE lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 using a newly developed "disease-in-a-dish" RPE model from confirmed STGD1 patients. Induced pluripotent stem cell (iPSC)-derived RPE originating from three STGD1 patients exhibited elevated lysosomal pH, as previously reported in Abca4-/- mice. CatD protein maturation and activity were impaired in RPE from STGD1 patients and Abca4-/- mice. Consequently, STGD1 RPE cells have reduced photoreceptor outer segment degradation and abnormal accumulation of α-synuclein, the natural substrate of CatD. Furthermore, dysfunctional ABCA4 in STGD1 RPE cells results in intracellular accumulation of autofluorescent material and phosphatidylethanolamine (PE). The altered distribution of PE associated with the internal membranes of STGD1 RPE cells presumably compromises LC3-associated phagocytosis, contributing to delayed endo-lysosomal degradation activity. Drug-mediated re-acidification of lysosomes in the RPE of STGD1 restores CatD functional activity and reduces the accumulation of immature CatD protein loads. This preclinical study validates the contribution of CatD deficiencies to STGD1 pathology and provides evidence for an efficacious therapeutic approach targeting RPE cells. Our findings support a cell-autonomous RPE-driven pathology, informing future research aimed at targeting RPE cells to treat ABCA4-mediated retinopathies.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Catepsina D , Lisossomos , Epitélio Pigmentado da Retina , Doença de Stargardt , Catepsina D/metabolismo , Catepsina D/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Doença de Stargardt/metabolismo , Doença de Stargardt/patologia , Doença de Stargardt/genética , Animais , Humanos , Camundongos , Lisossomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genéticaRESUMO
MicroRNAs (miRs) are short, evolutionarily conserved noncoding RNAs that canonically downregulate expression of target genes. The miR family composed of miR-204 and miR-211 is among the most highly expressed miRs in the retinal pigment epithelium (RPE) in both mouse and human and also retains high sequence identity. To assess the role of this miR family in the developed mouse eye, we generated two floxed conditional KO mouse lines crossed to the RPE65-ERT2-Cre driver mouse line to perform an RPE-specific conditional KO of this miR family in adult mice. After Cre-mediated deletion, we observed retinal structural changes by optical coherence tomography; dysfunction and loss of photoreceptors by retinal imaging; and retinal inflammation marked by subretinal infiltration of immune cells by imaging and immunostaining. Single-cell RNA sequencing of diseased RPE and retinas showed potential miR-regulated target genes, as well as changes in noncoding RNAs in the RPE, rod photoreceptors, and Müller glia. This work thus highlights the role of miR-204 and miR-211 in maintaining RPE function and how the loss of miRs in the RPE exerts effects on the neural retina, leading to inflammation and retinal degeneration.
Assuntos
Camundongos Knockout , MicroRNAs , Degeneração Retiniana , Epitélio Pigmentado da Retina , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/metabolismo , Camundongos , Deleção de Genes , Tomografia de Coerência ÓpticaRESUMO
Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.
Assuntos
Células Fotorreceptoras Retinianas Cones , Células Fotorreceptoras Retinianas Bastonetes , Transducina , Animais , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Camundongos , Transducina/metabolismo , Transducina/genética , Retina/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genéticaRESUMO
A major limitation to developing chimeric antigen receptor (CAR)-T cell therapies for solid tumors is identifying surface proteins highly expressed in tumors but not in normal tissues. Here, we identify Tyrosinase Related Protein 1 (TYRP1) as a CAR-T cell therapy target to treat patients with cutaneous and rare melanoma subtypes unresponsive to immune checkpoint blockade. TYRP1 is primarily located intracellularly in the melanosomes, with a small fraction being trafficked to the cell surface via vesicular transport. We develop a highly sensitive CAR-T cell therapy that detects surface TYRP1 in tumor cells with high TYRP1 overexpression and presents antitumor activity in vitro and in vivo in murine and patient-derived cutaneous, acral and uveal melanoma models. Furthermore, no systemic or off-tumor severe toxicities are observed in an immunocompetent murine model. The efficacy and safety profile of the TYRP1 CAR-T cell therapy supports the ongoing preparation of a phase I clinical trial.
