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Autosomal dominant optic atrophy (ADOA) is frequently caused by mutations in the optic atrophy 1 (OPA1) gene, with haploinsufficiency being the major genetic pathomechanism. Almost 30% of the OPA1-associated cases suffer from splice defects. We identified a novel OPA1 mutation, c.1065+5G>A, in patients with ADOA. In patient-derived fibroblasts, the mutation led to skipping of OPA1 exon 10, reducing the OPA1 protein expression by approximately 50%. We developed a molecular treatment to correct the splice defect in OPA1 using engineered U1 splice factors retargeted to different locations in OPA1 exon 10 or intron 10. The strongest therapeutic effect was detected when U1 binding was engineered to bind to intron 10 at position +18, a position predicted by bioinformatics to be a promising binding site. We were able to significantly silence the effect of the mutation (skipping of exon 10) and simultaneously increase the expression level of normal transcripts. Retargeting U1 to the canonical splice donor site did not lead to a detectable splice correction. This proof-of-concept study indicates for the first time the feasibility of splice mutation correction as a treatment option for ADOA. Increasing the amount of correctly spliced OPA1 transcripts may suffice to overcome the haploinsufficiency.

Autosomal dominant optic atrophy (ADOA) is frequently caused by mutations in the optic atrophy 1 (OPA1) gene, with haploinsufficiency being the major genetic pathomechanism. Almost 30% of the OPA1-associated cases suffer from splice defects. We identified a novel OPA1 mutation, c.1065+5G>A, in patients with ADOA. In patient-derived fibroblasts, the mutation led to skipping of OPA1 exon 10, reducing the OPA1 protein expression by approximately 50%. We developed a molecular treatment to correct the splice defect in OPA1 using engineered U1 splice factors retargeted to different locations in OPA1 exon 10 or intron 10. The strongest therapeutic effect was detected when U1 binding was engineered to bind to intron 10 at position +18, a position predicted by bioinformatics to be a promising binding site. We were able to significantly silence the effect of the mutation (skipping of exon 10) and simultaneously increase the expression level of normal transcripts. Retargeting U1 to the canonical splice donor site did not lead to a detectable splice correction. This proof-of-concept study indicates for the first time the feasibility of splice mutation correction as a treatment option for ADOA. Increasing the amount of correctly spliced OPA1 transcripts may suffice to overcome the haploinsufficiency., Graphical abstract, Optic atrophy is one of the most common inherited optic neuropathies, with progressive loss of vision starting in childhood. In patient-derived fibroblasts, the authors identified a mutation in OPA1 causing aberrant precursor mRNA splicing concomitant with reduced protein expression. Using engineered small nuclear RNAs, they could partially restore normal splicing.

Mutations in OPA1 are responsible of 32-89% cases of Autosomal Dominant Optic Atrophy (ADOA). OPA1 ADOA usually presents in childhood with bilateral, progressive visual loss due to retinal ganglion cells neurodegeneration, but environmental factors are supposed to influence onset and phenotype. Sixty Italian OPA1 mutations carriers (fifty-two symptomatic), belonging to thirteen families, underwent neuro-ophthalmologic evaluation. Visual acuity (n=60) and Optical Coherence Tomography (OCT) (n=12) were compared in missense mutations (OPA-M) versus haploinsufficiency-inducing mutations (OPA-H) and correlated with age. Presence of plus phenotypes was investigated. We found four known mutations, the most common being missense c.1034G>A, and a new missense mutation, c1193A>C, the latter in a 54-yrs old female with late-onset phenotype. Visual acuity, colour sensitivity, and optic disc atrophy were sensitive indicators of disease. OCT RNFL thickness was reduced in OPA1 compared to controls. OPA-M showed worst visual acuity than OPA-H, but not more frequent plus-phenotype, observed only in four OPA-H patients. In both groups, visual acuity worsened with age. Our data confirm worst vision in OPA-M, but not increased plus-phenotype. Since most patients belonged to nine families from south-eastern Sicily (a famous region for the cult of St. Lucy, patron of the blinds) local genetic and environmental factors might have accounted for the low occurrence of plus-phenotypes.

