Investigating Mutations to Reduce Huntingtin Aggregation by Increasing Htt-N-Terminal Stability and Weakening Interactions with PolyQ Domain

Huntington’s disease is a fatal autosomal genetic disorder characterized by an expanded glutamine-coding CAG repeat sequence in the huntingtin (Htt) exon 1 gene. The Htt protein associated with the disease misfolds into toxic oligomers and aggregate fibril structures. Competing models for the misfolding and aggregation phenomena have suggested the role of the Htt-N-terminal region and the CAG trinucleotide repeats (polyQ domain) in affecting aggregation propensities and misfolding. In particular, one model suggests a correlation between structural stability and the emergence of toxic oligomers, whereas a second model proposes that molecular interactions with the extended polyQ domain increase aggregation propensity. In this paper, we computationally explore the potential to reduce Htt aggregation by addressing the aggregation causes outlined in both models. We investigate the mutation landscape of the Htt-N-terminal region and explore amino acid residue mutations that affect its structural stability and hydrophobic interactions with the polyQ domain. Out of the millions of 3-point mutation combinations that we explored, the (L4K E12K K15E) was the most promising mutation combination that addressed aggregation causes in both models. The mutant structure exhibited extreme alpha-helical stability, low amyloidogenicity potential, a hydrophobic residue replacement, and removal of a solvent-inaccessible intermolecular side chain that assists oligomerization

Computational approaches to understanding protein aggregation in neurodegeneration

The generation of toxic non-native protein conformers has emerged as a unifying thread among disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Atomic-level detail regarding dynamical changes that facilitate protein aggregation, as well as the structural features of large-scale ordered aggregates and soluble non-native oligomers, would contribute significantly to current understanding of these complex phenomena and offer potential strategies for inhibiting formation of cytotoxic species. However, experimental limitations often preclude the acquisition of high-resolution structural and mechanistic information for aggregating systems. Computational methods, particularly those combine both all-atom and coarse-grained simulations to cover a wide range of time and length scales, have thus emerged as crucial tools for investigating protein aggregation. Here we review the current state of computational methodology for the study of protein self-assembly, with a focus on the application of these methods toward understanding of protein aggregates in human neurodegenerative disorders

Site-Specific Structure and Dynamics of Polyglutamine-Containing Amyloid Fibrils and the Caveolin Scaffolding Domain by Magic Angle Spinning Solid-State NMR

Protein assemblies and membrane associations play important roles in health and disease. Structural studies of these biological complexes are thus vital, but are often unfeasible by conventional tools in structural biology. Solid-state NMR (ssNMR) has made possible atomic-level structural studies of large, insoluble, and non-crystalline biological systems, such as amyloid fibrils and membrane proteins. Amyloid fibrils are associated with at least 20 human diseases, making structural aspects of their formation crucial to understanding their aggregation pathways. Huntington’s disease (HD) is caused by an expansion beyond a threshold of the polyglutamine (polyQ) domain in the Exon 1 of the huntingtin protein (htt). The 17-residue N-terminal segment of the Exon 1 (httNT) initiates the fibril aggregation and helps stabilize oligomers and fibrils. On the contrary, a polyPro segment C-terminal to the polyQ reduces fibril aggregation. In this thesis, magic-angle spinning (MAS) ssNMR was used to elucidate atomic-resolution structure and dynamics of the polyQ and its flanking domains in polyQ-containing amyloid-like fibrils. More than 25% of human proteins are membrane proteins. Caveolin-1 (Cav1) is a protein found associated with cholesterol-rich membranes that forms caveolae, curved cave-like invaginations in the plasma membrane, and is implicated in muscular diseases and cancers. The caveolin-scaffolding domain (CSD) of Cav1 is responsible for cholesterol-recognition and oligomerization to form the caveolae. In this thesis, MAS and static ssNMR are used to elucidate molecular structure of the CSD and its perturbation of the lipid bilayer in a cholesterol-rich lipid environment. These results allow for a more thorough understanding of the role of CSD in caveolae formation

