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Los fotorreceptores (FRs) son las neuronas que capturan la luz en el ojo, por lo que juegan un rol central en la visión. La pérdida progresiva de estas células durante ciertas enfermedades neurodegenerativas de la retina, como la retinitis pigmentosa o la degeneración macular, conduce a déficits de la visión y eventualmente a la ceguera. Estas patologías, así como otras que afectan al sistema nervioso central, están caracterizadas por la degeneración gradual, selectiva e irreversible de una población neuronal específica. La deficiencia de factores tróficos ha sido involucrada en muchos de estos procesos neurodegenerativos y es característica de la denominada muerte celular programada, tal como la que ocurre al momento de la sinaptogénesis en el desarrollo del sistema nervioso. En efecto, en la retina, así como también en otras partes del sistema nervioso, las neuronas requieren para su supervivencia, de factores tróficos, los cuales provienen de su entorno. El requerimiento de factores tróficos varía según el tipo celular y la etapa del desarrollo. En particular, para los FRs ya se han identificado varios de ellos, incluyendo el Factor Neurotrófico Derivado de la Glía (GDNF), el Factor Neutrófico Ciliar (CNTF), el Factor de Crecimiento Fibroblástico (FGF), el ácido docosahexaenoico (DHA), la esfingosina 1-fosfato (S1P) y, más recientemente, uno de los principales, el Factor Derivado del Epitelio Pigmentario (PEDF), una proteína con funciones neurotróficas y antiangiogénicas, asociadas a dominios separados de la proteína. La identificación de las secuencias de estos dominios ha permitido diseñar y sintetizar químicamente péptidos estables, como los fragmentos neurotróficos 44-mer y 17-mer, que conservan las propiedades de la proteína nativa y, por ende, de potencial valor médico. Sin embargo, estas características ventajosas requieren ser evaluadas en un modelo experimental adecuado. La mayoría del conocimiento actual sobre el PEDF se ha obtenido gracias a modelos in vivo, donde, debido su inherente complejidad, sumado a la cantidad de interacciones que ocurren entre las células y moléculas de tejidos circundantes, resulta difícil analizar los procesos involucrados. Una alternativa a este obstáculo son los cultivos primarios elegidos para realizar esta tesis, compuestos solo de neuronas amacrinas y FRs, las que, creciendo en medios químicamente definidos, permiten estudiarlas en un entorno mucho más controlado que en el organismo entero. En estos cultivos, los FRs se desarrollan independientemente, sin requerir la suplementación de factores tróficos, pero, una vez establecidas sus conexiones sinápticas, se tornan dependientes de los mismos para continuar con su desarrollo y prolongar supervivencia. La dependencia por estos factores hace de este sistema in vitro un modelo adecuado para evaluar el efecto de distintas moléculas sobre la supervivencia o diferenciación celular. Por ello, los resultados reseñados en el primer capítulo de esta tesis tuvieron como objetivo principal evaluar el efecto de PEDF y los péptidos derivados de su dominio neurotrófico y angiogénico en este modelo de cultivo primario de neuronas de retina. En estos cultivos, tanto los FRs como las neuronas amacrinas exhibieron el receptor de PEDF (PEDF-R), el cual es una fosfolipasa del tipo A2, localizado principalmente en la membrana celular. Por otro lado, la expresión del transcripto para PEDF-R mostró un patrón decreciente durante los primeros 5 días de cultivo, así como también en la retina in vivo, en el mismo periodo de desarrollo. Este patrón se observó también a nivel de la proteína, aunque su descenso en el tiempo fue más atenuado. Tanto el PEDF como los dos péptidos derivados de su dominio neurotrófico, protegieron a los FRs en cultivo de la muerte celular, caracterizada por ensayos de TUNEL y Anexina V. Además, previnieron la pérdida de la función mitocondrial evaluada mediante Mitotracker, y preservaron la integridad estructural de la membrana plasmática, analizada indirectamente por medio de ioduro de propidio y DAPI; dado que estos marcadores se visualizan una vez que se altera la permeabilidad de la membrana plasmática. Esta protección se debió principalmente a la interacción de PEDF con PEDF-R, y, en parte, al aumento constatado en la transcripción de factores antiapoptóticos como Bcl2 y Bcl2a1. El efecto del PEDF fue específico para la supervivencia de los FRs dado que el mismo no alteró la viabilidad de las neuronas amacrinas, la cual se mantuvo constante durante los días de cultivo analizados. Por el contrario, el PEDF y los péptidos 44-mer