Programa de Doctorado: Biotecnología en ciencias de la salud (Biotecnología y Bíomedicina)

Las distroglicanopatías (DGPs) constituyen un grupo heterogéneo de distrofias neuromusculares congénitas con herencia autosómica recesiva que afectan al músculo, cerebro y retina, y están originadas por deficiencias en la O-glicosilación del α-distroglicano (α-DG). Esta modificación postraduccional es esencial para el anclaje de las células musculares y nerviosas a la matriz extracelular, así como para la formación de las sinapsis en ‘cinta’ entre los fotorreceptores y las células bipolares y horizontales en la retina. Mutaciones en los genes POMT1, POMT2, POMGnT1, FKTN, FKRP y LARGE están asociadas a DGPs, originando una pérdida de función de las glicosiltransferasas que codifican y causando así una hipoglicosilación del α-DG. En esta Tesis Doctoral se demuestra que los genes POMT1, POMT2, POMGnT1, FKTN, FKRP y LARGE se expresan a nivel de ARNm mediante RT-PCR, y a nivel de proteína mediante Western blotting, en la retina neural ratón adulto y en fotorreceptores 661W en cultivo. Las proteínas codificadas por estos seis genes se localizan tanto en el citoplasma como en el núcleo de esta línea celular. Mediante microscopía confocal de inmunofluorescencia se ha determinado que POMT1, POMT2 y fukutina se localizan parcialmente en el retículo endoplásmico, mientras que POMGnT1 y FKRP están presentes en el aparato de Golgi de células 661W. En cuanto a LARGE, hemos observado que se encuentra disperso por todo el citoplasma, sin acumulación en ningún orgánulo en particular. Todas las proteínas estudiadas se acumulan en el núcleo de fotorreceptores 661W, aunque únicamente POMT1 y POMT2 se encuentran asociadas a las regiones de eucromatina, colocalizándose entre sí tanto en dicha fracción nuclear como en el citoplasma de estas células. También se ha demostrado en este trabajo mediante Western blotting que el factor de transcripción epigenético p38IP/FAM48A se expresa en la retina neural de ratón adulto y en la línea celular de fotorreceptores 661W, localizándose en la fracción nuclear. Esta proteína posee un dominio funcional denominado Spt20 y múltiples sitios potenciales de glicosilación y fosforilación, los cuales se encuentran muy conservados en todos los vertebrados analizados. En la retina de ratón p38IP/FAM48A se localiza en el núcleo de conos, células horizontales, bipolares, amacrinas y ganglionares, mientras que en el núcleo de las células 661W se localiza en la porción de eucromatina, donde se colocaliza con las proteínas POMT1 y POMT2. Dicho activador transcripcional interacciona físicamente con el heterodímero POMT1-POMT2 mediante su unión a la proteína POMT2. Estos resultados sugieren que dicho heterodímero podría ejercer una función como regulador indirecto de la expresión génica mediante la O-glicosilación de factores de transcripción y/o reguladores epigenéticos como p38IP/FAM48A en fotorreceptores de la línea 661W.