Developing genetic therapies for polyglutamine disorders

In this thesis various genetic therapies to reduce polyglutamine-induced toxicity are discussed. Although polyglutamine disorders are caused by CAG triplet repeat expansions in different genes, they all result in gain of toxic polyglutamine protein function and subsequently neurodegeneration. The polyglutamine disorders have a monogenic cause and thus far no therapies are available to delay the age of onset or slow the disease progression. These expanded polyglutamine proteins are known to undergo proteolytic processing and this results in polyglutamine-containing protein fragments that are considered to be the main toxic entities in polyglutamine disorders. In this thesis various novel genetic therapies for polyglutamine disorders are proposed. By targeting the polyglutamine-encoding transcripts, translation of mutant polyQ protein is reduced or the polyglutamine protein is modified. This is achieved by targeting the CAG repeat directly (chapter 3), removing the motifs that are implicated in the formation of polyglutamine fragments (chapter 4), or by removal of the CAG repeat-encoding exon (chapter 5).Vereniging van Huntington Prosensa Therapeutics B.V.UBL - phd migration 201

Modelling functional and structural impact of non-synonymous single nucleotide polymorphisms of the DQA1 gene of three Nigerian goat breeds

The DQA1 gene is a member of the highly polymorphic MHC class II locus that is responsible for the differences among individuals in immune response to infectious agents. In this study, the authors performed a comprehensive computational analysis of the functional and structural impact of non-synonymous or amino acid-changing single nucleotide polymorphisms (SNPs) (nsSNPs) that are deleterious to the DQA1 protein in Nigerian goats. A 310-bp fragment of exon 2 of the DQA1 gene was amplified and sequenced in 27 unrelated animals that are representative of three major Nigerian goat breeds (nine each of West African Dwarf, Red Sokoto, and Sahel of both sexes) using genomic DNA. Forty-two nsSNPs were identified from the alignment of the deduced amino acid sequences. Based on the PANTHER, PROVEAN and PolyPhen-2 algorithms, there was consensus in identifying the mutants I26D, E114V and V115F as being deleterious. Further, differences between the native and the mutant proteins in the subsequent molecular trajectory analysis (stabilizing and flexible residue composition, total grid energy, solvation energy, coulombic energy, solvent accessibility, and protein-protein interaction properties) revealed E114V and V115F to be highly deleterious. Combined mutational analysis comparing the amutant (I26D, E114V and V115F mutations collectively) with the native protein also showed changes that could affect protein function and structure. Further wet-lab confirmatory analysis in a pathological association study involving a larger population of goats is required at the DQA1 locus. This would lay a sound foundation for breeding disease-resistant individuals in the future. Keywords: Goats, in silico, mutants, protein, tropic

The regulation and induction of clathrin-mediated endocytosis through a protein aqueous-aqueous phase separation mechanism