L’atrofia ottica dominante (ADOA) è una malattia mitocondriale caratterizzata da difetti visivi, che si manifestano durante l’infanzia, causati da progressiva degenerazione delle cellule gangliari della retina (RGC). ADOA è una malattia genetica associata, nella maggior parte dei casi, a mutazioni nel gene OPA1 che codifica per la GTPasi mitocondriale OPA1, appartenente alla famiglia delle dinamine, principalmente coinvolta nel processo di fusione mitocondriale e nel mantenimento del mtDNA. Finora sono state identificate più di 300 mutazioni patologiche nel gene OPA1. Circa il 50% di queste sono mutazioni missenso, localizzate nel dominio GTPasico, che si pensa agiscano come dominanti negative. Questa classe di mutazioni è associata ad una sindrome più grave nota come “ADOA-plus”. Nel lievito Saccharomyces cerevisiae MGM1 è l’ortologo del gene OPA1: nonostante i due geni abbiano domini funzionali identici le sequenze amminoacidiche sono scarsamente conservate. Questo costituisce una limitazione all’uso del lievito per lo studio e la validazione di mutazioni patologiche nel gene OPA1, infatti solo poche sostituzioni possono essere introdotte e studiate nelle corrispettive posizioni del gene di lievito. Per superare questo ostacolo è stato pertanto costruito un nuovo modello di S. cerevisiae, contenente il gene chimerico MGM1/OPA1, in grado di complementare i difetti OXPHOS del mutante mgm1Δ. Questo gene di fusione contiene una larga parte di sequenza corrispondente al gene OPA1, nella quale è stato inserito un set di nuove mutazioni trovate in pazienti affetti da ADOA e ADOA-plus. La patogenicità di queste mutazioni è stata validata sia caratterizzando i difetti fenotipici associati agli alleli mutati, sia la loro dominanza/recessività nel modello di lievito. A tutt’oggi non è stato identificato alcun trattamento farmacologico per la cura di ADOA e ADOA-plus. Per questa ragione abbiamo utilizzato il nostro modello di lievito per la ricerca di molecole che agiscono come soppressori chimici, ossia composti in grado di ripristinare i difetti fenotipici indotti da mutazioni nel gene OPA1. Attraverso uno screening fenotipico high throughput sono state testate due differenti librerie di composti chimici. Questo approccio, noto con il nome di drug discovery, ha permesso l’identificazione di 23 potenziali molecole attive. Autosomal Dominant optic atrophy (ADOA) is a mitochondrial disease characterized by an insidious onset of visual impairment in early childhood due to selective degeneration of retinal ganglion cells. ADOA is a genetically inherited disorder associated, in most cases, with mutations in the OPA1 gene encoding the mitochondrial GTPase of the dynamin family OPA1, primarily involved in mitochondrial fusion and in mtDNA maintenance. More than 300 pathogenic mutations spread throughout the entire OPA1 gene have been identified so far. About 50% of these are missense mutations, mostly clustered in the GTPase domain, envisaged to exert a severe dominant negative effect. These latter mutations are often associated with a more severe syndromic disorder named “ADOA-plus”. In the yeast Saccharomyces cerevisiae MGM1 is the orthologous of OPA1 gene. Mgm1 and OPA1 own equivalent functional domains; however, their amino acid sequences are poorly conserved. This constitutes a limitation to the use of yeast for the study and validation of OPA1 pathological mutations, since only few pathological substitutions found in patients can be introduced in the corresponding positions of the yeast gene. To overcome this obstacle a novel S. cerevisiae model has been constructed, by generating a MGM1/OPA1 chimeric gene able to complement the OXPHOS defect of the mgm1Δ mutant. This fusion gene contains a large part of OPA1 in which a set of new DOA and DOA plus associated mutations have been introduced. The pathogenicity of these mutations has been validated, both by characterizing the phenotypic defects associated with the mutant alleles, and investigating their dominance/recessivity in the yeast model. Since no therapeutic treatment is currently available for ADOA and ADOA-plus, we exploited our S. cerevisiae model for the search of molecules acting as chemical suppressors, able to rescue the mitochondrial defective phenotypes induced by pathogenic mutations in the OPA1 gene. A yeast-based phenotype high throughput screening has been set up and two different chemical libraries have been tested. This drug discovery approach led to the identification of 23 potential active molecules.