y 17-mer promovieron el desarrollo de neuritas en las neuronas amacrinas, e indujeron la diferenciación de los FRs, al promover la polarización de la rodopsina hacia el extremo apical de estas neuronas, tal como ocurre en los FRs maduros de la retina in vivo. Estos efectos fueron anulados eficientemente mediante el secuestro de PEDF o de sus péptidos por medio del péptido bloqueante P1, o por la inhibición de la actividad enzimática del PEDF-R mediante el inhibidor enzimático selectivo atglistatin. Todos los efectos del PEDF y sus péptidos neurotróficos fueron asociados a la interacción de los mismos con el PEDF-R. Por su parte, el fragmento derivado del dominio antiangiogénico del PEDF no tuvo ningún efecto. Por otro lado, también se indagó sobre el potencial del epitelio pigmentario de la retina (EPR) derivado de células madre pluripotentes (CMP) en la producción y liberación del PEDF. Dado que la deficiencia del PEDF ha sido correlacionada con una mayor incidencia de ciertas retinopatías y el hecho que el EPR puede estar comprometido en algunas de estas patologías, ha impulsado el desarrollo de estrategias orientadas a reemplazar el EPR dañado. Entre ellas se destaca la estrategia de generar EPR por medio de CMP obtenidas a partir de la inducción de células somáticas mediante la transducción de factores de pluripotencia por medio de un sistema episomal. Este tipo de estrategia requiere sortear múltiples obstáculos antes de ser viable, particularmente el de garantizar la identidad del nuevo EPR. El objetivo de esta línea de investigación, descripta en el segundo capítulo de esta tesis, fue evaluar los aspectos funcionales del EPR derivado de células madre, comparándolos con los del EPR nativo. El EPR derivado de CMP humanas recapituló las características distintivas del EPR nativo, como la polarización baso-apical, la capacidad de fagocitar y metabolizar segmentos externos de FRs y la producción de PEDF. Todas estas características se mantuvieron aun hasta 50 días después de su inducción, con la producción de PEDF incrementándose significativamente en función del tiempo. En conclusión, el PEDF y los péptidos derivados de su dominio neurotrófico ejercieron efectos citoprotectores y de diferenciación sobre los fotorreceptores y promovieron el crecimiento de neuritas en las neuronas amacrinas. Todos estos efectos fueron dependientes de la interacción entre PEDF y PEDF- R. Por otro lado, el EPR derivado de CMP examinado en este trabajo de tesis mostró un comportamiento similar al del EPR en la retina intacta, lo que permite considerar sus posibles capacidades terapéuticas para estas enfermedades / Photoreceptors (PHRs) are the retinal neurons, which react to light, making them a central component in the visual process. Their loss during certain retinal neurodegenerative diseases, such as retinitis pigmentosa or age-related macular degeneration, leads to a gradual decline of vision and ultimately to blindness. These pathologies, as well as others that target the central nervous system, are characterized by the gradual, selective and irreversible degeneration of specific neuronal cell types. The lack of trophic factors has been involved in many of these neurodegenerative processes and it is characteristic of the so- called programmed cell death, such as the one that occurs during the period of synaptogenesis within the developing nervous system. In the retina, just as any other portions of the nervous system, neurons are dependent on trophic factors, which are produced by their environment. Trophic factor requirements vary according to each cell type and its developmental stage. For the PHRs, many trophic factors have been already identified, including the Glial cell line-derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF), Fibroblastic Growth Factor (FGF), docosahexaenoic acid (DHA), sphingosine-1-phosphate (S1P) and, perhaps one of the most important among them, the Pigment Epitheliumderived Factor (PEDF). PEDF is a protein exhibiting both neurotrophic and antiangiogenic properties, which are conferred by two spatially-separated domains of the PEDF polypeptide. The identification of the sequences of these domains has allowed for the design and chemical synthesis of stable peptides, such as the neurotrophic fragments 44-mer and 17-mer, which could potentially retain the neurotrophic properties of the native protein, and therefore having a potential therapeutic value. However, these advantages should be first evaluated on an adequate experimental model. Up to now, most of our knowledge regarding PEDF has been obtained through in vivo models, which, due to their inherent