La morphologie des cellules et leurs interactions avec l’environnement découlent de divers procédés mécaniques qui contribuent à la richesse et à la diversité de la vie qui nous entoure. À titre d’exemple, les cellules mammifères se conforment à différentes géométries en fonction de l’architecture de leur cytosquelette tandis que les bactéries et les levures adoptent une forme circulaire par turgescence. Je présente, dans cette thèse, la découverte d’un mécanisme de morphogénèse supplémentaire, soit la déformation de surface cellulaire via l’assemblage de protéines par démixtion de phases aqueuses non miscibles et l’adhésion entre les matériaux biologiques. J’expose de façon spécifique comment ce mécanisme régule le recrutement et le mouvement dynamique des protéines qui induisent l’invagination de la membrane plasmique lors de l’endocytose clathrine-dépendante (CME). Le phénomène de démixtion des protéines dans le cytoplasme est analogue à la séparation de phase de l’huile en solution aqueuse. Il constitue un mécanisme cellulaire important et conservé, où les protéines s’agglomèrent grâce aux interactions intermoléculaires qui supplantent la tendance du système à former un mélange homogène. Plusieurs exemples de compartiments cellulaires dépourvus de membrane se forment par démixtion de phase, tels que le nucléole et les granules de traitement de l’ARN [1-6]. Ces organes ou compartiments dénommés NMO, du terme anglais « non-membranous organelles », occupent des fonctions de stockage, de traitement et de modification chimique des molécules dans la cellule. J’explore ici les questions suivantes : est-ce que les NMO occupent d’autres fonctions à caractère morphologique ? Quels signaux cellulaires régulent la démixtion de phase des protéines dans la formation des NMO ? Fondée sur la physique mécanique du contact entre les matériaux, j’émets l’hypothèse que des compartiments cellulaires nanoscopiques, formés par démixtion de phase, génèrent des forces mécaniques par adhésion interfaciale. Le travail mécanique ainsi obtenu déforme le milieu cellulaire et les surfaces membranaires adjacents au NMO nouvellement créé. Le but de mon doctorat est de comprendre comment les cellules orchestrent, dans le temps et l’espace, la formation des NMO associés au CME et comment ceux-ci génèrent des forces mécaniques. Mes travaux se concentrent sur les mécanismes de démixtion de phase et d’adhésion de contact dans le processus d’endocytose chez la levure Saccharomyces cerevisiae. Pour enquêter sur le rôle des modifications post-traductionnelles dans ces mécanismes, nous avons premièrement analysé la cinétique de phosphorylation des protéines en conditions de stress. Mes résultats démontrent que le recrutement et la fonction de certaines protéines impliquées dans le CME se régulent via des mécanismes de phosphorylation. Outre les processus de contrôle post-traductionnel, nous avons élucidé le rôle des domaines de faible complexité dans l’assemblage de plusieurs protéines associées avec le CME. De concert avec les modifications de phosphorylation, des domaines d’interaction protéine-protéine de type PrD (du terme « prion-like domains ») modulent directement le recrutement des protéines au sein des NMO associés au CME. La nature intrinsèquement désordonnée de ces PrD favorise un mécanisme d’assemblage des protéines par démixtion de phase tel que postulé. Finalement, mes travaux confirment que la formation de ces NMO spécifiques génère des forces mécaniques qui déforment la membrane plasmique et assurent le processus de CME. D’un point de vue fondamental, mes recherches permettent de mieux comprendre l’évolution d’une stratégie cellulaire pour assembler des compartiments cellulaires sans membrane et pour fixer les dimensions biologiques associées au CME. De manière plus appliquée, cette étude a le potentiel de générer des retombées importantes dans la compréhension et le traitement de maladies neurodégénératives souvent associées à une séparation de phase aberrante et à la formation d’agrégats protéiques liés à la pathologie.Evolution has resulted in distinct mechanical processes that determine the shapes of living cells and their interactions