Retinitis Pigmentosa (RP) is an incurable inherited retinal degeneration affecting approximately 1 in 4,000 individuals globally. The aim of this dissertation was to identify drugs that can help patients suffering from the disease. To accomplish this goal, the zebrafish was utilized as a model for RP to perform <i>in vivo</i> drug screening. The zebrafish RP model expresses a human rhodopsin transgene which contains a premature stop codon at position 344 (<i>Tg</i>(<i>rho:Hsa.RH1_Q344X</i>)). This zebrafish model exhibits significant rod photoreceptor degeneration beginning at 7 days post fertilization (dpf). To assess the visual consequence of this rod degeneration the zebrafish behavior visual motor response (VMR) was assayed under scotopic conditions. The Q344X RP model larvae displayed a deficit in this VMR in response to a scotopic light offset. This deficit in behavior was utilized to perform a drug screen to identify compounds that could ameliorate the deficient Q344X VMR. The ENZO SCREEN-WELL® REDOX library was chosen to be screened since oxidative stress may increase RP progression in a non-specific manner. From this library, a β-blocker, carvedilol, was identified as a compound that improved the Q344X VMR behavior. This drug was also able to increase rod number in the Q344X retina. Carvedilol was shown to be capable of working directly on rods by demonstrating that the drug can signal through the adrenergic pathway in the rod-like human Y79 cell line. Since carvedilol is an FDA-approved drug, this screening paradigm was utilized to screen the Selleckchem FDA-approved library to identify more drugs that can potentially be repurposed to treat RP like carvedilol. Additionally, this scotopic VMR assay was used to demonstrate that it can identify behavioral deficits in the P23H RP model zebrafish<i> (Tg</i>(<i>rho:Hsa.RH1_P23H</i>)). This dissertation work provides a potential FDA-approved drug for RP treatment and sets the foundation for future drug screening to identify more drugs to treat different forms of RP.

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.

<p>For many years, researchers have developed ways to diagnose degenerative disease in the retina by utilizing multiple gene analysis techniques. Retinitis pigmentosa (RP) disease can cause either partially or totally blindness in adults. For that reason, it is crucial to find a way to pinpoint the causes in order to develop a proper medication or treatment. One of the common methods is genome-wide analysis (GWA). However, it cannot fully identify the genes that are indirectly related to the changes in eye size. In this research, RNA sequencing (RNA-seq) analysis is used to link the phenotype to genotype, creating a pool of candidate genes that might associate with the RP. This will support future research in finding a therapy or treatment to cure such disease in human adults.</p> <p><br></p> <p>Using the Drosophila Genetic Reference Panel (DGRP) – a gene reference panel of fruit fly – two types of datasets are involved in this analysis: eye-size data and gene expression data with two replicates for each strain. This allows us to create a phenotype-genotype map. In other words, we are trying to trace the genes (genotype) that exhibit the RP disease guided by comparing their eye size (phenotype). The basic idea of the algorithm is to discover the best replicate combination that maximizes the correlation between gene expression and eye-size. Since there are 2N possible replicate combinations, where N is the number of selected strains, the original implementation of sequential algorithm was computationally intensive.</p> <p><br></p> <p>The original idea of finding the best replicate combination was proposed by Nguyen et al. (2022). In this research, however, we restructured the algorithms to distribute the tasks of finding the best replicate combination and run them in parallel. The implementation was done using the R programming language, utilizing doParallel and foreach packages, and able to execute on a multicore machine. The program was tested on both a laptop and a server, and the experimental results showed an outstanding improvement in terms of the execution time. For instance, while using 32 processes, the results reported up to 95% reduction in execution time when compared with the sequential version of the code. Furthermore, with the increment of computational capabilities, we were able to explore and analyze more extreme eye-size lines using three eye-size datasets representing different phenotype models. This further improved the accuracy of the results where the top candidate genes from all cases showed connection to RP.</p>

