Autofagia selectiva en la retina: fisiología y patología

En esta tesis doctoral hemos investigado el papel de la autofagia selectiva durante el desarrollo y degeneración del ojo de vertebrados. En primer lugar, nos hemos centrado en la mitofagia o la eliminación selectiva de mitocondrias por autofagia. Para este estudio hemos tenido primero que optimizar una serie de herramientas y métodos que nos permiten determinar de una manera cuantitativa, robusta, y lo más importante, in vivo, este proceso. Entre estos métodos destacamos el uso de dos animales reporteros para determinar autofagia y mitofagia en tejido. También se han optimizado diferentes inductores e inhibidores de autofagia y mitofagia, controlando sus cinéticas que nos permiten modular el proceso. Nuestros datos indican que la autofagia predomina en las diferentes partes del ojo durante su desarrollo. En el ojo de adulto, sin embargo, encontramos en la capa nuclear externa (CNE), donde se encuentran los fotorreceptores y la glía de Müller, el epitelio pigmentario y nervio óptico, que toda la actividad autofágica corresponde a mitofagia, mientras que en las otras partes del ojo hay autofagia pero no mitofagia. Por otro lado, hemos demostrado que para que tenga lugar la mitofagia en la CNE, hay un transporte desde el segmento interno a la zona de los somas tanto de los lisosomas como de los mitolisosomas..

Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal

17 p.-6 figRelatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand,replace and regenerate. Resident stem/progenitor cells are key in this process because, upon activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly,autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.F.R. is supported by Marie Skłodowska-Curie Cofund [713660], Marie Skłodowska-Curie ITN [765912], ALW Open Program [ALWOP.310]and ZonMW VICI [016.130.606] grants. R.C. and C.R. are supported by the Dutch Cancer Society [Grant number 5792 and 12458]. Patricia Boya is supported by the Ministerio de Ciencia, Innovación y Universidades (MCIU), the Agencia Estatal de Investigación (AEI),the Fondo Europeo de Desarrollo Regional (FEDER) [PGC2018-098557-B-I00] and a Marie Skłodowska-Curie ITN grant [765912]. Idil Orhon is a recipient of a FEBS postdoctoral fellowship and Beatriz Villarejo-Zori of a Fundacion Tatiana Perez de Guzman el Bueno predoctoral fellowhip.Peer reviewe

A comparative map of macroautophagy and mitophagy in the vertebrate eye

Photoreception is pivotal to our experience and perception of the natural world; hence the eye is of prime importance for most vertebrate animals to sense light. Central to visual health is mitochondrial homeostasis, and the selective autophagic turnover of mitochondria (mitophagy) is predicted to play a key role here. Despite studies that link aberrant mitophagy to ocular dysfunction, little is known about the prevalence of basal mitophagy, or its relationship to general autophagy, in the visual system. In this study, we utilize the mito-QC mouse and a closely related general macroautophagy reporter model to profile basal mitophagy and macroautophagy in the adult and developing eye. We report that ocular macroautophagy is widespread, but surprisingly mitophagy does not always follow the same pattern of occurrence. We observe low levels of mitophagy in the lens and ciliary body, in stark contrast to the high levels of general MAP1LC3-dependent macroautophagy in these regions. We uncover a striking reversal of this process in the adult retina, where mitophagy accounts for a larger degree of the macroautophagy taking place, specifically in the photoreceptor neurons of the outer nuclear layer. We also show the developmental regulation of autophagy in a variety of ocular tissues. In particular, mitophagy in the adult mouse retina is reversed in localization during the latter stages of development. Our work thus defines the landscape of mitochondrial homeostasis in the mammalian eye, and in doing so highlights the selective nature of autophagy in vivo and the specificity of the reporters used.Peer reviewe

Small molecules targeting glycogen synthase kinase 3 as potential drug candidates for the treatment of retinitis pigmentosa

6 p.-5 fig.Retinitis pigmentosa (RP) is an inherited retinal dystrophy that courses with progressive degeneration of retinal tissue and loss of vision. Currently, RP is an unpreventable, incurable condition. We propose glycogen synthase kinase 3 (GSK-3) inhibitors as potential leads for retinal cell neuroprotection, since the retina is also a part of the central nervous system and GSK-3 inhibitors are potent neuroprotectant agents. Using a chemical genetic approach, diverse small molecules with different potency and binding mode to GSK-3 have been used to validate and confirm GSK-3 as a pharmacological target for RP. Moreover, this medicinal chemistry approach has provided new leads for the future disease-modifying treatment of RP.This work has been partially funded by the Spanish MINECO grants (SAF2012-37979-C03-01 to AM and SAF2013-41059-R to EJdlR).Peer reviewe

The mito-QC reporter for quantitative mitophagy assessment in primary retinal ganglion cells and experimental glaucoma models

This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 and European Union’s Horizon 2020 research and innovation programme under grant agreement No 765912. BVZ is a recipient of PhD contract from the Fundación Tatiana Pérez de Guzmán el Bueno (Spain), PT from H2020-MSCA-ITN-2017, NRM a Juan de la Cierva Grant from Ministerio Ciencia e Innovación (Spain) and KB from DFG (Deutsche Forschungsgemeinschaft, Germany, 6619/1-1).Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.publishersversionpublishe

