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Nachweis von α-Synuclein und phosphoryliertem α-Synuclein im Gastrointestinaltrakt von Morbus-Parkinson-Patienten: Eine Post-mortem-StudieHarapan, Biyan Nathanael 27 September 2021 (has links)
Der Morbus Parkinson bzw. das idiopathische Parkinson-Syndrom ist pathophysiologisch durch eine progressive Degeneration der dopaminergen Neurone in der Substantia nigra und im Locus coeruleus charakterisiert. Obwohl die genaue Ursache der Erkrankung bis heute unbekannt ist, wird eine multifaktorielle Genese bei polygenetischer Prädisposition angenommen. Des Weiteren liefert die sogenannte Braak-Hypothese eine mögliche Kausalkette. Diese Hypothese besagt, dass unbekannte Erreger die Fehlfaltung von physiologischem α-Synuclein im peripheren Nervensystem, insbesondere auch im enterischen Nervensystem, initiieren können, welche sich dann retrograd durch axonalen Transport über den Nervus vagus zu dem dorsalen motorischen Kern des Nervus vagus ausbreitet. Von dort werden zusätzliche Hirnregionen, einschließlich der Substantia nigra, beeinträchtigt. Es wird vermutet, dass diese neuronale α-Synuclein-Aggregation im Gehirn ein zentraler Bestandteil der Pathogenese des Morbus Parkinson darstellt. Als histopathologisches Korrelat des Morbus Parkinson können in der Substantia nigra sogenannte Lewy-Körperchen im Zytoplasma der Nervenzellen nachgewiesen werden, die aus intrazellulären Proteinablagerungen und hauptsächlich aus unlöslichem und aggregiertem α-Synuclein bestehen.
α-Synuclein-Aggregate im Magen-Darm-Trakt wurden als potenzieller Biomarker für die Früherkennung eines Morbus Parkinson vorgeschlagen. Postuliert wurde hier, dass die α-Synuclein-Pathologie im Darm zeitlich bereits vor der Pathologie in der Substantia nigra nachweisbar ist. Studien, die diese Hypothese weiter untersuchten, führten jedoch zu divergierenden Ergebnissen.
Die Zielsetzung der vorgelegten Studie war es, Ablagerungen des α-Synucleins und des phosphorylierten α-Synucleins im menschlichen Gastrointestinaltrakt mithilfe von Immunhistochemie zu untersuchen, um eine Aussage darüber treffen zu können, ob α-Synuclein als potenzieller prädiktiver Biomarker für die Erkrankung geeignet sein könnte. Verwendet wurden hierzu einerseits Antikörper gegen natives α-Synuclein und andererseits Antikörper gegen das an Serin 129 phosphorylierte α-Synuclein, welche die pathologische, aggregierte Form des Proteins besser darstellt. Es sollte festgestellt werden, ob sich die Prävalenz der (phosphorylierten) α-Synuclein-Ablagerungen im Darm von Parkinson-Patienten und Kontrollpatienten unterscheiden. Eine mangelnde Spezifität des Antikörpers gegen das phosphorylierte α-Synuclein wurde dabei durch die Implementierung eines Kontrollexperiments mit einer proteolytischen Vorbehandlung an Gewebeproben histopathologisch bestätigter Parkinson-Patienten ausgeschlossen.
In dieser retrospektiven Studie wurde die α-Synuclein-Expression in post-mortem entnommenen Hirn-, Dünn- und Dickdarmproben von 25 neuropathologisch bestätigten Parkinson-Patienten und 20 alters- und geschlechtsspezifisch abgestimmten Kontrollpatienten untersucht. Die Immunoreaktivität wurde durch einen neuen Ansatz quantifiziert, der die detaillierte Bewertung von a-Synuclein-positiven morphologischen Strukturen des enterischen Nervensystems beinhaltet. Alle immungefärbten Schnitte wurden von zwei Untersuchern unabhängig evaluiert. Beide waren bezüglich der Gruppenzuordnung (Gewebeproben von Parkinson-Patienten oder von Kontrollpatienten) verblindet. Zu diesem Zweck wurden von jedem Präparat jeweils 10 zufällig ausgewählte, nicht selektive mikroskopische Bilder/HPF (High-power Fields) in einer 100-fachen Vergrößerung aufgenommen. Insgesamt wurden 1620 HPFs von jedem Untersucher hinsichtlich einer positiven Immunoreaktivität in den drei morphologischen Strukturen „Nervenfasern/Einzelfasern“, „Plexus“ und „Ganglienzellen“ beurteilt.