with each other and with the environment. These molecular mechanisms have contributed to the wide variety of life we observe today. For example, mammalian cells rely on a complex cytoskeleton to adapt specific shapes whereas bacteria, yeast and plants use a combination of turgor pressure and cell walls to have their characteristic bloated form. In this dissertation, I describe my discovery of an unforeseen additional mechanism of morphogenesis: protein aqueous-aqueous phase separation and adhesive contact between biomaterials as a simple and efficient ways for cells to organize internal matter and accomplish work to shape internal structures and surfaces. I specifically describe how a fundamental process of phospholipid membrane and membrane-embedded protein recycling, clathrin-mediated endocytosis (CME), is driven by this mechanism. Analogous to water and oil emulsions, proteins, and biopolymers in general, can phase separate from single to a binary aqueous phase. For proteins that de-mix from the bulk environment, the intermolecular interactions (or cohesive energy) that favors protein condensation only needs to overcome the low mixing entropy of the system and represents a conserved and energy efficient cellular strategy [2, 3, 7, 8]. So far, various examples of phase separated cellular compartments, termed non-membranous organelles (NMOs), have been discovered. These include the nucleoli, germ line P granules and P bodies, to name a few [1-6]. NMOs are involved in many conserved biological processes and can function as storage, bioreactor or signaling bodies. Cells use phase separation as a scheme to organize internal matter, but do NMOs occupy other complex functions, such as morphogenesis? What specific signals trigger protein phase separation? Based on mechanical contact theory, I proposed that hundreds of nanometer- to micron-scale phase separated bodies can deform the cellular environment, both cytoplasm and membranes, through interfacial adhesion. I studied how mechanical contact between a phase-separated protein fluid droplet and CME nucleation sites on membranes drive endocytosis in the model organism budding yeast, Saccharomyces cerevisiae. Specifically, this dissertation describes first, my investigations of post-translational modifications (phosphorylation) of several CME-mediating proteins and the implications of these modifications in regulating CME. I then describe how my efforts to understand what was distinct about the proteins that are phosphorylated led me to propose their phase separation into droplets capable of driving invagination and vesicle formation from plasma membrane. I used fluorescence microscopy, mass spectrometry and micro rheology techniques to respectively determine the spatiotemporal dynamics, phosphorylation modifications and material properties of coalesced CME-mediating proteins. I further investigated how phase separation of these proteins might generate mechanical force. I demonstrate that changes in the phosphorylation of some endocytic proteins regulates their recruitment to CME nucleation sites. We achieved reliable predictions of functional phosphosites by combining information on the conservation of the post-translational modifications with analysis of the proportion of a protein that is dynamically phosphorylated with time. The same dynamically phosphorylated proteins were enriched for low amino acid compositional complexity “prion-like domains”, which we demonstrated were essential to these proteins undergoing aqueous-aqueous phase separation on CME nucleation sites. I then demonstrate how phase separated droplet can produce mechanical work to invaginate membranes and drive CME to completion. In summary, I have discovered a fundamental molecular mechanism by which phase separated biopolymers and membranes could apply work to shape each other. This mechanism determines the natural selection of spatial scale and material properties of CME. Finally, I discuss broader implications of this dissertation to mechanistic understandings of the origins of neurodegenerative diseases, which likely involve pathological forms of protein phase separation and/or aggregation