A GSK-3b modulator delays photoreceptor cell death and preserves visual function in the rd10 mouse model of retinitis pigmentosa

1 p.Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies that lead to blindness. Photoreceptor cell death, reactive gliosis and retinal inammation are common features in animal models of the disease. The enzyme Glycogen Synthase Kinase-3 Beta (GSK-3b) is involved in inammatory processes associated to diverse neurodegenerative pathologies. The aim of our study is to test in the rd10 mouse whether the GSK-3b inhibitor VP3.15 (a small heterocyclic molecule) is a potential therapeutic treatment for RP.SAF2013-41059-RPeer reviewe

Autofagia selectiva en la retina: Fisiología y patología

221 p.-114 fig.-11 tab.-anexo.[EN]In this doctoral thesis we have investigated the role of selective autophagy during the development and degeneration of the vertebrate eye. First, we have focused on the mitophagy or the selective elimination of mitochondria by autophagy. For this study, we first had to optimize a series of tools and methods that allow us to determine this process in a quantitative and robust form, and most important way, in vivo. Among these methods we highlight the use of two reporter animals to determine autophagy and mitophagy in tissue. Different inductors and inhibitors of autophagy and mitophagy have also been optimized, controlling their kinetics that allow us to modulate the process. Our data indicate that autophagy predominates in different parts of the eye during its development. In the adult eye, however, we find in the outer nuclear layer (ONL), where the photoreceptors and Müller glia, the pigmentary epithelium and the optic nerve are located, that all the autophagic activity is mitophagy, while in the other parts of the eye there is autophagy but no mitophagy. On the other hand, we have defined that to take place the mitophagy in the ONL, there is a transport from the internal segment to the area of the somas of both lysosomes and mitolysosomes. To understand the role of these processes in retinal stress conditions, we have used a pharmacological model of photoreceptor death, N-methyl-N-nitrosourea (MNU). We have discovered that with one day of in vivo treatment, the loss of photoreceptors begins in the central area of the retina, both of cones and rods, and Müller glia is activated. We also produce the alteration of different cellular organelles such as the endoplasmic reticulum and mitochondria, and a disorganization and vacuolization of the cytoplasm. The pH of the lysosomes is also affected thereby altering its functionality. The MNU modulates the mitophagy via the PINK1/Parkin-dependent pathway. MNU induces mitophagy at low doses, while at high doses reduces it. Before a blockage of mitophagy in the outer nuclear layer, the mitolysosomes decrease in the ONL and are accumulated in the lower part of the internal segments of the photoreceptors, presenting a size larger than the mitolysosomes of the control retinas. Finally, the mitophagy has a neuroprotective role of photoreceptors in the face of the damage caused by the MNU, extending its survival. On the other hand, we also wanted to understand the role of canonical and non-canonical autophagy in the elimination of naturally occurring apoptotic cells during retinal neurogenesis. We have seen that Atg5 is essential for the detection of apoptotic cells during embryonic retinal development. Retinas deficient in Atg5 have a blockage in the degradation of apoptotic cells by the microglia, as the fusion between autophagosomes and lysosomes is diminished. In addition, we demonstrate that LC3 associated phagocytosis (LAP) is essential for homeostatic maintenance and neurogenesis during embryonic development of the retina, presenting retinas deficient in LAP a phenotype similar to Atg5 deficient retinas. It also plays an important role in the maintenance of RPE and photoreceptors in the adult retina. We conclude that selective autophagy maintains the survival of photoreceptors in the face of damage that causes their degeneration, and therefore proposes the induction of mitophagy as a possible therapy for neurodegenerative diseases. We also highlight the important role that LC3 associated phagocytosis plays in the removal of apoptotic cells during development for the maintenance of retinal homeostasis.[ES] En esta tesis doctoral hemos investigado el papel de la autofagia selectiva durante el desarrollo y degeneración del ojo de vertebrados. En primer lugar, nos hemos centrado en la mitofagia o la eliminación selectiva de mitocondrias por autofagia. Para este estudio hemos tenido primero que optimizar una serie de herramientas y métodos que nos permiten determinar de una manera cuantitativa, robusta, y lo más importante, in vivo, este proceso. Entre estos métodos destacamos el uso de dos animales reporteros para determinar autofagia y mitofagia en tejido. También se han optimizado diferentes inductores e inhibidores de autofagia y mitofagia, controlando sus cinéticas que nos permiten modular el proceso. Nuestros datos indican que la autofagia predomina en las diferentes partes del ojo durante su desarrollo. En el ojo de adulto, sin embargo, encontramos en la capa nuclear externa (CNE), donde se encuentran los fotorreceptores y la glía de Müller, el epitelio pigmentario y nervio óptico, que toda la actividad autofágica corresponde a mitofagia, mientras que en las otras partes del ojo hay autofagia pero no mitofagia. Por otro lado, hemos demostrado que para que tenga lugar la mitofagia en la CNE, hay un transporte desde el segmento interno a la zona de los somas tanto de los lisosomas como de los mitolisosomas. Para entender el papel de estos procesos en condiciones de estrés retiniano, hemos utilizado un modelo farmacológico de muerte de fotorreceptores, N-metil-N-nitrosourea (MNU). Hemos descubierto que con un día de tratamiento in vivo, empieza la pérdida de fotorreceptores en la zona central de la retina, tanto de conos como de bastones, y se activa la glía de Müller. También produce la alteración de diferentes orgánulos celulares como el retículo endoplasmático y las mitocondrias, y una desorganización y vacuolización del citoplasma. El pH de los lisosomas también se ve afectado alterando por tanto su funcionalidad. El MNU modula la mitofagia por la vía dependiente de PINK1/Parkin. Induce mitofagia a dosis bajas mientras que a dosis altas disminuye la mitofagia. Ante un bloqueo de mitofagia en la CNE, los mitolisosomas disminuyen en la CNE y se encuentran acumulados en la zona inferior de los segmentos internos de los fotorreceptores, presentando un tamaño mayor que los mitolisosomas de las retinas control. Por último, la mitofagia presenta un papel neuroprotector de los fotorreceptores ante el daño provocado por el MNU, alargando su supervivencia. Por otro lado, también hemos querido entender el papel de la autofagia canónica y no canónica en la eliminación de las células apoptóticas que se producen naturalmente durante la neurogénesis retiniana. Hemos visto que Atg5 es esencial para la eliminación de las células apoptóticas durante el desarrollo embrionario de la retina. Las retinas deficientes en Atg5 muestran un bloqueo en la degradación de las células apoptóticas por parte de la microglía, viéndose disminuida la fusión entre autofagosomas y lisosomas. Además, demostramos que la fagocitosis asociada a LC3 (LAP) es esencial para el mantenimiento homeostático y para la neurogénesis durante el desarrollo embrionario de la retina, presentando las retinas deficientes en LAP un fenotipo similar a las retinas deficientes en Atg5. También desarrolla un papel importante en el mantenimiento del epitelio pigmentario de la retina (RPE) y de los fotorreceptores en la retina adulta. Concluimos que la autofagia selectiva mantiene la supervivencia de los fotorreceptores ante un daño que provoca su degeneración, por lo que proponemos la inducción de mitofagia como una posible terapia para las enfermedades neurodegenerativas. También destacamos el importante papel que tiene la autofagia asociada a LC3 en la eliminación de las células apoptóticas durante el desarrollo para el mantenimiento de la homeostasis de la retina.Beca predoctoral Fundación Tatiana Pérez de Guzmán el Bueno.Peer reviewe