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Vision impairment and blindness are in industrialized countries primarily caused by the degeneration of the retina, the light sensing tissue inside the eye. The degeneration, occurring in diseases like age-related macular degeneration (AMD) or retinitis pigmentosa (RP), can be caused by environmental factors as well as genetic defects and thus shows diverse pathologies. In all conditions, the light detecting photoreceptors (rods and/or cones) are dying caused by either direct photoreceptor damage or as a secondary effect following degeneration of supporting cells. Although promising treatment approaches are currently under investigation, up to date it is not possible to cure these diseases. Amongst these therapeutic strategies, pre-clinical studies evaluating the replacement of degenerated cells by transplantation of new photoreceptors demonstrated promising results. First studies conducted the specific enrichment and transplantation of primary photoreceptors derived from postnatal mice and their sufficient integration and differentiation into mature photoreceptors in wild-type as well as degenerated mouse retinae. Recent experiments additionally proved the recovery of some dim-light vision after transplantation in mice lacking night sight. The in vitro differentiation of whole eye cups containing photoreceptors, out of human or mouse ES or iPS cells, peaked in the transplantation of ES-derived photoreceptors into wild-type as well as degenerated mice and the integration and maturation of these cells. These observations are encouraging, but prior to a save implementation of this strategy into a clinical routine, several further hurdles need to be challenged. Collection of photoreceptors out of whole retinal tissues prior to transplantation was shown to be an important step to reach high integration rates. Additionally, transplantation of photoreceptors derived from stem cells comprises the risk of tumor formation after transplantation and thus also requires depletion of inadvertent cells. Therefore, we established the enrichment of photoreceptors using the cell surface marker dependent method magnetic-activated cell sorting (MACS). For identification of suitable target-specific surface markers, we characterized young transplantable mouse photoreceptors using microarray analysis and screened their transcriptome. Amongst others, ecto-5´nucleotidase (Nt5e, termed CD73) was identified being a rod photoreceptor specific cell surface protein. Thus, we enriched young photoreceptors with CD73-dependent MACS with sufficient purity and transplanted these cells into the subretinal space of wild-type mice. In contrast to unsorted retinal cells, enriched photoreceptors integrated in significantly higher number into the host retina, proving that MACS is a suitable alternative for specific photoreceptor enrichment. Testing other proteins, identified as photoreceptor specific, for MACS suitability and the translation of this approach to photoreceptors, derived from mouse as well as human iPS or ES cells, should be the focus of consecutive investigations. The integration of grafted cells into the retina is a complex process dependent on a variety of influencing factors. Transplantation experiments in aging wild-type mice and a rod-depleted mouse model, containing a retina composed of cone and cone-like photoreceptors, indicated that the activation of Müller glia cells facilitates integration of transplanted photoreceptors. Besides that, reduced outer limiting membrane (OLM) integrity, increased subretinal graft distribution or reduced retinal cell density are further suggested as potential cell engraftment enhancers. These factors might open up important possibilities of host retina manipulation to increase cell integration rates. Although retinal transplantation experiments were in addition to mice also performed using pigs or rats as hosts, the transplantation of enriched single photoreceptors, following the protocols successfully established in mice, has not been performed in other species. Nevertheless, transferring this technique is important and would allow better predictions for future application in human patients. Therefore, we transferred our protocol, using CD73 based