Mitochondria are double membrane organelles of prokaryotic origin that are historically associated with cellular respiration, being the main site of ATP production through oxidative phosphorylation. Apart from this function, mitochondria are also crucial participants in mediating intrinsic apoptosis as host of numerous apoptogenic factors such as cytochrome c. These are stored within the cristae of mitochondria, which are sac-like structures that are formed by the inner mitochondrial membrane that are connected to the periphery of the membrane by the narrow tubular cristae junctions. Intact, cristae keep apoptogenic within the mitochondria. The disruption of cristae junctions and cristae remodeling allows contained molecules, including cytochrome c, to gain access to cellular cytosol and to initiate a cascade resulting in cell death. Numerous mechanisms control cristae structure, among them being the large GTPase OPA1 (Optic atrophy protein 1). OPA1 forms complexes at the site of cristae junctions to maintain narrow openings and thus prevent efflux of cristae contents. During apoptosis, OPA1 oligomers are disrupted, coinciding with remodeling of the cristae, opening of their junctions and the release of apoptogenic factors. The role of OPA1 in this process was affirmed by previous data showing that the absence of OPA1, and its oligomers facilitate the induction of intrinsic apoptosis. Conversely, increased levels of OPA1 reduce cristae width and raise the threshold for inducing apoptosis. Through its control of cristae morphology, OPA1 also regulates mitochondrial metabolism by stabilizing the respiratory chain supercomplexes that reside on the cristae membranes. Apart from the aforementioned functions, OPA1 mediates mitochondrial fusion together with the outer mitochondrial membrane protein Mitofusin1. Mitochondrial fusion and fission are crucial because they allow these highly dynamic organelles to rapidly adapt to cellular demands and challenges. The machineries driving fusion and fission are located at the inner and outer mitochondrial membrane. The enzymes driving these membrane modulations are members of the family of large GTPases, which comprises dynamin-like proteins that commonly drive membrane fusion or fission in a GTP- and oligomerization- dependent manner. The physiological importance of OPA1 is underlined by the fact that mutant forms of it are associated with the development of autosomal dominant optic atrophy (ADOA) in humans, the leading cause for inherited blindness worldwide originating from the selective death of retinal ganglion cells (RGCs). Interestingly, protein domains that are associated with the OPA1 GTPase domain and GTPase effector domain are hotspots for ADOA-associated mutations. The GTPase effector domain (GED) is thought to function similarly to the classic guanine nucleotide exchange factor of small GTPases to support GTP hydrolysis. The aim of this thesis was to investigate the impact of mutations that are associated with the development of ADOA on the function of OPA1 through genetic, biochemical, and chemical biology approaches. First, we established a yeast-based assay to characterize OPA1 mutations by expressing different disease variants of OPA1 in a yeast strain lacking Mgm1, the yeast homologue of OPA1. Growth on a non-fermentable carbon source (glycerol) forces yeast cells to rely on respiration for energy production, an Mgm1-dependent process. Expression of an Mgm1-OPA1 chimeric protein restores the growth defect observed in the absence of Mgm1. After introducing ADOA-associated OPA1 mutations into the Mgm1- OPA1 fusion protein, we can assess the extent to which growth on a non-fermentable carbon source is rescued and determine their impact on the OPA1 function in this process. To complement this genetic approach, we developed an in vitro system to characterize the enzyme kinetics of wild type and mutant OPA1. To do so, we generated a protocol for the purification of recombinant OPA1 (rOPA1) using an inducible bacterial expression system and affinity based purification. rOPA1 was used to elucidate the enzymatic kinetics of OPA1, which shows positive cooperative hydrolysis behavior. Further biochemical characterization suggests that the cooperative behavior is due to OPA1 oligomerization. We used both systems to analyze the impact of mutations that are associated with ADOA classic and ADOA plus, a more severe multi-systemic form of the disease, in the GTPase domain and GED. Combining the data of both approaches allowed us to correlate mutations associated with ADOA classic with a loss of function phenotype and the ones associated with ADOA plus with a dominant negative effect on the wild type protein in vivo. Based on the kinetics data we obtained for the individual mutant proteins this effect is likely due to changes in the hydrolysis capacity and the cooperative kinetics of OPA1. While ADOA classic mutations lose their cooperative GTPase activity which correlates with a lower affinity for GTP, ADOA plus mutations maintain their cooperativity but have a severely