Die Funktionsweise des invertierten Aufbaus der Netzhaut im Wirbeltierauge ist ein altes Rätsel der Wissenschaft. Das beim Sehvorgang auf die Netzhaut einfallende Licht muss erst alle Netzhautschichten durchdringen, bevor es die Photorezeptorzellen erreicht, welche sich auf der lichtabgewandten Seite des Gewebes befinden. Die vorgelagerten Gewebsschichten enthalten zahlreiche lichtstreuende Bestandteile und müssten den Sehvorgang der Wirbeltiere theoretisch negativ beeinflussen. Diese Annahme steht jedoch im Widerspruch zu dem beeindruckenden Sehvermögen der meisten Wirbeltiere. Die Müllerschen Radialgliazellen stellen eine Lösung für diesen scheinbaren Widerspruch dar. Aufgrund der auffälligen morphologischen Struktur dieser Gliazellen, welche die gesamte Dicke der Netzhaut säulenförmig durchspannen, wurde die Hypothese aufgestellt, dass Müllerzellen nach dem Prinzip der Lichtleitung arbeiten und so das Licht zu den Photorezeptoren transportieren. Diese Theorie konnte jedoch bisher noch nicht bewiesen werden, da die bisherigen experimentellen Messmethoden auf der Basis von isolierten Müllerzellen ungeeignet sind, um diese Funktion im lebenden Gewebe nachzuweisen. Die vorliegende Arbeit beweist erstmalig, dass die Müllerschen Gliazellen als lebende Lichtleiter im Netzhautgewebe funktionieren. Um diese Aufgabe den Müllerzellen eindeutig zuzuordnen, wurde eine neuartige Methode entwickelt, welche gleichzeitig mehrere für den Nachweis unverzichtbare Parameter erfassen kann. Aufgrund einer fluoreszenzbasierten Visualisierung der Müllerzellen in der intakten Netzhaut konnte mit Hilfe eines auf Glasfaseroptik basierenden Aufbaus die Beleuchtung einzelner Müllerzellen erfolgen. Zeitgleich war es möglich, sowohl den Weg des Lichtes von der lichtzugewandten Seite bis zu den Photorezeptoren als auch die Transmission hinter dem Gewebe zu detektieren. Die Komplexität dieses Messverfahrens erlaubte eine detaillierte Charakterisierung des Einflusses der Müllerzelle auf die Streueigenschaften der verschiedenen retinalen Schichten sowie des sich ergebenden Lichtsignals an den Rezeptorzellen. Mittels eines speziellen Analyseverfahrens konnte umfassendes Datenmaterial erhoben und so die Müllerzelle eindeutig als Lichtleiter identifiziert werden. Darauf aufbauend wird in dieser Arbeit außerdem gezeigt, dass alle Müllerzellen gemeinsam und damit in ihrer Gesamtheit mittels ihrer Lichtleitfunktion das an den Photorezeptoren ankommende Lichtmuster beeinflussen, was zu einer verbesserten Bildqualität führt. Dies wird zusätzlich durch morphologische Untersuchungen gestützt, die zeigen, dass die für das Kontrastsehen verantwortlichen Zapfen-Photorezeptorzellen lokal hinter den Müllerzellen angeordnet sind. Demnach ist jeder Zapfen mit einem ihm vorgelagerten Lichtleiter ausgestattet. Zusammenfassend liefert diese Arbeit eine Erklärung, wie trotz des invertierten Aufbaus der Netzhaut die visuelle Information als Grundlage für das Sehen der Wirbeltiere erhalten bleibt.

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.

Neurturin (NRTN) is a neurotrophic factor required for the development of many parasympathetic neurons and normal cholinergic innervation of the heart, lacrimal glands and numerous other tissues. Previous studies with transgenic mouse models showed that NRTN is also essential for normal development and function of the retina (J. Neurosci. 28:4123-4135, 2008). NRTN knockout (KO) mice exhibit a marked thinning of the outer plexiform layer (OPL) of the retina, with reduced abundance of horizontal cell dendrites and axons, and aberrant projections of horizontal cells and bipolar cells into the outer nuclear layer. The effects of NRTN deletion on specific neurotransmitter systems in the retina and on cholinergic innervation of the iris are unknown. To begin addressing this deficiency, we used immunohistochemical methods to study cholinergic and noradrenergic innervation of the iris and the presence and localization of cholinergic and dopaminergic neurons and nerve fibers in eyes from adult male wild-type (WT) and NRTN KO mice (age 4-6 months). Mice were euthanized, and eyes were removed and fixed in cold neutral buffered formalin or 4% paraformaldehyde. Formalin-fixed eyes were embedded in paraffin, and 5μm cross-sections were collected. Representative sections were stained with hematoxylin and eosin or processed for fluorescence immunohistochemistry after treatment for antigen retrieval. Whole mount preparations were dissected from paraformaldehyde fixed eyes and used for immunohistochemistry. Cholinergic and catecholaminergic nerve fibers were labeled with primary antibodies to the vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH), respectively. Cholinergic and dopaminergic cell bodies were labeled with antibodies to choline acetyltransferase (ChAT) and TH, respectively. Cholinergic innervation of the mouse iris was restricted to the sphincter region, and noradrenergic fibers occurred throughout the iris and in the ciliary processes. This pattern was unaffected by deletion of NRTN. Furthermore, functional experiments demonstrated that cholinergic regulation of the pupil diameter was retained in NRTN KO mice. Hematoxylin and eosin stains of the retina confirmed a marked thinning of the OPL in KO mice. VAChT and ChAT staining of the retina revealed two bands of cholinergic processes in the inner plexiform layer, and these were unaffected by NRTN deletion. Likewise, NRTN deletion did not affect the abundance of ChAT-positive ganglion and amacrine cells. In marked contrast, staining for TH showed an increased abundance of dopaminergic processes in the OPL of retina from KO mice. Staining of retinal whole mounts for TH showed no difference in the abundance of dopaminergic amacrine cells between WT and KO mice. These findings demonstrate that the neurotrophic factor NRTN is not required for the development or maintenance of cholinergic innervation of the iris, cholinergic control of pupil diameter, or for development of cholinergic and dopaminergic amacrine cells of the retina. However, NRTN deficiency causes a marked reduction in the size of the OPL and aberrant growth of dopaminergic processes into this region.