complexity and the multiplicity of interactions between cells and their surrounding tissues, makes it difficult to analyze the specific processes occurring at a smaller scale. An alternative to overcome these limitations is the use of primary cultures chosen for the present thesis, composed solely of PHRs and amacrine neurons cultured in a chemically defined medium. These cultures allow the study of these neurons in a much more controlled environment when compared to a whole organism. PHRs in these cultures, initially develop and replicate without requiring trophic factor supplementation, but once they establish their synaptic connections, become reliant on them for their survival. This reliance makes this in vitro system an adequate testbed to evaluate the effects of different trophic factors on cell survival and differentiation. Therefore, the results of the first part of the thesis, which are shown in the first chapter, have the main objective of evaluating the effects of PEDF and peptides derived from its neurotrophic and antiangiogenic domains in a primary retinal cell culture-based model. In these cultures, both neuronal types exhibited the PEDF receptor (PEDF-R), which was primarily localized in the cell membrane. Additionally, the expression patterns for the PEDF-R transcript showed a decreasing trend on the first 5 days in culture, which was also observed in the in vivo environment. This pattern was also observed at the protein level, albeit in a less dramatic fashion. PEDF as well as its neurotrophic domain-derived peptides protected cultured PHRs from cell death, which was measured by TUNEL and Annexin V assays. Furthermore, they also prevented the loss of mitochondrial function, as evaluated by Mitotracker, and preserved the structural integrity of the plasma membrane analyzed by propidium iodide and DAPI staining, given that these markers are visualized once the plasma membrane permeability is altered. This protection was exerted through PEDF/PEDF-R interaction, along with the upregulation of antiapoptotic factors such as Bcl2 and Bcl2a1. This protective effect was PHR-specific, given that there was no significant difference in the survival rate of amacrine neurons, which was constant throughout the observed timeframe. Furthermore, PEDF and the 44-mer and 17-mer peptides promoted neurite outgrowth in amacrine neurons and induced PHR differentiation by promoting apical rhodopsin polarization, mimicking the same process in the retina in vivo. These effects were readily annulled either by sequestering PEDF or its derived neurotrophic peptides with the blocking P1 peptide, or by inhibiting the enzymatic activity of PEDF-R with the selective enzymatic inhibitor atglistatin. Every previously observed effect, exerted by PEDF or its neurotrophic peptides, was linked to their interaction with PEDF-R. The antiangiogenic domain-derived peptide showed no effects whatsoever. In a second line of research carried out in this thesis and described in the second chapter of this thesis, I delved on the potential PEDF production and secretion of induced pluripotent cell-derived retinal pigmented epithelium (RPE). Due to the fact that PEDF deficit has been correlated with an increased incidence of retinopathies, coupled with the observation that the pigmented epithelium itself is compromised in several of them; has led to the emergence of novel therapeutic strategies based on replacing the damaged retinal pigmented epithelium. One of the main approaches relies on generating RPE by the differentiation of induced pluripotent stem cells, obtained by the transduction of pluripotency factors in somatic cells by means of an episomal system. This approach must clear several hurdles before becoming viable, starting with confirming the identity and properties of this new RPE and how it compares to native RPE. The stem cell-derived human RPE was able to replicate the main hallmarks of native RPE, such as basalapical polarization, the capacity to phagocyte and metabolize PHR outer segments and to secrete PEDF. All these features were consistent even at 50 days post-induction, with PEDF secretion showing a significant increase over time. In conclusion, PEDF and its neurotrophic peptides exerted cytoprotective effects and stimulated neuronal development on photoreceptors and promoted neurite outgrowth amacrine neurons. All of these effects were driven by PEDF/PEDF-R interaction. Furthermore, stem cell-derived RPE showed a similar behavior to native RPE, allowing this approach of RPE replacement to be further considered as another potential therapeutic approach for the treatment of these diseases.