MACS, to the rat and successfully enriched rat photoreceptors with sufficient purity. We subsequently transplanted these cells into the subretinal space of rats as well as mice and observed limited integration capacity of grafted cells. Only few transplanted rat photoreceptors were localized in the rat retina, lacking proper photoreceptor morphology. Especially regarding a perspective clinical application in humans, these data are remarkable. They imply the question, whether low integration in rat represents a general problem and might thus also be relevant for treatment in humans, or whether the rat retina forms just an exception. Thus, further detailed analysis of the cellular and molecular mechanisms underlying the integration process of transplanted photoreceptors represent an essential prerequisite for the development of a safe and efficient therapy, aiming to treat retinal degenerative diseases characterized by photoreceptor loss. / Degenerationserkrankungen der Netzhaut (Retina) sind in Industrieländern die Hauptursache für verminderte Sehfähigkeit und Blindheit. Sowohl Umweltfaktoren als auch vererbte Mutationen können Defekte wie altersbedingte Makuladegeneration (AMD) oder Retinitis pigmentosa (RP) auslösen und führen zu einem sehr variablen Krankheitsbild. Eine Gemeinsamkeit aller Formen ist das Absterben der lichtdetektierenden Fotorezeptoren (Stäbchen und/oder Zapfen). Dieses kann entweder durch direkte Schädigung, oder als Sekundäreffekt nach Degeneration der unterstützenden Zellen erfolgen. Obwohl im Moment vielversprechende Behandlungsansätze untersucht werden, ist es zurzeit nicht möglich, retinale Degenerationserkrankungen dieser Art zu heilen. Ein erfolgversprechender Ansatz könnte jedoch der Ersatz der degenerierten Zellen durch transplantierte Fotorezeptoren sein. Erste Studien demonstrierten die spezifische Anreicherung von primären Fotorezeptoren aus der Netzhaut neugeborener Mäuse und deren subretinale Transplantation in Wildtyp-Mäuse und Mausmodelle mit retinaler Degeneration. Die transplantierten Zellen integrierten in die Empfängernetzhaut und entwickelten sich in ausgereifte Fotorezeptoren und konnten unter anderem bei nachtblinden Mäusen die Sehfähigkeit bei Dunkelheit verbessern. Die Differenzierung von humanen oder murinen ES- und iPS-Zellen in vitro in vollständige Retinae und die Transplantation daraus gewonnener Fotorezeptoren in Mäuse, bilden vorläufig den Höhepunkt dieser Entwicklung. Obwohl die Fortschritte der jüngsten Vergangenheit beeindruckend sind, sollten vor der sicheren und effektiven Anwendung einer retinalen Zellersatztherapie als therapeutische Maßnahme beim Menschen noch einige wissenschaftliche Fragestellungen beantwortet werden. Studien zeigen, dass Zellpopulationen, die direkt aus der Spendernetzhaut entnommen und transplantiert wurden, auf Grund ihrer Heterogenität in geringeren Zahlen in die Empfängerretina einwandern als angereicherte Fotorezeptoren. Zusätzlich besteht bei unsortierten Zellen, die aus Stammzellpopulationen gewonnen wurden, das Risiko einer Tumorbildung. Daher haben wir die magnetisch-aktivierte Zellsortierung (MACS) zur Anreicherung junger Fotorezeptoren etabliert. Die dabei benötigten, für Fotorezeptoren spezifischen, Oberflächenproteine wurden mit Hilfe von Microarray-Analysen des Transkriptoms junger Stäbchen von Mäusen identifiziert. Dabei wurde unter anderem die 5\'-Nukleotidase (Nt5e, CD73) entdeckt, die uns die erfolgreiche Anreicherung junger Mausfotorezeptoren mit Hilfe von CD73-vermitteltem MACS erlaubte. Die Transplantation dieser angereicherten Zellpopulation in die Netzhaut von Empfängertieren resultierte in einer signifikant erhöhten Integrationsrate im Vergleich zu nicht-angereicherten retinalen Zellen. Die Überprüfung der Nutzbarkeit weiterer identifizierter Oberflächenproteine zur Zellanreicherung bzw. die Übertragung der etablierten Protokolle zur Zellsortierung und Transplantation auf Fotorezeptoren aus ES- und iPS-Zellkulturen, sollten im Fokus nachfolgender Experimente stehen. Die Integration transplantierter Zellen in die Empfängernetzhaut ist ein komplexer Prozess und von unterschiedlichen Einflussfaktoren abhängig. Durch Transplantationsexperimente in alternden Wildtyp-Mäusen und einem Mausmodell, dessen Fotorezeptorschicht keine Stäbchen und stattdessen nur Zapfen und zapfenähnlichen Fotorezeptoren aufweist, konnte gezeigt werden, dass vor allem die Aktivierung von Müllerzellen die Integrationsrate der Fotorezeptoren