RESUMEN
Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Regulación Enzimológica de la Expresión Génica , Proteínas HSP90 de Choque Térmico , Células Fotorreceptoras Retinianas Bastones , Animales , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas HSP90 de Choque Térmico/deficiencia , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/enzimología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Subunidades de Proteína , Supervivencia CelularRESUMEN
Retinitis pigmentosa (RP) is a debilitating blinding disease affecting over 1.5 million people worldwide, but the mechanisms underlying this disease are not well understood. One of the common models used to study RP is the retinal degeneration-10 (rd10) mouse, which has a mutation in Phosphodiesterase-6b (Pde6b) that causes a phenotype mimicking the human disease. In rd10 mice, photoreceptor cell death occurs with exposure to normal light conditions, but as demonstrated in this study, rearing these mice in dark preserves their retinal function. We found that inactivating rhodopsin signaling protected photoreceptors from degeneration suggesting that the pathway activated by this G-protein-coupled receptor is causing light-induced photoreceptor cell death in rd10 mice. However, inhibition of transducin signaling did not prevent the loss of photoreceptors in rd10 mice reared under normal light conditions implying that the degeneration caused by rhodopsin signaling is not mediated through its canonical G-protein transducin. Inexplicably, loss of transducin in rd10 mice also led to photoreceptor cell death in darkness. Furthermore, we found that the rd10 mutation in Pde6b led to a reduction in the assembled PDE6αßγ2 complex, which was corroborated by our data showing mislocalization of the γ subunit. Based on our findings and previous studies, we propose a model where light activates a non-canonical pathway mediated by rhodopsin but independent of transducin that sensitizes cyclic nucleotide gated channels to cGMP and causes photoreceptor cell death. These results generate exciting possibilities for treatment of RP patients without affecting their vision or the canonical phototransduction cascade.
Asunto(s)
Muerte Celular , Luz , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/patología , Rodopsina/metabolismo , Transducina/fisiología , cis-trans-Isomerasas/fisiología , Animales , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/efectos de la radiación , Retinitis Pigmentosa , Transducción de SeñalRESUMEN
Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor-specific chaperone that stabilizes the effector enzyme of phototransduction, cGMP phosphodiesterase 6 (PDE6). Mutations in the AIPL1 gene cause a severe inherited retinal dystrophy, Leber congenital amaurosis type 4 (LCA4), that manifests as the loss of vision during the first year of life. In this study, we generated three-dimensional (3D) retinal organoids (ROs) from human induced pluripotent stem cells (hiPSCs) derived from an LCA4 patient carrying a Cys89Arg mutation in AIPL1. This study aimed to (i) explore whether the patient hiPSC-derived ROs recapitulate LCA4 disease phenotype, and (ii) generate a clinically relevant resource to investigate the molecular mechanism of disease and safely test novel therapies for LCA4 in vitro. We demonstrate reduced levels of the mutant AIPL1 and PDE6 proteins in patient organoids, corroborating the findings in animal models; however, patient-derived organoids maintained retinal cell cytoarchitecture despite significantly reduced levels of AIPL1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Ojo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Organoides/metabolismo , Retina/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas del Ojo/genética , Humanos , Amaurosis Congénita de Leber/genética , Amaurosis Congénita de Leber/metabolismo , Mutación/genéticaRESUMEN
The outer segment (OS) of photoreceptor cells is an elaboration of a primary cilium with organized stacks of membranous disks that contain the proteins needed for phototransduction and vision. Though ciliary formation and function has been well characterized, little is known about the role of cilia in the development of photoreceptor OS. Nevertheless, progress has been made by studying mutations in ciliary proteins, which often result in malformed OSs and lead to blinding diseases. To investigate how ciliary proteins contribute to OS formation, we generated a knockout (KO) mouse model for ARL2BP, a ciliary protein linked to retinitis pigmentosa. The KO mice display an early and progressive reduction in visual response. Before photoreceptor degeneration, we observed disorganization of the photoreceptor OS, with vertically aligned disks and shortened axonemes. Interestingly, ciliary doublet microtubule (MT) structure was also impaired, displaying open B-tubule doublets, paired with loss of singlet MTs. On the basis of results from this study, we conclude that ARL2BP is necessary for photoreceptor ciliary doublet formation and axoneme elongation, which is required for OS morphogenesis and vision.