In this study, we report a detailed analysis of the different variants of amyloid-␤ (A␤) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one-and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the A␤ peptides A␤(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified A␤ (A␤(N3pE)) and endogenous murine A␤ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of A␤ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of A␤ peptides, such as A␤(N3pE), A␤(1-42), and N-terminally truncated A␤(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.The major histopathological hallmarks of Alzheimer disease (AD) 2 are neuritic plaques and neurofibrillary tangles that are composed of aggregates of the amyloid-␤ (A␤) peptide and hyperphosphorylated Tau protein, respectively (1-5). A␤ peptides are generated from the amyloid precursor protein (APP) by two proteolytic enzymes called ␤-and ␥-secretases (for reviews see Refs. 6 -8). The resulting A␤ peptides represent a heterogeneous group of peptides with different lengths. A␤(1-40) is the predominant form released from cultured cells (9) and in biological fluids, such as blood (10) and cerebrospinal fluid (CSF) (11). Longer forms ending at amino acid 42 are believed to be particularly important in the pathogenesis of AD due to a higher propensity to aggregate (12). A␤ accumulation in the AD brain likely starts many years before cognitive deficits become evident (13,14). The reported inverse association of in vivo cortical binding of Pittsburgh Compound-B and CSF A␤42 suggests that brain amyloid deposition and low CSF A␤42 are mechanistically related (13,15). Based on the amyloid cascade hypothesis stating a central role of A␤ in AD, several therapeutic strategies targeting A␤ and aiming for inhibition of disease progression are pursued. The more advanced of these potential therapeutic approaches include inhibition or modulation of cellular A␤ production and A␤-targeted immunotherapy (16,17).Animal models of the human disease are important research tools to facilitate the study of the pathophysiology, and they have been widely used for testing potential therapeutic approaches. The APP23 mouse line (18) represents one out of several well established transgenic mouse models that display key features of ...

Covalent coupling of β-secretase inhibitors to a raftophilic lipid anchor via a suitable spacer by using solid-phase peptide synthesis leads to tripartite structures displaying substantially improved inhibition of cellular secretion of the β-amyloid peptide (Aβ). Herein, we describe a series of novel tripartite structures, their full characterization by NMR spectroscopy and mass spectrometry, and the analysis of their biological activity in cell-based assays. The tripartite structure concept is applicable to different pharmacophores, and the potency in terms of β-secretase inhibition can be optimized by adjusting the spacer length to achieve an optimal distance of the inhibitor from the lipid bilayer. A tripartite structure containing a transition-state mimic inhibitor was found to be less potent on Aβ generation from Swedish-mutant amyloid precursor protein (APP) than from the wild-type protein. Moreover, our observations suggest that specific variants of Aβ are generated from wild-type APP but not from Swedish-mutant APP and are resistant to β-secretase inhibition. Efficient inhibition of Aβ secretion by tripartite structures in the absence of appreciable neurotoxicity was confirmed in a primary neuronal cell culture, thus further supporting the concept.

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).

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.

BackgroundFirst 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.MethodsSTART 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.ResultsThirty-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.ConclusionsContinuous 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.Trial registration NCT01585298; registered April 23, 2012.

BackgroundSo far, clinical studies in primary progressive MS (PPMS) have failed to meet their primary efficacy endpoints. To some extent this might be attributable to the choice of assessments or to the selection of the study population.ObjectiveThe aim of this study was to identify outcome influencing factors by analyzing the design and methods of previous randomized studies in PPMS patients without restriction to intervention or comparator.MethodsA systematic literature search was conducted in MEDLINE, EMBASE, BIOSIS and the COCHRANE Central Register of Controlled Trials (inception to February 2015). Keywords included PPMS, primary progressive multiple sclerosis and chronic progressive multiple sclerosis. Randomized, controlled trials of at least one year’s duration were selected if they included only patients with PPMS or if they reported sufficient PPMS subgroup data. No restrictions with respect to intervention or comparator were applied. Study quality was assessed by a biometrics expert. Relevant baseline characteristics and outcomes were extracted and compared.ResultsOf 52 PPMS studies identified, four were selected. Inclusion criteria were notably different among studies with respect to both the definition of PPMS and the requirements for the presence of disability progression at enrolment. Differences between the study populations included the baseline lesion load, pretreatment status and disease duration. The rate of disease progression may also be an important factor, as all but one of the studies included a large proportion of patients with a low progression rate. In addition, the endpoints specified could not detect progression adequately.ConclusionOptimal PPMS study methods involve appropriate patient selection, especially regarding the PPMS phenotype and progression rate. Functional composite endpoints might be more sensitive than single endpoints in capturing progression.

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.

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