erhöht. Neben dieser sogenannten Gliose werden weitere Faktoren, wie die reduzierte Stabilität der äußeren Grenzmembran, die flächenmäßig größere Verteilung der transplantierten Zellen im subretinalen Raum oder die reduzierte Dichte der Zellen in der äußeren Körnerschicht, als potentielle integrationsfördernde Komponenten in Betracht gezogen. Diese bilden interessante Schwerpunkte für weitere Forschungen, um eine ausreichende Zellintegration durch Manipulation der Empfängernetzhaut, auch in der klinischen Anwendung, zu erreichen. Obwohl Transplantationsexperimente zusätzlich zur Maus auch in anderen Empfängerspezies, wie Ratten und Schweinen, durchgeführt wurden, liegen bis jetzt keine Studien vor, die die in der Maus erfolgreich etablierten Protokolle der Zellanreicherung und Transplantation von Fotorezeptor-Suspensionen in diesen Spezies reproduzierte. Der Transfer dieser Technik und eine Generalisierung der Anwendbarkeit eines Fotorezeptorersatzes durch Transplantation in verschiedenen Säugetierarten geben jedoch wichtige Hinweise für eine mögliche Translation dieser Technologie für klinische Anwendungen. Deshalb haben wir unser bereits an der Maus getestetes Protokoll auf die Ratte übertragen und erfolgreich Fotorezeptoren der Ratte mit Hilfe von CD73-vermitteltem MACS angereichert. Nach deren Transplantation in die Netzhaut von Ratten und Mäusen zeigten die Rattenfotorezeptoren aber eine stark verminderte Integrationsfähigkeit und das Fehlen einer reifen Fotorezeptormorphologie. Speziell in Hinsicht auf eine zukünftige klinische Anwendung sind diese Ergebnisse relevant, da sie die Frage aufwerfen, ob die mangelnde Integration in der Ratte ein generelles Problem darstellt und daher auch beim Menschen zu erwarten ist, oder ob sie nur eine Ausnahme im Rattenmodell bildet. Aus diesem Grund bildet die weitere Erforschung der zellulären und molekularen Mechanismen der Integration transplantierter Fotorezeptoren eine wichtige Grundlage für die Entwicklung einer sicheren und effizienten Therapie mit dem Ziel, degenerative Netzhauterkrankungen zu heilen.

The retina is the ocular tissue responsible for the detection of light. Its extensive demand for oxygen, coupled with a concomitant elevated supply, renders this tissue prone to both hypoxia and hyperoxia. In this thesis, we construct mathematical models of the retina, formulated as systems of reaction-diffusion equations, investigating its oxygen-related dynamics in healthy and diseased states. In the healthy state, we model the oxygen distribution across the human retina, examining the efficacy of the protein neuroglobin in the prevention of hypoxia. It has been suggested that neuroglobin could prevent hypoxia, either by transporting oxygen from regions where it is rich to those where it is poor, or by storing oxygen during periods of diminished supply or increased uptake. Numerical solutions demonstrate that neuroglobin may be effective in preventing or alleviating hypoxia via oxygen transport, but that its capacity for oxygen storage is essentially negligible, whilst asymptotic analysis reveals that, contrary to the prevailing assumption, neuroglobin's oxygen affinity is near optimal for oxygen transport. A further asymptotic analysis justifies the common approximation of a piecewise constant oxygen uptake across the retina, placing existing models upon a stronger theoretical foundation. In the diseased state, we explore the effect of hyperoxia upon the progression of the inherited retinal diseases, known collectively as retinitis pigmentosa. Both numerical solutions and asymptotic analyses show that this mechanism may replicate many of the patterns of retinal degeneration seen in vivo, but that others are inaccessible to it, demonstrating both the strengths and weaknesses of the oxygen toxicity hypothesis. It is shown that the wave speed of hyperoxic degeneration is negatively correlated with the local photoreceptor density, high density regions acting as a barrier to the spread of photoreceptor loss. The effects of capillary degeneration and treatment with antioxidants or trophic factors are also investigated, demonstrating that each has the potential to delay, halt or partially reverse photoreceptor loss. In addition to answering questions that are not accessible to experimental investigation, these models generate a number of experimentally testable predictions, forming the first loop in what has the potential to be a fruitful experimental/modelling cycle.

