IST Austria Thesis

The aim of this thesis was the development of new strategies for optical and optogenetic control of proliferative and pro-survival signaling, and characterizing them from the molecular mechanism up to cellular effects. These new light-based methods have unique features, such as red light as an activator, or the avoidance of gene delivery, which enable to overcome current limitations, such as light delivery to target tissues and feasibility as therapeutic approach. A special focus was placed on implementing these new light-based approaches in pancreatic β-cells, as β-cells are the key players in diabetes and especially their loss in number negatively affects disease progression. Currently no treatment options are available to compensate the lack of functional β-cells in diabetic patients. In a first approach, red-light-activated growth factor receptors, in particular receptor tyrosine kinases were engineered and characterized. Receptor activation with light allows spatio-temporal control compared to ligand-based activation, and especially red light exhibits deeper tissue penetration than other wavelengths of the visible spectrum. Red-light-activated receptor tyrosine kinases robustly activated major growth factor related signaling pathways with a high temporal resolution. Moreover, the remote activation of the proliferative MAPK/Erk pathway by red-light-activated receptor tyrosine kinases in a pancreatic β-cell line was also achieved, through one centimeter thick mouse tissue. Although red-light-activated receptor tyrosine kinases are particularly attractive for applications in animal models due to the deep tissue penetration of red light, a drawback, especially with regard to translation into humans, is the requirement of gene therapy. In a second approach an endogenous light-sensitive mechanism was identified and its potential to promote proliferative and pro-survival signals was explored, towards light-based tissue regeneration without the need for gene transfer. Blue-green light illumination was found to be sufficient for the activation of proliferation and survival promoting signaling pathways in primary pancreatic murine and human islets. Blue-green light also led to an increase in proliferation of primary islet cells, an effect which was shown to be mostly β-cell specific in human islets. Moreover, it was demonstrated that this approach of pancreatic β-cell expansion did not have any negative effect on the β-cell function, in particular on their insulin secretion capacity. In contrast, a trend for enhanced insulin secretion under high glucose conditions after illumination was detected. In order to unravel the detailed characteristics of this endogenous light-sensitive mechanism, the precise light requirements were determined. In addition, the expression of light sensing proteins, OPN3 and rhodopsin, was detected. The observed effects were found to be independent of handling effects such as temperature differences and cytochrome c oxidase dependent ATP increase, but they were found to be enhanced through the knockout of OPN3. The exact mechanism of how islets cells sense light and the identity of the photoreceptor remains unknown. Summarized two new light-based systems with unique features were established that enable the activation of proliferative and pro-survival signaling pathways. While red-light-activated receptor tyrosine kinases open a new avenue for optogenetics research, by allowing non-invasive control of signaling in vivo, the identified endogenous light-sensitive mechanism has the potential to be the basis of a gene therapy-free therapeutical approach for light-based β-cell expansion

Lack of netrin-4 modulates pathologic neovascularization in the eye

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4−/− mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4−/− mice. In OIR, Ntn-4−/− mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4−/− mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4−/− mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization

Lack of netrin-4 modulates pathologic neovascularization in the eye

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4−/− mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4−/− mice. In OIR, Ntn-4−/− mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4−/− mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4−/− mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization

The optogenetic promise for oncology: Episode I

As light-based control of fundamental signaling pathways is becoming a reality, the field of optogenetics is rapidly moving beyond neuroscience. We have recently developed receptor tyrosine kinases that are activated by light and control cell proliferation, epithelial–mesenchymal transition, and angiogenic sprouting—cell behaviors central to cancer progression

A phytochrome sensory domain permits receptor activation by red light

Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases—the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light. Go deep with red: The sensory domain (S) of the cyanobacterial phytochrome 1 (CPH1) was repurposed to induce the homodimerization of proteins in living cells by red light. By using this domain, light-activated protein kinases were engineered that can be activated orthogonally from many fluorescent proteins and through mammalian tissue. Pr/Pfr=red-/far-red-absorbing state of CPH1

Light-assisted small-molecule screening against protein kinases

High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes

Grünlicht-induzierte Rezeptorinaktivierung durch Cobalamin-bindende Domänen

Optogenetik und Photopharmakologie ermöglichen präzise räumliche und zeitliche Kontrolle von Proteinwechselwirkung und -funktion in Zellen und Tieren. Optogenetische Methoden, die auf grünes Licht ansprechen und zum Trennen von Proteinkomplexen geeignet sind, sind nichtweitläufig verfügbar, würden jedoch mehrfarbige Experimente zur Beantwortung von biologischen Fragestellungen ermöglichen. Hier demonstrieren wir die Verwendung von Cobalamin(Vitamin B12)-bindenden Domänen von bakteriellen CarH-Transkriptionsfaktoren zur Grünlicht-induzierten Dissoziation von Rezeptoren. Fusioniert mit dem Fibroblasten-W achstumsfaktor-Rezeptor 1 führten diese im Dunkeln in kultivierten Zellen zu Signalaktivität durch Oligomerisierung, welche durch Beleuchten umgehend aufgehoben wurde. In Zebrafischembryonen, die einen derartigen Rezeptor exprimieren, ermöglichte grünes Licht die Kontrolle über abnormale Signalaktivität während der Embryonalentwicklung