Structure and mechanism of kynurenine 3-monooxygenase, a candidate huntington’s disease drug target

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2013Huntington’s disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. There are currently no effective therapeutics available to treat this disorder despite intense research in the field. Recently, however, the flavoenzyme kynurenine 3-monooxygenase (KMO) emerged as a promising candidate therapeutic target for HD. KMO is an FAD-dependent outer mitochondrial membrane protein which catalyses the conversion of L-kynurenine (L-KYN) to 3- hydroxykynurenine (3-HK). It has been shown that inhibition of KMO activity is protective in yeast, fruit fly, and mouse models of HD [1–5]. Additionally, it has been also implicated in the pathophysiology of several other neurological conditions such as Alzheimer’s and Parkinson’s Disease, AIDS-dementia complex, amyotrophic lateral sclerosis, depression and schizophrenia [6, 7].Despite major interest in pharmacological targeting of KMO, only a few potent inhibitors are currently available, and none are known to appreciably penetrate the bloodbrain barrier in adult animals [3, 8]. Furthermore, the molecular basis of KMO inhibition by available lead compounds has remained unknown and for that reason KMO crystal structures in complex with tight binding inhibitors would be of undeniable interest for the future design of new small molecule inhibitors that can penetrate the blood-brain barrier and could ultimately have major therapeutic value.The aim of this thesis was to produce high levels of KMO protein for structural, functional, and mechanistic studies, with the final goal of developing novel inhibitors that possess the selectivity and affinity to open up new opportunities for therapeutic intervention and inform the development of brain-penetrant KMO inhibitors.Several constructs, including both full length and truncated forms of human KMO (HsKMO), were efficiently overexpressed and purified and kinetic analysis of pure recombinant KMO showed a Km value for L-kynurenine of 22.62± 4 M which is very similar to that observed for the rat liver mitochondria preparations (16 M) [9] and human liver enzyme (13.0 ± 3.3 M) [10]. The tight-binding substrate-like inhibitor UPF 648 was found to bind recombinant KMO tightly (Ki 56.7 nM). The poor stability and low expression yield of human KMO however prevented crystallisation. We thus turned our attention to Saccharomyces cerevisiae KMO (ScKMO), which is highly related to human KMO (38 % identity and 51 % similarity). The biochemical characterisation of ScKMO was carried out by using a combination of UV/Visible absorbance spectroscopy, fluorescence spectroscopy, HPLC-based assays and stopped-flow analyses and revealed that Sc enzyme was active as a flavin-dependent monooxygenases, generated authentic 3-HK in HPLC-based assays and was inhibited by UPF 648 (Ki 74 nM) with potency similar to that with HsKMO.The structure of ScKMO was determined using selenomethionine single anomalous diffraction and subsequent crystal structures were solved to 1.85 Å resolution. We were unable to obtain a complex with the kynurenine substrate but succeeded in cocrystallising the enzyme with UPF 648, a tight-binding substrate-like inhibitor. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate kynurenine.Functional assays and targeted mutagenesis revealed that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the ScKMO– UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood–brain barrier in targeted therapies against HD and other neurological diseases.A doença de Huntington é uma doença neurodegenerativa causada por uma mutação no gene que codifica a proteína huntingtina. Apesar do grande desenvolvimento cientifico no campo das neurociências não existe actualmente nenhum tratamento capaz de tratar ou retardar o progresso desta doença. A enzima quinurenina mono oxigenase (KMO) surgiu recentemente como importante alvo terapêutico para a doença de Huntington. KMO é uma flavoproteína mitocondrial que catalisa a conversão do substrato quinorinina em 3-hidroxiquinorinina. Vários estudos mostraram que a inibição da actividade da KMO é neuroprotectora em modelos animais de Huntington, incluindo modelos de levedura, de Drosophila e de rato [1–5]. Esta enzima está igualmente relacionada com outras doenças neurológicas como por exemplo Alzheimer, Parkinson, complexo AIDS demência, esclerose lateral amiotrófica, depressão e esquizofrenia [6, 7]. Apesar do grande interesse terapêutico da KMO, até à data foram desenvolvidos poucos inibidores especificos para esta enzima, e nenhum deles comprovou ser eficiente a atravessar a barreira hemoto-encefálica em modelos animais.O conhecimento da estrutura molecular da KMO em complexo com substratos ou potenciais inibidores é desta forma fundamental para o desenvolvimento de novas moléculas capazes de penetrar a barreira hemoto-encefálica. O objectivo principal da presente tese é produzir quantidades significativas da proteína KMO para o desenvolvimentos de estudos estruturais, funcionais e mecanisticos com o objectivo futuro de desenvolver novos fármacos que possuam a selectividade e afinidade necessárias para intervenções terapêuticas.A proteina humana KMO foi eficientemente expressa e purificada, e estudos cinéticos da respectiva proteina revelaram um valor de Km para quinorinina (22.62± 4 M) bastante semelhante ao valor calculado para preparações mitocondriais de fígado de rato (16 M) [9] e humano (13.0 ± 3.3 M) [10]. Estudos de inibição enzimática com o inibidor forte da KMO (substrato análogo) UPF 648, revelaram também que este se liga com uma afinidade nanomolar (Ki 56.7 nM). Os baixos niveis de expressão proteica em conjunto com a instabilidade da proteina impossibilitaram o processo de crsitalização e futuros estudos estruturais. Consequentemente, o projecto foi direccionado para a proteína homologa KMO de Saccharomyces cerevisiae (ScKMO), que apresenta 38 % de identidade e 51 % de similaridade com a proteina humana.A caracterização bioquimica da proteina Sc foi realizada utilizando uma combinação de várias técnicas biofisicas, como espectroscopia de UV-visivel e de fluorescência, HPLC e ensaios de stopped-flow, mostrando que o mecanismo enzimático da ScKMO se assemelha a maioria das flavoproteinas, e mostrando que esta enzima é inibida pela molécula UPF 648 de modo semelhante a homóloga humana (Ki 74 nM). A estrutura da ScKMO foi inicialmente determinada usando o método da dispersão anómala a um unico comprimento de onda e estruturas subsequentes foram determinadas com uma resolução de 1.85 Å. Foi igualmente determinada a estrutura da ScKMO em complexo com a molécula UPF 648. Este inibidor liga-se na proximidade do cofactor FAD e perturba a estrutura do sitio activo, impossibilitando a ligação do substrato quinorinina.Estudos de mutagenese sitio-dirigida em aminoácidos do sitio activo e respectivos ensaios enzimáticos revelaram que a arquitectura do sitio activo é identico à proteina humana, validando a estrutura ScKMO–UPF 648 como base para o futuro desenvolvimento de moléculas baseadas na estrutura da proteina, e consequentemente a pesquisa de novos farmacos capazes de atravessar a barreira hemato-encefalica e com potencial terapêutico para a doença de Huntington e outras doenças neurológicas.Fundação para a Ciência e a Tecnologia (FCT