Malgré des recherches intensives portant sur l’hérédité et les aspects biologiques de la rétinite pigmentaire (RP), peu de recherches fondées ont porté sur les aspects psychologiques. Ces quelques études suggèrent que les personnes atteintes de rétinite pigmentaire s’adaptent différemment à la déficience visuelle. Le but de la présente étude était donc de vérifier si les personnes atteintes de rétinite pigmentaire s’adaptaient différemment d’un point de vue psychologique par rapport à des personnes ayant une déficience visuelle causée par une autre pathologie. Des entrevues téléphoniques incluant des personnes ayant la rétinite pigmentaire, la rétinopathie diabétique (RD) et l’albinisme ont été menées. Cinq questionnaires ont été utilisés afin d’évaluer le bien-être psychologique et de recueillir les données démographique. Les résultats de la première étude démontrent qu’il n’existe aucune différence entre les individus atteints de rétinite pigmentaire et ceux ayant d’autres pathologies visuelles d’un point de vue « bien-être psychologique ». En fait, les facteurs démographiques, la baisse de vision, les fluctuations et le type de perte de vision semblent être les seuls facteurs directement corrélés à l’adaptation et au bien-être psychologique. Dans la deuxième étude, aucune différence n’a pu être établie entre les trois types de pathologies. Ce sont plutôt, des facteurs comme la perception des capacités fonctionnelles, l’identité personnelle, l’appréhension de la perception sociale et le niveau d’indépendance qui étaient davantage reliés au bien-être psychologique associé à la déficience visuelle. Les résultats de cette étude suggèrent que les personnes atteintes de Rétinite pigmentaire ne présentent pas de différences au niveau du bien-être psychologique et de l’adaptation. Les facteurs démographiques et psychologiques sont plus importants que la pathologie elle-même. / While there is extensive research on retinitis pigmentosa (RP) focusing on biological and hereditary aspects of the disease, little research regarding psychological adjustment has been conducted. These few studies suggest that people with RP adapt differently to vision impairment. This study investigated whether those with RP adapt differently to vision loss/impairment than those with other vision disorders. Telephone interviews of those with RP, diabetic retinopathy (DR), and albinism were conducted. Demographic information was gathered and psychological wellbeing was assessed using the Visual Function-14, Centre of Epidemiology Studies Depression-10 symptoms index, Impact of Vision Impairment Profile, Brief COPE, and Adaptation to Vision Loss Scale. In Experiment I it was found that individuals with RP did not differ from those with other diagnoses on any of the measures of psychological wellbeing and adaptation. Rather, demographic factors, visual factors such as declining and fluctuating vision, and pattern of vision loss, were better correlates of adaptation to and psychological wellbeing associated with vision loss/impairment. In Experiment II there was no difference found between those with RP and other diagnoses on any of the measures. Rather, factors such as perceived visual ability, self-identity, fear of social stigma and level of dependence were more closely related to adaptation to and psychological wellbeing associated with vision loss/impairment. The results of this study suggest that individuals with RP do not differ from those with other vision disorders in their adaptation to and psychological wellbeing associated with vision loss/impairment, but that other demographic, visual and psychological factors are more important.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

La complexité de l’étude des neuropathies héréditaires provient de leur hétérogénéité clinique et génétique et de la diversité des fibres composant les nerfs périphériques. Cette complexité se reflète dans les nombreuses classifications différentes. Les neuropathies héréditaires se classifient entre autres selon leur mode de transmission et leur atteinte sensitive, autonomique et motrice. Les neuropathies héréditaires sensitives et autonomiques (NHSA) se présentent avec une perte de la sensation distale aux membres, accompagnée d’autres manifestations selon le type de NHSA. L’étude des NHSA est facilitée lorsqu’il existe des grappes de familles originaires de régions du Québec où des effets fondateurs pour des maladies récessives ont déjà