Die Auswertung der Ergebnisse ergab, dass die Immunoreaktivität von α-Synuclein und phosphoryliertem α-Synuclein bei Parkinson-Patienten im Vergleich zu den Kontrollen in fast jeder der untersuchten morphologischen Strukturen signifikant reduziert war. Bis auf die Einzelfasern im Dickdarm bei der immunhistochemischen Färbung mit Antikörper gegen nativem α-Synuclein, sah man durchgehend signifikant weniger Immunoreaktivität in den verschiedenen morphologischen Strukturen im Darmgewebe der Parkinson-Patienten verglichen zu denen der Kontrollpatienten.
Der immunhistochemische Nachweis von α-Synuclein und phosphoryliertem α-Synuclein scheint demzufolge ein häufiger und potenziell normaler Befund zu sein. Weder α-Synuclein noch phosphoryliertes α-Synuclein kann daher als molekularer Biomarker der Parkinson-Pathologie betrachtet werden. Die reduzierte intestinale Immunoreaktivität bei Parkinson-Patienten spiegelt eher die Parkinson-bedingte neuronale Degeneration wider.
Das Resultat dieser Studie legt nahe, dass der Nachweis von α-Synuclein- und phosphoryliertem α-Synuclein in intestinalen Nervenfasern, dem intestinalen Plexus und intestinalen Ganglienzellen einem Normalbefund entspricht und nicht einer Morbus Parkinson-assoziierten Pathologie. Als bis heute größte Post-mortem-Studie, welche α-Synuclein und phosphoryliertes α-Synuclein in Gewebeproben des Gastrointestinaltraktes von histopathologisch bestätigten Parkinson-Patienten untersuchte, liefert die vorliegende Studie einen wichtigen Beitrag zur Erforschung von α-Synuclein im Gastrointestinaltrakt bei Parkinson-Patienten. Der Nachweis von α-Synuclein in gastrointestinalem Gewebe erscheint für die Diagnostik, insbesondere für die klinische Prädiktion einer Parkinson-Krankheit, ungeeignet.:1. Einführung
1.1. Morbus Parkinson
1.2. Ätiologie und Epidemiologie des idiopathischen Parkinson-Syndroms
1.3. Neuropathologie des idiopathischen Parkinson-Syndroms
1.4. α-Synuclein und phosphoryliertes α-Synuclein
2. Die Bedeutung von (phosphoryliertem) α-Synuclein als molekularer Biomarker bei Morbus Parkinson: aktuelle Studienlage
3. Ableitung der Rationale für die publizierte Studie
3.1. Grundlagen
3.2. Studienziele
3.2.1. Nachweis von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten
3.2.2. Vergleich von α-Synuclein und phosphoryliertem α-Synuclein im Dünndarm und Dickdarm von Parkinson-Patienten und Kontrollpatienten
4. Methodik
4.1. Patientenkollektiv
4.2. Gewebeverarbeitung
4.3. Immunhistochemie
4.3.1. α-Synuclein
4.3.2. phosphoryliertes α-Synuclein
4.4. Beurteilung der immunhistochemischen Färbung und Quantifizierung der morphologischen Strukturen
5. Publikationsmanuskript
6. Zusammenfassung der Arbeit
7. Literaturverzeichnis
Erklärung über die eigenständige Abfassung der Arbeit
Lebenslauf
Danksagung
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Alpha-synuclein spreading pathology in Parkinson's disease: the influence of iron and the Rho-kinase inhibitor fasudilJoppe, Karina 10 March 2020 (has links)
No description available.