Assuntos
Melanoma , Receptores de Antígenos Quiméricos , Neoplasias Uveais , Humanos , Camundongos , Animais , Melanoma/terapia , Melanoma/tratamento farmacológico , Imunoterapia Adotiva , Neoplasias Uveais/terapia , Neoplasias Uveais/tratamento farmacológico , Terapia Baseada em Transplante de Células e Tecidos , Glicoproteínas de Membrana , OxirredutasesRESUMO
Recessive Stargardt disease (STGD1) is an inherited retinopathy caused by mutations in the ABCA4 gene. The ABCA4 protein is a phospholipid-retinoid flippase in the outer segments of photoreceptors and the internal membranes of retinal pigment epithelial (RPE) cells. Here, we show that RPE cells derived via induced pluripotent stem-cell from a molecularly and clinically diagnosed STGD1 patient exhibited reduced ABCA4 protein and diminished activity compared to a normal subject. Consequently, STGD1 RPE cells accumulated intracellular autofluorescence-lipofuscin and displayed increased complement C3 activity. The level of C3 inversely correlated with the level of CD46, an early negative regulator of the complement cascade. Persistent complement dysregulation led to deposition of the membrane attack complex on the surface of RPE cells, decrease in transepithelial resistance, and subsequent cell death. These findings are strong evidence of complement-mediated RPE cell damage in STGD1, in the absence of photoreceptors, caused by reduced CD46 regulatory protein.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento , Epitélio Pigmentado da Retina , Humanos , Doença de Stargardt , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas do Sistema Complemento/metabolismo , Morte CelularRESUMO
RAB28 is a farnesylated, ciliary G-protein. Patient variants in RAB28 are causative of autosomal recessive cone-rod dystrophy (CRD), an inherited human blindness. In rodent and zebrafish models, the absence of Rab28 results in diminished dawn, photoreceptor, outer segment phagocytosis (OSP). Here, we demonstrate that Rab28 is also required for dusk peaks of OSP, but not for basal OSP levels. This study further elucidated the molecular mechanisms by which Rab28 controls OSP and inherited blindness. Proteomic profiling identified factors whose expression in the eye or whose expression at dawn and dusk peaks of OSP is dysregulated by loss of Rab28. Notably, transgenic overexpression of Rab28, solely in zebrafish cones, rescues the OSP defect in rab28 KO fish, suggesting rab28 gene replacement in cone photoreceptors is sufficient to regulate Rab28-OSP. Rab28 loss also perturbs function of the visual cycle as retinoid levels of 11-cRAL, 11cRP, and atRP are significantly reduced in larval and adult rab28 KO retinae (p < .05). These data give further understanding on the molecular mechanisms of RAB28-associated CRD, highlighting roles of Rab28 in both peaks of OSP, in vitamin A metabolism and in retinoid recycling.
Assuntos
Proteômica , Peixe-Zebra , Animais , Cegueira/metabolismo , Humanos , Fagocitose , Células Fotorreceptoras Retinianas Cones/metabolismo , Retinoides/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Purpose: Modern molecular genetics has revolutionized gene discovery, genetic diagnoses, and precision medicine yet many patients remain unable to benefit from these advances as disease-causing variants remain elusive for up to half of Mendelian genetic disorders. Patient-derived induced pluripotent stem (iPS) cells and transcriptomics were used to identify the fate of unsolved ABCA4 alleles in patients with Stargardt disease. Methods: Multiple independent iPS lines were generated from skin biopsies of three patients with Stargardt disease harboring a single identified pathogenic ABCA4 variant. Derived retinal pigment epithelial cells (dRPE) from a normal control and patient cells were subjected to RNA-Seq on the Novaseq6000 platform, analyzed using DESeq2 with calculation of allele specific imbalance from the pathogenic or a known linked variant. Protein analysis was performed using the automated Simple Western system. Results: Nine dRPE samples were generated, with transcriptome analysis on eight. Allele-specific expression indicated normal transcripts expressed from splice variants albeit at low levels, and missense transcripts expressed at near-normal levels. Corresponding protein was not easily detected. Patient phenotype correlation indicated missense variants expressed at high levels have more deleterious outcomes. Transcriptome analysis suggests mitochondrial membrane biodynamics and the unfolded protein response pathway may be relevant in Stargardt disease. Conclusions: Patient-specific iPS-derived RPE cells set the stage to assess non-expressing variants in difficult-to-detect genomic regions using easily biopsied tissue. Translational Relevance: This "Disease in a Dish" approach is likely to enhance the ability of patients to participate in and benefit from clinical trials while providing insights into perturbations in RPE biology.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Células Epiteliais , Transportadores de Cassetes de Ligação de ATP/genética , Humanos , Fenótipo , Pigmentos da Retina , Doença de StargardtRESUMO
The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the ß subunit (PDEB) to â¼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.
Assuntos
GMP Cíclico , Diester Fosfórico Hidrolases , Animais , GMP Cíclico/metabolismo , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Reprodutibilidade dos Testes , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismoRESUMO
Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.