Indiana University-Purdue University Indianapolis (IUPUI) / Glaucoma is a multi-factorial ocular disease associated with death of retinal ganglion cells and irreversible vision loss. Many risk factors contribute to glaucomatous damage, including elevated intraocular pressure (IOP), age, genetics, and other diseases such as diabetes and systemic hypertension. Interestingly, alterations in retinal hemodynamics have also been associated with glaucoma. A better understanding of the factors that contribute to these hemodynamic alterations could lead to improved and more appropriate clinical approaches to manage and hopefully treat glaucoma patients. In this thesis, we develop several mathematical models aimed at describing ocular hemodynamics and oxygenation in health and disease. Precisely we describe: (i) a time-dependent mathematical model for the retinal circulation that includes macrocirculation, microcirculation, phenomenological vascular regulation, and the mechanical effect of IOP on the retinal vasculature; (ii) a steady-state mathematical model for the retinal circulation that includes macrocirculation, microcirculation, mechanistic vascular regulation, the effect of IOP on the central retinal artery and central retinal vein, and the transport of oxygen in the retinal tissue using a Krogh cylinder type model; (iii) a steady-state mathematical model for the transport of oxygen in the retinal microcirculation and tissue based on a realistic retinal anatomy; and (iv) a steady-state mathematical model for the production and drainage of aqueous humor (AH). The main objective of this work is to study the relationship between IOP, systemic blood pressure, and the functionality of vascular autoregulation; the transport and exchange of oxygen in the retinal vasculature and tissue; and the production and drainage of AH, that contributes to the level of IOP. The models developed in this thesis predict that (i) the autoregulation plateau occurs for different values of IOP in hypertensive and normotensive patients. Thus, the level of blood pressure and functionality of autoregulation affect the changes in retinal hemodynamics caused by IOP and might explain the inconsistent outcomes of clinical studies; (ii) the metabolic and carbon dioxide mechanisms play a major role in the vascular regulation of the retina. Thus, the impairment of either of these mechanisms could cause ischemic damage to the retinal tissue; (iii) the multi-layer description of transport of oxygen in the retinal tissue accounts for the effect of the inner and outer retina, thereby improving the predictive ability of the model; (iv) a greater reduction in IOP is obtained if topical medications target AH production rather that AH drainage and if IOP-lowering medications are administrated to patients that exhibit a high initial level of IOP. Thus, the effectiveness of IOP-lowering medications depend on a patient’s value of IOP. In conclusion, the results of this thesis demonstrate that the insight provided by mathematical modeling alongside clinical studies can improve the understanding of diseases and potentially contribute to the clinical development of new treatments.

Indiana University-Purdue University Indianapolis (IUPUI) / Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.

En la presente Tesis Doctoral se describen los cambios celulares que ocurren en la retina en la enfermedad de Parkinson y su posible uso como biomarcadores tempranos de la enfermedad. Los pacientes con enfermedad de Parkinson poseen acumulaciones de alfa sinucleína fosforilada en la retina similares a las que se encuentran en el cerebro de los mismos pacientes. De hecho, la cantidad de alfa-sinucleína fosforilada en la retina correlaciona con la cantidad de alfa-sinucleína fosforilada en el cerebro, con el estadio de progresión de la enfermedad y con la severidad de los síntomas motores. Además, en la retina de enfermos de párkinson se describe una degeneración de las células ganglionares melanopsínicas de la retina, lo que podría explicar las alteraciones en los ritmos circadianos y los desórdenes del sueño que aparecen en pacientes. Finalmente, también se muestra la degeneración de las células amacrinas dopaminérgicas, que se reducen en un 45%. Este fallo en el sistema dopaminérgico de la retina provoca alteraciones morfológicas en las células amacrinas AII, sus principales postsinápticas, y podría explicar algunas alteraciones visuales descritas en la enfermedad como la disminución de la sensibilidad al contraste o de la agudeza visual. En global, los resultados muestran que la retina reproduce los procesos degenerativos que ocurren en el cerebro en la enfermedad de Parkinson y, por tanto, que es un tejido idóneo para el estudio de la enfermedad. Además, el estudio de la retina aporta información sobre el estadio de la enfermedad y puede ser empleado como un biomarcador temprano que ayude al diagnóstico y seguimiento de la misma.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18904号 / 医科博第60号 / 新制||医科||4(附属図書館) / 31855 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 吉村 長久, 教授 髙橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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