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The role of alpha-synuclein on transcriptional deregulation in Parkinson’s diseaseCastro, Isabel Paiva de 24 April 2018 (has links)
No description available.
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The relationship between alpha-synuclein and the proteasome and its role in Parkinson's pathologyDyllick-Brenzinger, Melanie 14 March 2013 (has links)
Morbus Parkinson (PD) ist eine Bewegungsstörung die durch intrazelluläre Einschlüsse, sogenannte Lewy Bodies, charakterisiert ist. Das synaptische Protein alpha-Synuclein (alpha-Syn) und Polyubiquitin sind Hauptkomponenten von Lewy Bodies. Das Ziel dieser Arbeit war es erstens herauszufinden ob alpha-Syn die proteolytische Aktivität des Proteasoms beeinflusst und zweitens die Auswirkungen der Proteasominhibition auf die Löslichkeit und Lokalisation von alpha-Syn zu prüfen. alpha-Syn inhibierte in vitro reversibel die 20S Proteasomenaktivität während in vivo alpha-Syn Überexpression weder in Neuroblastoma- (SH-SY5Y und SK-N-BE), HEK293 Zellen, noch in alpha-Syn transgenen Mäusen die Aktivität beeinflusste. Umgekehrt wurde der Einfluss pharmakologischer Proteasominhibition (mit MG132 und Epoxomicin) auf die Löslichkeit von alpha-Syn in SH-SY5Y Zellen untersucht. Diese Behandlung führte nicht zur Aggregation von alpha-Syn, wie sie in PD beobachtet wird, jedoch zu einer Verschiebung von mehr löslichem zu mehr membrangebundenem alpha-Syn in Zellen mit endogenem (mock) und mit WT alpha-Syn transfizierten Zellen. Diese Behandlung führte auch zur Aussparung von alpha-Syn und Polyubiquitin im Zellkern und zu cytoplasmatischen alpha-Syn-Einschlüssen ohne Polyubiquitin in einem geringen Anteil der Zellen. Die Kombination aus Proteasominhibition und Serum-Depletion, welches in den Zellen zu oxidativem Stress führen kann, verursachte die Bildung von höhermolekularem alpha-Syn im Western Blot, vergleichbar mit jenem, was in Lewy Bodies gefunden wird. Diese Daten zeigen, dass hohe Konzentrationen an alpha-Syn in vitro die Proteasomfunktion beeinflussen kann, dass dies in unseren Modellsystemen aber nicht in vivo geschieht. Außerdem führt Proteasominhibition zu strukturellen Veränderungen von alpha-Syn. Jedoch muss mehr als eine krankhafte Bedingung (z.B. oxidativer Stress und Proteasominhibition) vorhanden sein, um pathologische Veränderungen zu induzieren. / Parkinson’s disease (PD) is a movement disorder characterized by the appearance of inclusions known as Lewy bodies. The synaptic protein alpha-synuclein (alpha-syn) and polyubiquitin are major components of Lewy bodies. The objective of this study was, firstly, to evaluate whether alpha-syn inhibits proteolytic function of the proteasome and, secondly, to determine the effects of proteasome inhibition on alpha-syn solubility and localization. alpha-Syn inhibited 20S proteasome activity reversibly in vitro, while alpha-syn overexpression did not affect activity in neuroblastoma (SH-SY5Y and SK-N-BE) or HEK293 cells nor in alpha-syn transgenic mice in vivo. A reciprocal approach was used to analyze the effects of pharmacological proteasome inhibition (with MG132 and epoxomicin) on alpha-syn solubility in SH-SY5Y cells. This treatment did not lead to alpha-syn aggregation, as seen in PD. However, it induced a shift from more soluble alpha-syn toward more membrane bound alpha-syn in endogenous (mock) and WT alpha-syn transfected cells. This treatment also led to the clearing of nuclei of alpha-syn and ubiquitin, as well as to cytoplasmic alpha-syn inclusions devoid of polyubiquitin in a small percentage of the cells. The combination of proteasome inhibition with serum deprivation, which is known to produce oxidative dysfunction, caused the appearance of high molecular weight alpha-syn species, such as those found in Lewy bodies. So far these data suggest that, although not observed in our in vivo models, high concentrations of alpha-syn can interfere with proteasome function under certain conditions, while proteasome inhibition leads to structural changes in alpha-syn that may precede neurodegeneration. However, more than one condition (e.g. oxidative stress and proteasome inhibition) needs to be met to induce pathological changes.
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Neuroprotektion durch BAG1 in Modellen des idiopathischen Parkinson-Syndroms / Neuroprotection via BAG1 in models of Parkinson's diseaseBaumann, Anja Friederike 03 January 2011 (has links)
No description available.
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Protein Structure Characterization by Solid-State NMR: Structural Comparison of Mouse and Human alpha-Synuclein Fibrils, Sparse 13C Labeling Schemes, and Stereospecific Assignment of Val and Leu Prochiral Methyl GroupsLv, Guohua 28 March 2013 (has links)
No description available.
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Dopamine Induced Post-Translational Modifications of α-Synuclein and the Role of Arsenic in the Development of Parkinson's Disease and Other SynucleinopathiesCholanians, Aram B. January 2016 (has links)
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.
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Studies of α-synuclein Oligomers-with Relevance to Lewy Body DisordersFagerqvist, Therese January 2013 (has links)
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.
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Molecular and behavioural characterisation of novel α-synuclein BAC transgenic mouse models of Parkinson's diseaseJanezic, Stephanie January 2013 (has links)
Alterations in the expression levels of α-synuclein (SNCA) provide an important link between familial and sporadic forms of Parkinson’s disease (PD). Multiplications of the human wild-type SNCA locus give rise to early-onset autosomal-dominant forms of PD and elevated α-synuclein expression has been linked to an increased risk for late-onset sporadic PD. The identification of α-synuclein’s physiological and pathophysiological functions has been hindered by a lack of animal models that accurately recapitulate the key disease features. Traditional cDNA-based transgenic models fail to correctly reflect the spatiotemporal expression pattern of α-synuclein and consequently do not accurately model the disease. Bacterial artificial chromosome (BAC) technology allows transgene expression from the entire genomic locus under the control of native regulatory elements and therefore allows improved modelling of disease mechanisms and phenotypes. This thesis describes a longitudinal characterisation of the molecular and behavioural effects of overexpressing human wild-type α-synuclein in a novel BAC transgenic PD mouse model, the SNCA-OVX model. Firstly, the work investigates transgene expression and localisation, dopamine neuron loss, synaptic function and PD-related motor and non-motor phenotypes. At 3 months of age, deficits in gastrointestinal function were observed while normal levels of dopaminergic markers and neurons were maintained. At 18 months, mice displayed a 30% decrease in nigrostriatal dopamine neurons, accompanied by reduced motor coordination and function. This novel PD model, which reflects accurate transgene expression and displays progressive dopamine neuron loss accompanied by characteristic behavioural PD phenotypes, will aid the investigation of molecular disease mechanisms and the development of novel therapies. Secondly, this thesis describes the generation of Translating Ribosome Affinity Purification (TRAP) transgenic mice, which express an EGFP-tagged ribosomal protein L10a transgene under the control of the tyrosine hydroxylase promoter. Finally, double transgenic TH bacTRAP SNCA-OVX mice were generated to investigate changes in genome-wide gene expression in dopaminergic cell populations of SNCA-OVX mice to identify novel drug targets.
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Detection of alpha-synuclein conformational variants from gastro-intestinal biopsy tissue as a potential biomarker for Parkinson's diseaseRuffmann, Claudio January 2017 (has links)
Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson's disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.
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