The amyloid pathology in a mouse model of Alzheimer`s Disease and the modulation of the β-amyloid peptide secretion by membrane-anchored inhibitors

Die genaue Pathogenese der Alzheimer Demenz ist noch ungeklärt, doch wird angenommen, dass die Aggregation und Akkumulation von bestimmten Formen des Aβ-Peptides den Auslöser für die nachfolgenden pathologischen Mechanismen bildet. Als experimentelle Modelle für die charakteristische Amyloidpathologie der Alzheimer Demenz und für die Untersuchung von möglichen auf β-Amyloid (Aβ)abzielenden Therapieansätzen, werden häufig transgene Mäuse eingesetzt, die mutierte Varianten des Amyloid-Vorläufer-Proteins (APP) exprimieren. In einem ersten Teilprojekt dieser Arbeit sollte die altersabhängige Amyloidpathologie der APP23 Mäuse, die humanes APP mit der schwedischen Mutation exprimieren, biochemisch untersucht werden und mit humanen Alzheimer Proben verglichen werden. Ein Schwerpunkt sollte dabei der Analyse verschiedener Modifikationen des Aβ-Peptides gelten. In den APP23 Mäusen, wurde ein exponentieller zeitlicher Verlauf der Aβ-Akkumulation beobachtet, der im Einklang steht mit der „Keim-Theorie“ für die Aggregation von amyloidogenen Proteinen. Es zeigten sich grundlegende Unterschiede in der Zusammensetzung der amyloiden Ablagerungen zwischen Maus und Mensch. Während beim Menschen hautpsächlich Aβ42 abgelagert wurde, war die Hauptmenge bei der Maus das am C-Terminus um zwei Aminosäurereste kürzere Aβ40. Des Weiteren enthielten die humanen Plaques verschiedene N-terminal trunkierte Aβ Variationen, von denen eine Aβ-Form, die an Position 3 mit einem Pyroglutamat beginnt, die Hauptmenge darstellte. Diese konnten auch in den APP23 Mäusen nachgewiesen werden, jedoch in weitaus geringeren Mengen als beim Menschen. Dies verdeutlicht, dass die APP23 Mäuse die humane Amyloidpathologie nur unvollständig abbilden. Aufgrund der biophysikalischen Eigenschaften des hydrophoben, zur Aggregation neigenden Aβ-Peptides ergaben sich methodische Schwierigkeiten. Deshalb wurde eine Extraktionsmethode, die für die Extraktion von Aymloidproteinen bei peripheren Amyloidosen entwickelt wurde, modifiziert und erstmals auf die Gewebextraktion von Aβ angewendet. Dadurch gelang es, das Amlyoid aus dem Gewebe in Reinstwasser zu lösen und die fibrilläre Struktur im Elektronenmikroskop darzustellen. Kontaminationsproblemen bei der 2DE durch adhäsive Aβ-Peptide, wurden durch die Entwicklung eines mehrstufigen Reinigungsprotokolls, das die Behandlung mit Proteinase K einschließt, begegnet. In einem zweiten Teilprojekt sollten in primären neuronalen Zellen und in Zelllinien membranverankerte BACE Inhibitoren mit verschiedenen Pharmakophoren, unterschiedlichen Linkerlängen und Variationen von Lipid-Ankern charakterisiert werden. BACE ist die hauptsächliche β-Sekretase, die enzymatische Aktivität, die den ersten Schritt bei der Aβ-Generierung katalysiert. Der mittels Dihydrocholesterol und einem 89 Å Linker in der Membran verankerte Inhibitor war in allen Zellmodellen weitaus effektiver als der entsprechende freie Inhibitor in der Reduktion der Menge an sezernierten Aβ-Peptiden. Es zeigte sich, dass Inhibitoren, die einen Anker enthielten, der vornehmlich in „Lipid Raft“ inserierte, effektiver waren als andere, weniger oder nicht „raftophile“ Lipid-Anker. Zusätzlich konnte durch Anpassung des Abstandes des Inhibitors von der Membran, der Inhibitor weiter optimiert werden. Interessanterweise hatten die Inhibitoren unterschiedliche Effekte auf Wild-Typ APP und auf APP mit der schwedischen Doppelmutation, die sich direkt vor der β-Sekretase Schnittstelle im APP befindet. Auch entstanden in der Gegenwart