Optogenetic delivery of trophic signals in a genetic model of Parkinson's disease

Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair

Big bang et akirine, deux protéines impliquées dans la réponse inflammatoire de Drosophila melanogaster

La flore commensale endogène présente dans l intestin de tous les métazoaires doit être préservée afin d assurer le maintien de l homéostasie intestinale. La réponse immunitaire innée est donc finement régulée dans l intestin afin de permettre l élimination des microorganismes pathogènes tout en tolérant cette flore endogène. Dans ce contexte, la première partie de mon projet de thèse a porté sur l étude de la protéine Big bBang (BBG) localisée au niveau de l épithélium intestinal chez la drosophile adulte. J ai démontré que les mouches mutantes pour le gène bbg présentent un défaut de structure des jonctions septées (qui assurent l étanchéité de l épithélium intestinal) ainsi qu une activation constitutive de la réponse immunitaire innée dans l intestin, et qu elles meurent précocement. L étiologie des maladies inflammatoires chroniques de l intestin chez les mammifères, comme la maladie de Crohn, suggère que l inflammation constitutive du tube digestif est liée à un défaut des jonctions épithéliales de l intestin et à une réaction anormale contre la flore endogène, l administration d antibiotiques permettant de réduire l intensité de la réponse inflammatoire. D une manière similaire, l élimination de la flore intestinale endogène par l administration d antibiotiques permet de prévenir l inflammation constitutive de l intestin chez les mouches mutantes pour le gène bbg et leur mort précoce. Mes résultats suggèrent ainsi que BBG est une nouvelle protéine impliquée dans le maintien de la tolérance immunitaire envers la flore commensale.La seconde partie de mon projet de thèse a porté sur l étude de l Akirine, une protéine nucléaire impliquée dans la régulation différentielle de l expression des gènes cibles des facteurs de transcription NF- kB au cours de la réponse inflammatoire chez les insectes et chez les mammifères. Chez la drosophile, l Akirine agit dans la voie IMD. Afin de comprendre les mécanismes d action de l Akirine, j ai identifié 115 de ses partenaires protéiques potentiels par le biais de deux cribles protéomiques. J ai testé l implication de 47 d entre eux dans la voie IMD in vitro, et ainsi mis en évidence 5 activateurs et 1 répresseur de cette voie. Les fonctions de ces 6 protéines suggèrent que l Akirine pourrait contrôler la transcription des gènes cibles des facteurs de transcription NF- B en participant au remodelage de la chromatine avec le complexe Brahma.The endogenous flora present in the gut of all metazoans has to be preserved in order to maintain gut homeostasis. The gut innate immune response is then tightly regulated in order to eliminate pathogenic microorganisms and allow tolerance to this flora. In this context, the first part of my PhD project consisted in the study of the Big bBang (BBG) protein localized at the level of the Drosophila gut epithelium. My results showed that bbg null mutant flies have altered septate junctions (that are responsible for the maintenance of gut epithelium integrity) as well as constitutive activation of the gut innate immune response, and die prematurely. The etiology of inflammatory bowel diseases in mammals, such as Crohn s disease, suggests that gut chronic inflammation is linked to defects in gut epithelial junctions and to inappropriate reactions against the commensal flora as antibiotic treatment reduces inflammation. Similarly, the elimination of gut commensal flora by treatment with antibiotics can prevent constitutive inflammation of the gut of bbg mutant flies and rescues their impaired lifespan. Collectively my results suggest that BBG is a novel protein implicated in the maintenance of immune tolerance towards the commensal flora.The second part of my PhD project consisted in the study of Akirin, a nuclear protein implicated in the differential regulation of NF- B target genes expression in insects and mammals. In Drosophila, Akirin is implicated in the IMD pathway. In order to understand the function of Akirin, I identified 115 of its potential partners by using two proteomic screens. I tested the implication of 47 of them in the IMD pathway in vitro, and showed that five are activators and one is a repressor of this pathway. The function of these six proteins suggests that Akirin could control NF- B target genes expression by participating in the Brahma chromatin-remodeling complex.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF