No description available.

No description available.

No description available.

Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1-42) and pGlu-Aβ(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1-42) and pGlu-Aβ(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

In der vorliegenden Arbeit wird ein neuer Ansatz vorgestellt, um Lipiddomänen, die Bindungsorte peripherer und integraler Membranproteine darstellen können, zu charakterisieren. Insbesondere wurde die Analyse der Fluoreszenzlebenszeiten von NBD-markierten Lipidanaloga benutzt, um Lipiddomänen in Giant unilamellar vesicles (GUV) und darauf aufbauend, in der Plasmamembran von Säugerzellen zu untersuchen. Das typische Zeitfenster von Fluoreszenzlebenszeiten im Bereich von Nanosekunden ermöglicht es, auch sehr kurzlebige Lipiddomänen nachzuweisen. Mit Hilfe des Fluorescence lifetime imaging (FLIM) wurden für die liquid disordered (ld) und liquid ordered (lo) Domänen in GUV jeweils spezifische Werte für das Abklingen der Fluoreszenz gemessen. Sogar die Existenz von submikroskopischen Domänen in GUV konnte nachgewiesen werden. Die Fluoreszenzlebenszeit des Lipidanalogs C6-NBD-PC zeigte in der Plasmamembran von Säugerzellen eine breite Verteilung. Dies legt in Übereinstimmung mit FLIM-Experimenten an aus der Plasmamembran von HeLa-Zellen gewonnenen Giant vesicles nahe, dass in der Plasmamembran von Zellen eine Vielzahl verschiedener submikroskopischer Lipiddomänen existiert. Darauf aufbauend wurde die Fluoreszenzmikroskopie an GUV angewendet, um die Bindung von fluoreszenzmarkiertem alpha-Synuclein an mittels FLIM charakterisierte Lipiddomänen zu untersuchen. Die Experimente zeigten, dass das Protein mit hoher Affinität an negativ geladene Phospholipide unter der Vorraussetzung bindet, dass diese sich in ld Domänen befinden. Im Gegensatz dazu erfolgt keine Bindung wenn diese Lipide in lo Domänen lokalisiert sind. Im Vergleich zum wildtypischen alpha-Synuclein zeigte die Variante A30P eine geringere Affinität zur Membran, während die E46K-Variante eine stärkere Bindung zeigte. Dies deutet darauf hin, dass bei den erblichen Formen des Morbus Parkinson eine veränderte Assoziation des alpha-Synucleins mit der Membran eine Rolle spielen kann. / In the present study a novel approach to characterize lipid domains, which may provide binding sites for peripheral or integral membrane proteins, is demonstrated. In particular, analysis of fluorescence lifetimes of NBD-labeled lipid analogues was used to study lipid domains in Giant unilamellar vesicles (GUV) and – based on the GUV results – in the plasma membrane of mammalian cells. As fluorescence decays in a few nanoseconds it is possible to to detect also very short-lived lipid domains. Fluorescence Lifetime Imaging (FLIM) revealed that the fluorescence decay of NBD-lipid analogues showed domain dependent decay times in the liquid disordered (ld) and the liquid ordered (lo) phase of GUV. Even the existence of submicroscopic domains in lipid membranes could be detected by FLIM. A broad distribution of the fluorescence lifetime was found for C6-NBD-PC inserted in the plasma membrane of mammalian cells. In agreement with FLIM studies on lipid domain forming Giant vesicles derived from the plasma membrane of HeLa-cells this may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact mammalian cells. Based on that, fluorescence microscopy was used on GUV to study the binding of fluorescently labeled alpha-synuclein at lipid domains previously characterized by FLIM. The experiments suggested that alpha-synuclein binds with high affinity to negatively charged phospholipids, when they are embedded in a ld as opposed to a lo environment. When compared with wildtype alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that an altered association of alpha-synuclein with membranes may play a role in the inherited forms of Parkinson’s disease.

Synucleinopathies are a family of neurodegenerative diseases, with the distinctive pathological feature of Lewy bodies, which include Parkinson’s disease. Lewy bodies are intracellular inclusions filled with α-synuclein, a small neuronal protein with prion-like properties. The main function of α-synuclein is not fully understood, however, it plays a major role in disease progression. Dopamine interactions with α-synuclein have also been implicated in the progression of Parkinson’s disease. Dopamine crosslinks α-synuclein and causes generation of toxic oligomeric species of the protein. Little is known about dopamine-α-synuclein adducts, and one section of the current dissertation focuses on dopamine, levodopa, and α-synuclein interactions. Studies detailed herein demonstrated that lysine residues on α-synuclein have an essential role in the dopamine-induced oligomer formation. Evidence is also presented showing that removal of one of the reactive sites on dopamine by N-acetylcysteine and/or glutathione inhibits dopamine-induced oligomer formation, although the dopamine thiol-conjugates still bind to α-synuclein. In contrast, thiol-conjugates of the dopamine precursor levodopa, significantly increase α-synuclein oligomer formation. The data demonstrate the importance of the scavenging of dopamine and levodopa quinones by N-acetylcysteine and glutathione, and the subsequent changes in the interaction with α-synuclein and its oligomeric states. Environmental factors are key players in the development of synucleinopathies. Although arsenic pesticide exposure has been linked to elevated risk of Parkinson’s disease, there is a paucity of information on arsenic-induced pathological changes, which may be attributed to the onset of neurodegenerative processes. SH-SY5Y cells exposed to environmentally relevant levels of arsenic for 72 hours, develop α-synuclein oligomers and exhibitaugmented expression of stress markers. Thus, there is an increase in autophagy markers and other stress markers, including the accumulation and co-localization of LC3, major autophagy marker, and α-synuclein. Animals transiently exposed to arsenic through drinking water for 2 or 5 weeks, exhibited pathological features resembling synucleinopathies. Although animals were exposed at two-months of age and remained exposure free up to geriatric age (18 months), they still exhibited accumulation of α-synuclein and elevations in autophagy markers. The results demonstrated how even a short period of exposure to a toxicant can have detrimental neurological effects, which may contribute to the development of neurodegenerative disease years after exposure.

The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.       The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology. Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model. By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice. To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text] / Savitvarkės biomolekulės, gebančios formuoti beta-klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta-klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą]

Savitvarkės biomolekulės, gebančios formuoti beta klosčių struktūras, gali būti pritaikomos nanomedžiagų su naujomis elektrinėmis, optinėmis, katalitinėmis ir/ar mechaninėmis savybėmis, kūrimui. Šiame darbe buvo siekiama kurti nanodarinius, grįstus amiloidines fibriles formuojančiais baltymais (Abeta40 peptidas, a-sinukleinas (a-Syn), kumelės pieno lizocimas (EL)), kurie būtų pagrindas nano dydžio funkcinių sistemų gamybai. Šiam tikslui buvo sukonstruoti mutantiniai ir hibridiniai baltymai bei tiriamos jų fibrilinių struktūrų savybės. Sukurti Abeta40 ir a-Syn cisteino mutantai, kurie gali būti modifikuojami per cisteino tiolinę grupę. Pirmą kartą buvo pademonstruotas a-Syncys141 baltymo fibrilių modifikavimas biotinu ir aukso nanodalelėmis su neutravidinu. Sukonstruoti hibridiniai baltymai, kurie sudaryti iš Abeta40 ar a-Syn bei GDH, streptavidino ir hidrofobino. Buvo tikimasi, kad tokie baltymai formuos fibrilines struktūras, o funkciniai baltymai bus aktyvūs. Esant atitinkamoms sąlygoms, šie baltymai agregavo suformuodami skirtingos morfologijos beta klostines struktūras. Streptavidino ir Abeta40 hibridinis baltymas formavo fibrilinę struktūrą – tinklą, o streptavidinas buvo aktyvus. Šiame darbe pirmą kartą aprašoma rekombinantinio EL gamyba E. coli ląstelėse. Aktyvus EL gali formuoti panašias į natyvaus EL fibrilines struktūras. Sukonstruoti nauji hibridiniai ir mutantiniai baltymai, gebantys formuoti amiloidines fibriles, yra geras pagrindas, kuriant funkcionalizuotus... [toliau žr. visą tekstą] / Self-assembly of biomolecules into beta-sheet structures can be applied in the creation of nano-materials with novel electrical, optical, catalytical, or/and mechanical characteristics. This work was directed towards the construction of nano-derivatives based on amyloid fibrils forming proteins (Abeta40 peptide, a-Synuclein (a-Syn), equine lysozyme (EL)). Such nanostructures can be used to produce nanoscale functional systems. Herein, different mutant and hybrid proteins, which were able to form fibrillar structures, were constructed and the properties of fibrils were investigated. Designed cysteine mutants of Abeta40 and a-Syn can be modified through thiol group of cysteine. Herein, for the first time, it was demonstrated that a-Syncys141 fibrils could be modified with biotin and gold nanoparticles with neutravidin molecules. Hybrid proteins of Abeta40 or a-Syn and other non-amyloid proteins were designed on purpose to obtain fibrils with active functional non-amyloid proteins. Under appropriate conditions, these proteins aggregated into beta-sheet structures. Hybrid protein of streptavidin and Abeta40 formed a net-like fibrillar structure, and streptavidin was active. For the first time it was described the production of recombinant EL in E. coli. Moreover, active EL can form fibrils, which are similar to the fibrils formed by native EL. Constructed novel hybrids and mutants that are able to form amyloid fibrils, can be applied for the creation of functionalized nanodevices... [to full text]

No description available.