The retina is the neural tissue situated at the back of the eyes that samples the visual scene and sends the processed information to the brain. Millions of people worldwide suffer from retinal diseases that affect mainly the light sensing photoreceptors or retinal ganglion cells, the output neurons projecting to the brain. Despite promising attempts in the fields of gene therapy and cell transplantation, a definitive cure for retinal diseases is still missing. Research on highly regenerative organisms like zebrafish (Danio rerio) offers an attractive perspective to inform gene as well as cell transplantation-based therapies to treat retinal pathologies. Indeed, the zebrafish retina has the same structure and function as the human retina, including the presence of all retinal neurons as well as Müller glia, glial cells that provide structural and metabolic support. Remarkably, and differently from mammalian species, zebrafish Müller glia behave additionally as stem cells upon tissue damage. In this context, Müller glia re-enter the cell cycle and generate retinal progenitors that eventually differentiate to all retinal neurons. In the last twenty years, there has been a considerable effort to understand the molecular mechanisms underlying zebrafish Müller glia reprogramming to pro-regenerative stem cells and retinal progenitor production. However, a comprehensive study of the molecular identity of Müller glia, Müller glia-derived retinal progenitors as well as regenerated progeny in uninjured and lesioned conditions is still missing. Furthermore, although retinal progenitors regenerate all retinal neurons, independently of the specific retinal cell type that has been mostly affected by the tissue damage, it is not known whether all regenerated progeny integrate and rewire into the existing circuitry. The present study had two aims: • First, it aimed to provide a comprehensive description of Müller glia, Müller glia-derived progenitors as well as regenerated progeny in uninjured and light-lesioned retina at 44 hours as well as at 4 and 6 days post-lesion. • Second, it aimed to establish a CreERT2 recombinase-based strategy to allow genetic access to follow and manipulate Müller glia-derived progenitors and their progeny during regeneration. To achieve the first aim, a short-term lineage tracing strategy was devised using the two fluorescent reporters Tg(gfap:mCherry) and Tg(pcna:EGFP) labelling Müller glia and proliferating cells, respectively. Double transgenic animals were employed to sort for Müller glia, Müller glia-derived progenitors as well as regenerated progeny from the uninjured and light-lesioned retina. Subsequently, 10x Genomics, single cell RNA sequencing was performed to characterize their transcriptome and to deduct their differentiation trajectories during retina regeneration. The sequencing experiment showed the presence of a glial and a neurogenic trajectory in the regenerating retina up to 6 days post-lesion. The glial trajectory starts with non-reactive Müller glia, characterized by canonical glial markers, and continues with injury-reactive Müller glia at 44 hours post-lesion, which upregulate genes associated with glia reprogramming and inflammation as well as proliferation. These early reactive Müller glia divide and generate cells belonging to a population with a hybrid identity that becomes eminent at 4 days post-lesion and is characterized by marker genes of both Müller glia and progenitors. A glial self-renewal and a neurogenic trajectory depart from the hybrid cell population. While the glial self-renewal trajectory feeds back to the non-reactive Müller glia cell population, the neurogenic trajectory continues with neurogenic progenitors, which progressively express markers of restricted fate competence and eventually regenerate several retinal neurons. The birthdate order of the regenerated progeny recapitulates the order observed during retinal development to a great extent. Indeed, retinal ganglion cells and red cone photoreceptors are born at 4 days post-lesion, followed by blue cones, amacrine and bipolar cells at 6 day post-lesion. Regenerated rod photoreceptors as well as horizontal cells were not detected