Autophagie et vision

La rétine est un tissu de l’œil des vertébrés sensible à la lumière qui détecte et traite les images visuelles. L’œil et la rétine sont soumis à une variété d’agressions et de stress environnementaux, des modifications liées à l’âge et des mutations génétiques qui nuisent à leur fonction. L’autophagie est une voie catabolique qui permet la dégradation et le recyclage des composants cellulaires, en conditions physiologique ou de stress. Des protéines, des lipides, et même des organites entiers sont ainsi pris en charge par les autophagosomes et transportés dans les lysosomes afin de les éliminer. Dans le système visuel, le rôle de l’autophagie commence à être étudié. Nous relaterons dans cette revue les principales études montrant l’importance de l’autophagie dans la fonction visuelle

HIF1α or mitophagy: which drives cardiomyocyte differentiation?

4 p.-1 fig.Research in the P.B. lab is supported by grants PGC2018-098557-B-I00 from Spain’s Ministerio Ciencia e Innovación, and funding from the European Union’s Horizon 2020 re-search and innovation programme under grant agreement No. 765912.Peer reviewe

Ambra1 and its role during physiological ageing in the retina

Abstract from 2020 ARVO Annual Meeting, canceled because of COVID-19Purpose : Ambra1 (activating-molecule in Beclin-1-regulated autophagy) is a protein that regulates the early stages of autophagy initiation. Ambra1 haploinsufficiency is linked to autophagy under several pathological conditions such as Alzheimer Disease, autism or multiple system atrophy. In the retina, there is a decline in the autophagy response upon ageing thus we aimed to test if Ambragt/+ haploinsufficiency might affect retinal ageing.Methods : As Ambra1 homozigous animals are embryonic lethal, we have analised the phenotype of Ambra1gt/+ during aging and compared with Ambra1+/+ litermates. We have assessed retinal morphology and function with ERG in animals up to 24 months as well as compared the metabolic profile using mass spectrometry.Results : Our results show that Ambra1gt/+ display premature death in comparison to control littermates. We also observe symptoms of premature retinal ageing in Ambra1gt/+ mice at 15 months old. This tendency was increased in geriatric animals (>20 months) as Ambra1gt/+ displayed increased inflammatory response and a reduction in the photoreceptor layer thickness. Finally, aged Ambra1gt/+ mice displayed a different metabolic signature indicating a misbalance in the purine metabolism and redox status.Conclusions : This data suggest that autophagy deficiency results in a pro-ageing phenotype and impair retinal homeostasis and metabolism.Peer reviewe