von BACE-Inhibitoren aus APP Wild-Typ geringe Mengen an Aβ-Peptiden, die möglicherweise modifizierte N-Termini aufwiesen und nicht in den mit APPsw transfizierten Zellen detektiert wurden. Da N-terminal modifizierte und / oder verkürzte Aβ-Peptide einen großen Anteil in humanen Plaques ausmachen, könnte deren Entstehung pathophysiologische Bedeutung haben.The major histopathological hallmarks of Alzheimer`s Disease (AD) are neuritic plaques and neurofibrillary tangles which are composed of aggregates of the β-amyloid (Aβ) peptide and hyperphosphorylated tau protein, respectively. Based on the amyloid hypothesis the accumulation and aggregation of Aβ-peptides are the cause of the pathological events leading to neurodegeneration. Transgenic mice overexpressing mutant forms of the Amyloid-Precursor-Protein (APP) are a widely used tool to study potential therapeutic approaches. In a first project the age-dependent amyloid pathology of APP23 mice, which express human APP with the Swedish mutation, was analysed and compared with authentic human brain material from AD patients. 1-dimensional (1D) and 2D electrophoresis combined with Western-blots or mass spectrometry was applied. The mice displayed a time-dependent exponential Aβ-accumulation which is in line with the “seeded-aggregation” theory. The Aβ-patterns observed in the mouse brain extracts were strikingly different to those in the human AD brain samples. While in human brain, Aβ42 is the predominant deposited peptide, the shorter variation, Aβ40, is mainly found in brain samples of APP23 mice. Further, up to 50% of the Aβ-peptides from human brain samples were modified at the N-terminus, with Aβ-peptides starting at position 3 with a pyroglutamate (Aβ(N3pE)) being the most abundant one. These peptides could also be detected in APP23 mice, but were only a minor species. A modified version of an extraction method which was originally developed for the extraction of peripheral amyloidosis, was for the first time successfully applied for Aβ-peptides from brain samples. By applying this method, Aβ-fibrils could be prepared in pure water, without using harsh chemicals. This was confirmed by its fibrillar structure under electron microscopy. In order to prevent cross contaminations by Aβ-peptides which possess a high aggregation propensity, during 2DE, a multi-step cleaning procedure, involving the use of proteinase K was established. In a second project, membrane anchored BACE inhibitors, with variations in the pharmakophor, the spacer length or the lipid anchor were tested in primary neuronal cells and in cell lines. BACE is the main β-secretase, the enzymatic activity, catalysing the first cleavage step of APP for the generation of Aβ-peptides. The inhibitor which was anchored to the membrane by a dihydrocholesterol anchor via a 89 Å spacer was much more effective than the free inhibitor in reducing the concentration of Aβ-peptides. Inhibitors with lipid anchors which inserted preferentially in lipid rafts proved to be more efficient than inhibitors with non-raftophilic anchors. Additionally, the inhibitor could be further optimized, by adjusting the distance of the inhibitor to the membrane. Interestingly, the inhibitors had differentially effects on the generation of Aβ-peptides derived from APP wild-type (APPwt) and from APP with the Swedish mutation (APPsw). After BACE inhibition Aβ-peptides were generated from APPwt which differed at their N-termini and which could not be detected in cells expressing APPsw. As N-terminal modified Aβ-peptides are a predominant species in human amyloid plaques, their formation might have pathophysiological relevanc