Assuntos
Membranas Intracelulares/metabolismo , Lipofuscina/farmacologia , Lisossomos/metabolismo , Necroptose/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Envelhecimento , Oxirredutases do Álcool , Animais , Morte Celular , Humanos , Lipofuscina/metabolismo , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4-/-. These vitamin A-derived fluorophores have been shown to induce oxidative stress, stimulate complement activity, and cause chronic inflammation of the RPE. In vivo modulation of complement regulatory pathway in the STGD mouse model has partially rescued the STGD phenotype suggesting that complement attack on the RPE is an important etiologic factor in disease pathogenesis. While bisretinoid-dependent complement activation was further evidenced in cultured RPE cells, this pathway has never been investigated directly in the context of RPE from STGD donor eyes. In the current study, we evaluate the complement reactivity in postmortem donor eyes of clinically diagnosed STGD patients. All three STGD donor eyes RPE displayed strong immunoreactivity for an antibody specific to 4-Hydroxynonenal, a lipid peroxidation byproduct. Also, unlike the control eyes, all three STGD donor eyes showed significantly increased membrane attack complex deposition on the RPE cells. In STGD eyes, increased MAC accumulation was mirrored by elevated C3 fragments internalized by the RPE and inversely correlated with the levels of complement factor H, a major complement regulatory protein. Here, we report the first direct evidence of RPE complement dysregulation as a causative factor in developing Stargardt phenotype.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Transportadores de Cassetes de Ligação de ATP , Animais , Humanos , Degeneração Macular/genética , Camundongos , Retina , Doença de StargardtRESUMO
Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion. Methods: Enucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD. Results: Around the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells. Conclusions: Fluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.
Assuntos
Lâmina Basilar da Corioide/patologia , Angiofluoresceinografia/métodos , Atrofia Geográfica/patologia , Oftalmoscopia/métodos , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica/métodos , Acuidade Visual , Idoso , Idoso de 80 Anos ou mais , Feminino , Fundo de Olho , Humanos , MasculinoRESUMO
Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.
Assuntos
Retina/diagnóstico por imagem , Doença de Stargardt/diagnóstico por imagem , Doença de Stargardt/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Eletrorretinografia , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Lipofuscina/metabolismo , Masculino , Melaninas/metabolismo , Camundongos , Microscopia de Fluorescência , Retina/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Effective treatments and animal models for the most prevalent neurodegenerative form of blindness in elderly people, called age-related macular degeneration (AMD), are lacking. Genome-wide association studies have identified lipid metabolism and inflammation as AMD-associated pathogenic pathways. Given liver X receptors (LXRs), encoded by the nuclear receptor subfamily 1 group H members 2 and 3 (NR1H3 and NR1H2), are master regulators of these pathways, herein we investigated the role of LXR in human and mouse eyes as a function of age and disease and tested the therapeutic potential of targeting LXR. We identified immunopositive LXR fragments in human extracellular early dry AMD lesions and a decrease in LXR expression within the retinal pigment epithelium (RPE) as a function of age. Aged mice lacking LXR presented with isoform-dependent ocular pathologies. Specifically, loss of the Nr1h3 isoform resulted in pathobiologies aligned with AMD, supported by compromised visual function, accumulation of native and oxidized lipids in the outer retina, and upregulation of ocular inflammatory cytokines, while absence of Nr1h2 was associated with ocular lipoidal degeneration. LXR activation not only ameliorated lipid accumulation and oxidant-induced injury in RPE cells but also decreased ocular inflammatory markers and lipid deposition in a mouse model, thereby providing translational support for pursuing LXR-active pharmaceuticals as potential therapies for dry AMD.