among the sorted progeny, despite detection of their committed precursors. To achieve the second aim, genetic access to Müller glia-derived cells was established using the TgBAC(mmp9:CreETt2,cryaa:EGFP);Tg(Olactb:loxP-DsRed2-loxP-EGFP) double transgenic line. The injury-induced promoter mmp9 is expressed in reactive Müller glia and drives the expression of CreERT2. Upon administration of the metabolite 4-hydroxytamoxifen, CreERT2 catalyses recombination in the Cre-dependent reporter Tg(Olactb:loxP-DsRed2-loxP-EGFP), resulting in the expression of EGFP under the control of the broadly expressed Olactb promoter. Subsequently, recombined cells, which include progenitors and progeny, express EGFP permanently. Two time points of 4-hydroxytamoxifen intraperitoneal injection were tested to achieve efficient recombination: 6 hours post-lesion, corresponding to 2 hours prior to onset of mmp9 in reactive Müller glia, and 24 hours post-lesion. In both cases, a substantial number of EGFP-positive, Müller glia-derived cells was observed in the regenerating retina at 4 days post-lesion. The majority of the EGFP-positive cells co-localized with PCNA-positive nuclei and corresponded most likely to progenitors. Importantly, EGFP-positive cells were neither observed in the light-lesioned, ethanol injected controls nor in the uninjured, 4-hydroxytamoxifen-injected controls, indicating tight control of CreERT2. In conclusion, the current PhD thesis provides a comprehensive description of the transcriptome of Müller glia, Müller glia-derived retinal progenitors and regenerated progeny. Moreover, it establishes a CreERT2-based approach to study the composition as well as long-term integration of Müller glia-derived cells in the regenerated retina and allow their genetic manipulations in future studies.

BACKGROUND: The brain and retina are derived from the neuroectoderm and have structural and functional similarities. Researchers have separately analyzed brain and retinal perfusion in psychosis patients, but few studies have investigated the relationship between them. While the retina can serve as a proxy for brain disorders such as Alzheimer’s or Parkinson’s, less is known for psychosis. Thus, this study aims to examine the connection between retinal and brain perfusion in patients with psychosis. METHODS: A total of 48 participants, 17 healthy control and 31 probands, took part in the Bipolar and Schizophrenia Network on Intermediate Phenotype-2 (BSNIP-2) study at the Boston location at Beth Israel Deaconess Medical Center. Participants underwent arterial spin labeling MRI (magnetic resonance imaging) and retinal OCTA (optical coherence tomography angiography) imaging to determine brain and retinal perfusion, respectively. Whole retinal layer (superficial, deep, and choriocapillaris) and lobe-wise brain perfusion (frontal, temporal, parietal, occipital, and cingulate cortices) was used for analyses. Statistical analysis was performed in R and results were summarized using basic descriptive statistics. RESULTS: In probands, there was a significant positive correlation between vessel diameter index (VDI) and frontal lobe perfusion (r=0.74, p=0.000027) and between vessel diameter (VD) and frontal lobe perfusion (r=0.64, p=0.00077), but not for healthy controls. There was a significant negative correlation between VDI and temporal lobe perfusion (r=-0.56, p=0.0046), but not for healthy controls. There were no significant results for healthy controls or probands between retinal perfusion and occipital lobe perfusion. CONCLUSION: This study demonstrates that retinal perfusion may be a proxy marker for frontal lobe perfusion and could be used for predicting cognitive performance in a psychosis population given that the frontal lobe is primarily involved in executive functioning. There was an absence of a relationship between retinal perfusion and the occipital perfusion which suggests that retinal perfusion does not match visual neuronal pathway connections to the occipital cortex. These findings demonstrate a step towards appreciating how the retina can be leveraged to understand brain dysfunction in psychosis.

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