Optimisation of BACE1 inhibition of tripartite structures by modification of membrane anchors, spacers and pharmacophores – development of potential agents for the treatment of Alzheimer's disease

Systematic variation of membrane anchor, spacer and pharmacophore building blocks leads to an optimisation of the inhibitory effect of tripartite structures towards BACE1-induced cleavage of the amyloid precursor protein (APP).Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Evidence for Elevated Cerebrospinal Fluid ERK1/2 Levels in Alzheimer Dementia

Cerebrospinal fluid (CSF) samples from 33 patients with Alzheimer dementia (AD), 21 patients with mild cognitive impairment who converted to AD during followup (MCI-AD), 25 patients with stable mild cognitive impairment (MCI-stable), and 16 nondemented subjects (ND) were analyzed with a chemiluminescence immunoassay to assess the levels of the mitogen-activated protein kinase ERK1/2 (extracellular signal-regulated kinase 1/2). The results were evaluated in relation to total Tau (tTau), phosphorylated Tau (pTau), and beta-amyloid 42 peptide (Aβ42). CSF-ERK1/2 was significantly increased in the AD group as compared to stable MCI patients and the ND group. Western blot analysis of a pooled cerebrospinal fluid sample revealed that both isoforms, ERK1 and ERK2, and low amounts of doubly phosphorylated ERK2 were detectable. As a predictive diagnostic AD biomarker, CSF-ERK1/2 was inferior to tTau, pTau, and Aβ42

Patient satisfaction and healthcare services in specialized multiple sclerosis centres in Germany

Background: As patients with multiple sclerosis (MS) require lifelong treatment, optimization of therapy with respect to efficacy and safety is needed to limit long-term disease progression. Patients with MS also need a range of health-related services. Satisfaction with these as well as treatment is clinically relevant because satisfied patients are more likely to adhere to therapy. The aim of this study was to determine the status of patient satisfaction and of healthcare services in 70 specialized MS centres in Germany. Methods: In 2011, patients with MS responded to a questionnaire, which solicited clinical and demographic information, as well as patients’ perceptions of their overall situation and their satisfaction with treatment. Results: Of 2791 patients surveyed, 81.9% had relapsing-remitting MS with mild disability [mean (standard deviation) Expanded Disability Status Scale score: 2.6 (1.8)]. Disease activity data were collected from 2205 patients, of whom 57.6% had remained relapse-free during the preceding 12 months. However, 38.9% had experienced one or more relapses, most of whom (67.3%) while receiving immunomodulatory treatment. About one-third of the patients indicated that they were more dissatisfied with their overall situation compared with the time before diagnosis. However, many patients (58.3%) were satisfied with their existing medication. Overall, 72.8% of patients would prefer oral to injectable treatments, assuming there was no difference in their efficacy. Conclusions: A substantial proportion of patients experienced breakthrough disease on treatment and may potentially benefit from a change of therapy. Although largely satisfied with treatment, most patients with MS would choose oral over injectable treatments

Electrocardiographic assessments and cardiac events after fingolimod first dose - a comprehensive monitoring study

Background: First dose observation for cardiac effects is required for fingolimod, but recommendations on the extent vary. This study aims to assess cardiac safety of fingolimod first dose. Individual bradyarrhythmic episodes were evaluated to assess the relevance of continuous electrocardiogram (ECG) monitoring. Methods: START is an ongoing open-label, multi-center study. At the time of analysis 3951 patients were enrolled. The primary endpoints are the incidence of bradycardia (heart rate < 45 bpm) and second-/third-degree AV blocks during treatment initiation. The relevance of Holter was assessed by matching ECG findings with the occurrence of clinical symptoms as well as by rigorous analysis of AV blocks with regard to the duration of pauses and the minimal heart rate recorded during AV block. Results: Thirty-one patients (0.8%) developed bradycardia (< 45 bpm), 62 patients (1.6%) had second-degree Mobitz I and/or 2: 1 AV blocks with a lowest reading (i.e. mean of ten consecutive beats) of 35 bpm and the longest pause lasting for 2.6 s. No Mobitz II or third-degree AV blocks were observed. Only one patient complained about mild chest discomfort and fatigue. After 1 week, there was no second-/third-degree AV block. Conclusions: Continuous Holter ECG monitoring in this large real-life cohort revealed that bradycardia and AV conduction abnormalities were rare, transient and benign. No further unexpected abnormalities were detected. The data presented here give an indication that continuous Holter ECG monitoring does not add clinically relevant value to patients' safety