été identifiés. Nous avons recruté une grande famille canadienne-française originaire de Paspébiac dans la Baie-des-Chaleurs dans laquelle nous avons identifié quatre cas atteints d’une neuropathie héréditaire sensitive avec rétinite pigmentaire et ataxie (NHSRPA). Nous avons émis l’hypothèse que nous étions en présence d’une nouvelle forme de neuropathie héréditaire sensitive récessive à effet fondateur. Afin d’identifier la position chromosomique du gène muté responsable de la NHSRPA, nous avons tout d’abord complété un criblage du génome en génotypant des marqueurs microsatellites «single tandem repeat» (STR) sur des individus clés et nous avons ensuite procédé à une analyse de liaison génétique paramétrique. Ces études nous ont permis de lier cette famille au chromosome 1 et de définir un premier intervalle candidat de 6,7 Mb. Grâce à un génotypage de marqueurs «single nucleotide polymorphism» (SNP), nous avons réduit l’intervalle candidat à 5,3 Mb au locus 1q32,2-q32,3. Cette région contient 44 gènes candidats. Une revue plus fine de la littérature a fait ressortir qu’une famille espagnole et une américaine de souche hollandaise souffrant de la même maladie avaient déjà été liées au même locus. L’origine possiblement basque de notre famille gaspésienne nous a poussé à comparer l’haplotype porteur avec celui de la famille espagnole qui, quoi que gitane, provient du pays basque espagnol. Ces travaux ont démontré le partage d’une région de 203 kb. Afin de rétrécir davantage notre intervalle candidat, nous avons comparé les haplotypes des cas entre les deux familles et nous avons identifié un dernier intervalle candidat de 60 SNP au locus 1q32,3. Cette région ne contient que quatre gènes candidats dont le plus intéressant est le gène «activating transcription factor» (ATF3). À ce jour, aucune mutation n’a été trouvée dans le gène ATF3 et les gènes FAM71A, BATF3 et NSL1. Des expériences supplémentaires sont nécessaires afin d’identifier le gène muté responsable de la NHSRPA. / Hereditary neuropathies study’s complexity comes from their clinical and genetic heterogeneity and the peripheral nerves fibers’ diversity. This complexity leads to numerous different classifications. Hereditary neuropathies are classified based on the transmission mode and the sensitive, autonomic and motor affection. Hereditary sensory and autonomic neuropathies (HSAN) present themselves with members’ distal loss and other manifestations depending on the HSAN type. HSAN study can be facilitated when there are existing family grapes originating from Quebec regions where recessive diseases founder effects have been identified. We have recruited a large French-Canadian family originating from Paspébiac in the Baie-des-Chaleurs in which we have identified four cases affected by a hereditary sensory neuropathy with retinitis pigmentosa and ataxia (HSNRPA). We have hypothesized that we were in presence of a new form of recessive hereditary sensitive neuropathy with founder effect. In order to identify the HSNRPA causing mutated gene chromosomal position, we first completed a genome wide scan by genotyping microsatellite single tandem repeat (STR) markers on informative individuals and we have then proceeded to a parametric genetic linkage analysis. These studies allowed us to link this family to chromosome 1 and define a first candidate interval of 6.7 Mb. Second to the single nucleotide polymorphism (SNP) markers genotyping, we have reduced the candidate interval at 5.3 Mb on locus 1q32.2-q32.3. This region contains 44 genes. A finer literature review made us realize that a Spanish family and an American from Dutch origin suffering from the same disease had already been linked to the same locus. The possible Gaspesian family’s Basque origins brought us to compare their carrier haplotype with the Spanish family’s, although Gypsy but coming from the Spanish Basque country. This work has demonstrated a shared region of 203 kb. In order to further refine our candidate interval, we have compared the haplotypes of the cases between the two families and we have identified a last candidate interval of 60 SNP at locus 1q32.3. This region contains only four candidate genes, the activating transcription factor (ATF3) gene being the most interesting one. Until today, no mutation has been found in the ATF3 gene and in the FAM71A, BATF3 and NSL1 genes. Further experiments will be necessary in order to identify the HSNRPA causing mutated gene.