Assuntos
Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Animais , Modelos Animais de Doenças , Células Endoteliais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Inflamação/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Fenótipo , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina , Transcriptoma , Adulto JovemRESUMO
Epidemiological evidence correlates low serum vitamin A (retinol) levels with increased susceptibility to active tuberculosis (TB); however, retinol is biologically inactive and must be converted into its bioactive form, all-trans retinoic acid (ATRA). Given that ATRA triggers a Niemann-Pick type C2 (NPC2)-dependent antimicrobial response against Mycobacterium tuberculosis, we investigated the mechanism by which the immune system converts retinol into ATRA at the site of infection. We demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived dendritic cells (DCs), but not macrophages, express enzymes in the vitamin A metabolic pathway, including aldehyde dehydrogenase 1 family, member a2 (ALDH1A2) and short-chain dehydrogenase/reductase family, member 9 (DHRS9), enzymes capable of the two-step conversion of retinol into ATRA, which is subsequently released from the cell. Additionally, mRNA and protein expression levels of ALDH1A2 and DC marker CD1B were lower in tuberculosis lung tissues than in normal lung. The conditioned medium from DCs cultured with retinol stimulated antimicrobial activity from M. tuberculosis-infected macrophages, as well as the expression of NPC2 in monocytes, which was blocked by specific inhibitors, including retinoic acid receptor inhibitor (RARi) or N,N-diethylaminobenzaldehyde (DEAB), an ALDH1A2 inhibitor. These results indicate that metabolism of vitamin A by DCs transactivates macrophage antimicrobial responses.IMPORTANCE Tuberculosis (TB) is the leading cause of death by a single infectious agent worldwide. One factor that contributes to the success of the microbe is the deficiency in immunomodulatory nutrients, such as vitamin A (retinol), which are prevalent in areas where TB is endemic. Clinical trials show that restoration of systemic retinol levels in active TB patients is ineffective in mitigating the disease; however, laboratory studies demonstrate that activation of the vitamin A pathway in Mycobacterium tuberculosis-infected macrophages triggers an antimicrobial response. Therefore, the goal of this study was to determine the link between host retinol levels and retinoic acid-mediated antimicrobial responses against M. tuberculosis By combining established in vitro models with in situ studies of lung tissue from TB patients, this study demonstrates that the innate immune system utilizes transcellular metabolism leading to activation between dendritic cells and macrophages as a means to combat the pathogen.
Assuntos
Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Mycobacterium tuberculosis/imunologia , Vitamina A/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/imunologia , Adulto , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/imunologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Células Dendríticas/microbiologia , Humanos , Pulmão/microbiologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/microbiologia , Retinal Desidrogenase/genética , Retinal Desidrogenase/imunologia , Tuberculose/microbiologiaRESUMO
While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.
Assuntos
Células Ependimogliais/metabolismo , Proteínas do Olho/genética , Receptores Acoplados a Proteínas G/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Pigmentos da Retina/metabolismo , Ácido 2-Aminoadípico/farmacologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/efeitos da radiação , Animais , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/efeitos da radiação , Antagonistas de Aminoácidos Excitatórios/farmacologia , Proteínas do Olho/metabolismo , Proteínas do Olho/efeitos da radiação , Luz , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/efeitos da radiação , Células Fotorreceptoras Retinianas Cones/efeitos da radiação , Pigmentos da Retina/efeitos da radiação , Vitamina A/metabolismoRESUMO
Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo. Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo. Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion: This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Eletrorretinografia , Perfilação da Expressão Gênica , Histocitoquímica , Humanos , Metaboloma , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Tomografia de Coerência ÓpticaRESUMO
The recent approval in the United States of the first adeno-associated viral (AAV) vector for the treatment of an inherited retinal degeneration validates this approach for the treatment of many other diseases. A major limiting factor continues to be the size restriction of the AAV transgene at under 5 kb. Stargardt disease is the most prevalent form of recessively inherited blindness and is caused by mutations in ABCA4, the gene that codes for ATP-binding cassette transporter protein family member 4, which has a coding sequence length of 6.8 kb. Dual vector approaches increase the capacity of AAV gene therapy, but at the cost of substantially reduced levels of target protein, which may be insufficient to achieve a therapeutic effect. Here we show that the efficacy of recombination of dual vectors is dependent on the length of DNA overlap between two transgenes. With optimized recombination, full-length ABCA4 protein is expressed in the photoreceptor outer segments of Abca4-/- mice at levels sufficient to reduce bisretinoid formation and correct the autofluorescent phenotype. These observations support a dual vector approach in future clinical trials using AAV gene therapy to treat Stargardt disease.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Dependovirus/genética , Vetores Genéticos/genética , Fenótipo , Doença de Stargardt/genética , Transgenes , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Humanos , Camundongos Knockout , Fases de Leitura Aberta , Imagem Óptica , Células Fotorreceptoras/metabolismo , Doença de Stargardt/diagnóstico , Doença de Stargardt/terapiaRESUMO
Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4-/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk-/- but not Abca4-/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4-/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4-/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Degeneração Macular/congênito , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Transportadores de Cassetes de Ligação de ATP/biossíntese , Animais , Células Cultivadas , Modelos Animais de Doenças , Lipofuscina/metabolismo , Lisossomos/metabolismo , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fagocitose/imunologia , Retina/patologia , Degeneração Retiniana/patologia , Rodopsina/metabolismo , Doença de Stargardt , c-Mer Tirosina Quinase/genéticaRESUMO
Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had â¼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.