Stress granule recruitment and deposition of proteins of the FET family and TDP-43 in ALS and FTD

Neurodegenerative diseases such as Alzheimer´s disease, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are defined by progressive and selective loss of neurons. With increasing age the risk of developing a neurodegenerative disease exponentially rises. To date these diseases are untreatable, imposing a significant medical, social and financial burden onto our ageing society. Typical features of neurodegenerative diseases are abnormal aggregation of a disease characterizing protein and its deposition in pathological inclusions. A unifying feature in the majority of ALS cases and several subtypes of FTD is the pathological deposition of the TAR DNA-binding protein of 43kDa (TDP-43) or the Fused in Sarcoma (FUS) protein. Furthermore, stress granule (SG) marker proteins are consistently detected in FUS inclusions, suggesting that SGs might be involved in the formation of FUS inclusions. However, whether pathologic TDP-43 inclusions contain SG marker proteins is still controversially discussed. In this thesis I demonstrate that cytosolically mislocalized full-length TDP-43 is recruited into SGs, whereas C-terminal fragments of TDP-43 (TDP-CTFs) fail to localize to SGs. In accordance with these cell culture data, spinal cord inclusions in ALS and FTD patients contain full-length TDP-43 and SG marker proteins. In contrast, hippocampal inclusions are enriched for TDP-CTFs but are SG marker-negative. Thus, the protein composition of TDP-43 inclusions determines whether SG marker proteins are co-deposited in TDP-43 inclusions or not. By analyzing the prerequisites for SG recruitment of TDP-43 and FUS, I demonstrate that cytosolic mislocalization of TDP-43 and FUS is required for their localization in SGs. Additionally, I found that both proteins have the same requirements for SG recruitment, as their main RNA-binding domain and a glycine-rich domain are essential for SG localization. A detailed analysis of the protein composition of FUS inclusions in ALS and FTD cases unveiled that FUS inclusions in FTD cases contain not only FUS, but all FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) family proteins. Here, I provide evidence that this cytosolic deposition of FET proteins can be mimicked in cultured cells by inhibition of Transportin-mediated nuclear import, which causes cytosolic mislocalization of all FET proteins and recruitment of these proteins in SGs. In contrast to FTD cases, FUS inclusions in ALS cases contain only FUS, but not EWS and TAF15. In line with that, I show that ALS-associated FUS mutations result in cytosolic mislocalization of FUS that is upon subsequent cellular stress sequestered into SGs. These SGs then contain only FUS but not EWS or TAF15, demonstrating that mutant FUS is unable to co-sequester EWS or TAF15. In addition, I contributed to two studies that revealed that nuclear import defects are involved in the pathogenesis of ALS and FTD. ALS associated FUS mutations are frequently located within the proline-tyrosine nuclear localization signal (PY-NLS) of FUS and thus disrupt Transportin-mediated nuclear import and cause cytosolic mislocalization of FUS. EWS and TAF15 also contain a PY-NLS and thus are imported into the nucleus via Transportin. This interaction between Transportin and FET proteins can be modulated by arginine methylation that reduces Transportin binding. In FTD patients with FUS inclusions, this post-translational modification seems to be defective, as FUS inclusions in these cases contain hypomethylated FUS. Taken together, these data provide evidence that nuclear import defects and sequestration of FUS and TDP-43 in SGs are consecutive steps in the pathogenesis of ALS and several subtypes of FTD.Neurodegenerative Erkrankungen wie die Alzheimer-Erkrankung, die Amyotrophe Lateralsklerose (ALS) und die Frontotemporale Demenz (FTD) sind durch den progressiven und selektiven Verlust von Neuronen