altered catalytic turnover of GTP. Hence we hypothesize that retaining the cooperative behavior allows ADOA plus OPA1 variants to oligomerize with the wild type protein and thus impact negatively on their function. ADOA classic mutants however fail to cooperate with the wild type proteins. Similarly, co-expression of ADOA plus and wild type OPA1 in our yeast system revealed a dominant negative effect in respiration dependent growth and mitochondrial morphology while the ADOA classic mutations have no impact. Together, these data suggests that ADOA-associated mutations can be biochemically grouped into ADOA classic and ADOA plus mutations based on their impact on the cooperative GTPase activity of OPA1. We also sought to characterize the role of OPA1 in apoptosis. Given thatOPA1 has an anti-apoptotic role by controlling the release of cytochrome c from the intracristal space, we hypothesized that it inhibiting this function may yield a pharmacological drug to induce apoptosis in cancer cells. We made use of the rOPA1 to screen a library of small compounds. For that a semi-automated high-throughput screening (HTS) was set up in which the hydrolysis of GTP by rOPA1 served as readout to measure the inhibition of the GTPase activity of OPA1. OPA1 activity was determined using a colorimetric assay that allows the measurement of free organic phosphate that is released during the hydrolysis of GTP to GDP. Given that there is no existing inhibitor of OPA1 nor published model or structure of OPA1 to use in an in silico screen, we selected our HTS library based on diversity of compounds. Our screen revealed 8 compounds that inhibit OPA1 GTPase activity in two repetitions of the screen. The compounds’ effects were further confirmed in a dose-dependent response experiment, and also provided us with maximal inhibition (Imax), half maximal inhibition (IC50), and Hill slope values for each. Based on their potency and biochemical effect on OPA1 we grouped them into potent and moderate inhibitors. Potent compounds inhibit OPA1 activity by more than 50% while moderate ones inhibit up to 50% of the activity. How the inhibitors impact on the different species of OPA1 will be focus of future experiments that may serve as scaffold for the identification of an OPA1 inhibitor in vivo. In conclusion, the data presented in this doctoral thesis contributes to the field of ADOA research by correlating the mutations of ADOA classic and ADOA plus to functionally distinguished alteration on OPA1 GTPase function. Moreover, we developed a robust assay that can be used to characterize OPA1 disease mutation and phenotypes. Lastly, we set up and implemented a high-throughput screening that identified novel inhibitors of OPA1 GTPase activity in vitro.

Optic atrophy type 1 (OPA1) gene mutation causes autosomal dominant optic atrophy (ADOA, MIM #165500). Prevalence of ADOA ranges from 1:50,000 in most populations to 1:12,000 in Denmark. Seventy members of nine families were analysed for the presence of OPA1 gene mutations by polymerase chain reaction (PCR) and direct sequencing. We identified three OPA1 gene mutations in 48 patients with variable signs of optic atrophy. Two mutations, c.784-21_784-22insAluYb8 and c.876_878delTGT, were found in two different families. The third mutation, c.869G>A, was found in 28 patients from seven families. The haplotype analysis data suggested that the c.869G>A mutation is a founder mutation. Our main result suggests a higher ADOA prevalence in south-eastern Sicily than previously found in Denmark. This is because of not only the founder effect but also to the presence of three different mutations in the geographical area of the study. Our hypothesis is that a combination of social pressure because of blindness and migration factors is involved. In fact, in Siracusa, a provincial capital in south-eastern Sicily, St. Lucy, the patron saint of the blind was born and died.

Title Table of contents Summary i Zusammenfassung ii 1\. Introduction 1 2\. Materials 17 3 Methods 23 4\. Results 39 5\. Discussion 63 6\. References 75 7\. Appendix 83, The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for a eukaryotic cell. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. The entire mitochondrial population is in constant flux, driven by continual fusion and division of mitochondria. Defects in mitochondrial dynamics can cause deficits in mitochondrial respiration, morphology and motility leading to apoptosis under extreme conditions. Mutations in OPA1, a nuclear encoded mitochondrial protein, involved in the mitochondrial fusion mechanism cause autosomal dominant optic atrophy. OPA1 is expressed as eight mRNA splice variants in human cells generated by alternative splicing of exons 4, 4b and 5b (Delettre et al., 2001). This study was undertaken to characterize the mouse OPA1 GTPase. Four mRNA splice variants 1, 5, 7 and 8, were found in mouse tissues generated by alternative splicing of exons 4b and 5b. In contrast to an earlier report, alternative splicing of exon 4 was not confirmed. While the overall level of OPA1 gene