gekennzeichnet. Mit steigendem Alter nimmt das Risiko eine neurodegenerative Erkrankung zu entwickeln exponentiell zu. Bislang gelten diese Krankheiten als nicht behandelbar, was eine signifikante medizinische, soziale und finanzielle Belastung für unsere alternde Gesellschaft darstellt. Typische Charakteristika neurodegenerativer Erkrankungen sind die abnormale Aggregation eines Krankheits-assoziierten Proteins, sowie dessen Anhäufung in pathologischen Ablagerungen. Gemeinsames Merkmal der meisten ALS Fälle und bestimmter Untergruppen von FTD sind pathologische Ablagerungen, die hauptsächlich das TAR DNA-binding protein of 43kDa (TDP-43) oder das Fused in Sarcoma (FUS) Protein enthalten. In FUS Ablagerungen werden stets auch Markerproteine für Stress-Körnchen (engl. stress granules, SG) detektiert, was darauf schließen lässt, dass SGs an der Bildung von FUS Ablagerungen beteiligt sein könnten. Bei pathologischen TDP-43 Ablagerungen ist hingegen immer noch umstritten ob diese SG Markerproteine enthalten. In der vorliegenden Arbeit konnte ich zeigen, dass zytosolisch mislokalisiertes, unfragmentiertes TDP-43 in SGs rekrutiert wird, wohingegen C-terminale Fragmente von TDP-43 (TDP-CTFs) nicht in SGs lokalisieren. Diese Ergebnisse stimmen mit den Beobachtungen in ALS und FTD Patienten überein, wo TDP-43 Ablagerungen im Rückenmark unfragmentiertes TDP-43 und SG Markerproteine enthalten. Im Gegensatz dazu sind hippocampale Ablagerungen mit TDP-CTFs angereichert, enthalten jedoch keine SG Marker. Die Proteinzusammensetzung der TDP-43 Ablagerungen bestimmt also, ob SG Markerproteine darin abgelagert werden oder nicht. Bei der Bestimmung von Voraussetzungen für die Rekrutierung von TDP-43 und FUS in SGs konnte ich feststellen, dass eine zytosolische Umverteilung notwendig ist, damit TDP-43 und FUS in SGs sequestriert werden können. Des Weiteren konnte ich zeigen, dass beide Proteine ihre Haupt-RNA-bindende Domäne, sowie die Glycin-reiche Domäne für die Lokalisierung in SGs benötigen. Eine detaillierte Analyse der Proteinzusammensetzung von FUS Ablagerungen in ALS und FTD hat aufgedeckt, dass FUS Ablagerungen in FTD-Patienten nicht nur FUS, sondern alle FET (FUS, Ewing sarcoma protein (EWS), TATA binding protein-associated factor 15 (TAF15)) Familienproteine beinhalten. Ich konnte zeigen, dass diese cytosolische Ablagerung von FET Proteinen in Zellkultur durch eine Hemmung des Transportin-vermittelten Kerntransports nachgestellt werden kann, da dies zur zytosolischen Anhäufung aller FET Proteine und deren Rekrutierung in SGs führt. Im Gegensatz zu FTD Fällen enthalten FUS Ablagerungen in ALS nur FUS, nicht aber EWS und TAF15. In Zellkultur-Experimenten konnte ich zeigen, dass ALS-assoziierte FUS Mutationen zur zytosolischen Umverteilung von FUS führen, welches dann durch nachfolgenden zellulären Stress in SGs rekrutiert wird. Diese SGs enthalten FUS, jedoch nicht EWS oder TAF15, was beweist, dass mutiertes FUS nicht wildtypisches EWS oder TAF15 sequestrieren kann. Darüber hinaus habe ich an zwei Publikationen mitgearbeitet, in denen gezeigt wurde, dass Defekte im Kernimport an der Pathogenese von ALS und FTD beteiligt sind. ALS-assoziierte FUS Mutationen sind häufig im Prolin-Tyrosin Kernlokalisierungs-Signal (PY-NLS) lokalisiert und zerstören so den Transportin-vermittelten Kernimport und führen zur zytosolischen Misslokalisierung von FUS. EWS und TAF15 enthalten ebenfalls ein PY-NLS und werden daher über Transportin in den Kern importiert. Die Interaktion zwischen Transportin und den FET Proteinen kann durch Arginin-Methylierung moduliert werden, welche die Transportin-Bindung reduziert. In FTD Patienten mit FUS Ablagerungen scheint diese post-translationale Modifikation gestört zu sein, da FUS Ablagerungen in diesen Fällen hypomethyliertes FUS enthalten. Diese Daten liefern Beweise dafür, dass Defekte im Kernimport und die Sequestrierung von FUS und TDP-43 in SGs aufeinanderfolgende Schritte in der Pathogenese von ALS und verschiedenen Varianten von FTD sind