transcription seems to be constant throughout tissues, the individual expression level of the four splice variants in the mouse brain differs when compared to other tissues, indicating the existence of a post-transcriptional regulatory mechanism. Particularly, splice form 1 was predominantly expressed in the mouse brain. To study the expression of OPA1 protein isoforms, monoclonal antibodies were generated that identified six protein isoforms on western blots. Mass spectrometry and N-terminal microsequencing revealed the two longest forms to be OPA1 isoforms 1 and 7 which are cleaved by mitochondrial processing peptidase (MPP) during protein import into mitochondria. Furthermore, post-MPP processing by other unknown proteases in regions corresponding to exons 5, 5b and 6 generated three different short forms of OPA1. Only the long forms were tightly embedded into the mitochondrial inner membrane, whereas the short forms were extracted easily from the membranes presumably due to the loss of the transmembrane domain after post-MPP processing. The expression levels of OPA1 protein isoforms varied in a tissue dependent manner, though all the tissues contained identical set of isoforms. OPA1 isoform 1 was predominant in mouse nervous tissues. Gel filtration experiments showed that the longest of the three short forms distinctly formed a dimer of 184-kDa while all other OPA1 protein isoforms were part of a 285-kDa complex. The two coiled-coil domains present in the OPA1 protein isoforms showed a high affinity to self- associate (but not to hetero-associate) that likely mediated the complex formation. Yeast Two-Hybrid screens, co-immunoprecipitation and size exclusion chromatography experiments failed to identify proteins interacting with OPA1., Mitochondrien sind essentielle, cytoplasmatische Organelle, die den Hauptanteil der Energie generieren, den eine eukaryontischen Zelle benötigt. Die Struktur und Funktion der Mitochondrien wird durch mitochondrial- und kernkodierte Proteine aufrechterhalten. Die gesamte Mitochondrienpopulation einer Zelle befindet sichin einem konstanten Flux, der durch permanente Fusions- und Teilungsvorgänge dieser Organellen in Gang gehalten wird. Defekte in dieser Dynamik können Defizite in der Respiration, Morphologie und Beweglichkeit der Mitochondrien verursachen, die im Extremfall sogar zur Apoptose führen. Mutationen in OPA1, einem kernkodierten mitochondrialen Protein, welches in mitochondriale Fusionsprozesse involviert ist, können der Grund zur Ausprägung einer autosomal dominant erblichen Optikusatrophie sein. Die Expression des OPA1 Gens resultiert in 8 verschiedenen Spleissvarianten in humanen Zellen, die durch alternatives Spleissing der Exons 4, 4b und 5b entstehen (Delettre et al., 2001). Die vorliegende Studie befasst sich mit der Charakterisierung der OPA1 GTPase aus der Maus. Vier mRNA Spleissvarianten (1, 5, 7 und 8) wurden in Mausgewebe nachgewiesen, die durch alternatives Spleissing der Exons 4b und 5b entstehen. Im Gegensatz zu der oben genannten Veröffentlichung, wurde alternatives Spleissing von Exon 4 nicht bestätigt. Obwohl der generelle Level an OPA1 Gentranskription in vielen Geweben konstant zu sein scheint, unterscheidet sich das individuelle Expressionsniveau der 4 verschiedenen Spleissvarianten im Maushirn von den Niveaus in anderen Geweben. Dieser Befund deutet auf die Existenz eines post-transkriptionellen Regulationsmecha-nismusses hin. Eine besonders starke Expression der Spleissform 1 im Maushirn wurde festgestellt. Um die Expression von OPA1 Proteinisoformen studieren zu können, wurden monoklonale Antikörper hergestellt, die 6 verschiedene Proteinisoformen im western blot identifizierten. Massenspektroskopische Analysen und N-terminale Mikrosequenzie-rung zeigten, das es sich bei den beiden grössten OPA1 Formen um die OPA1 Isoformen 1 und 7 handelt, die durch die Mitochondriale Prozessierungspeptidase (MPP) während des mitochondrialen Proteinimports gespalten werden. Post-MPP Prozessierungsvor-gänge, die durch bisher unbekannte Proteasen in den Regionen ausgeführt werden, die von den Exons 5, 5b und 6 kodiert werden, lassen drei verschiedene kurze Formen von OPA1 entstehen. Diese kurzen Formen können leicht von mitochondrialen Membranen extrahiert werden, da sie ihre Transmembrandomäne nach der Post-MPP- Prozessierung verloren haben. Nur die langen OPA1 Polypeptide sind stabil in der inneren Mitochondrienmembran verankert. Obwohl die Expressionsnivieaus der OPA1 Proteinisoformen gewebespezifisch variieren, enthalten alle ii Zusammenfassung Gewebe das gleiche Set an Isoformen. Isoform1 war auch auf Proteinebene im Nervengewebe der Maus am stärksten exprimiert. Gelfiltrationsexperimente zeigten, dass die grösste der drei kurzen Formen einen Dimer von 184-kDa Grösse bildet, während alle anderen OPA1 Isoformen Teil eines 285-kDa Komplexes waren. Zwei Coiled-coil-Domänen des OPA1 Proteins zeigten eine hohe Affinität für Homo-Assoziation, gingen aber keine Hetero- Interaktionen ein. Sie tragen sehr wahrscheinlich zu der Komplexformation der OPA1 Moleküle bei.