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Introduction: Fast and minimally invasive approaches for early diagnosis of Alzheimer's disease (AD) are highly anticipated. Evidence of adaptive immune cells responding to cerebral β-amyloidosis has raised the question of whether immune markers could be used as proxies for β-amyloid accumulation in the brain. Methods: Here, we apply multidimensional mass-cytometry combined with unbiased machine-learning techniques to immunophenotype peripheral blood mononuclear cells from a total of 251 participants in cross-sectional and longitudinal studies. Results: We show that increases in antigen-experienced adaptive immune cells in the blood, particularly CD45RA-reactivated T effector memory (TEMRA) cells, are associated with early accumulation of brain β-amyloid and with changes in plasma AD biomarkers in still cognitively healthy subjects. Discussion: Our results suggest that preclinical AD pathology is linked to systemic alterations of the adaptive immune system. These immunophenotype changes may help identify and develop novel diagnostic tools for early AD assessment and better understand clinical outcomes., Alzheimer's & Dementia: The Journal of the Alzheimer's Association, ISSN:1552-5279, ISSN:1552-5260

In the brain capillaries, endothelial cells, pericytes, astrocytes and microglia form a structural and functional complex called neurovascular unit (NVU) which is critically involved in maintaining neuronal homeostasis. In the present study, we applied a comprehensive immunohistochemical approach to investigate the structural alterations in the NVU across different Alzheimer's disease (AD) neuropathological stages. Post‐mortem human cortical and hippocampal samples derived from AD patients and non‐demented elderly control individuals were immunostained using a panel of markers representing specific components of the NVU including Collagen IV (basement membrane), PDGFR‐β (pericytes), GFAP (astrocytes), Iba1 (microglia), MRC1 (perivascular macrophages) and lectin as an endothelial cell label. Astrocytes (GFAP) and microglia (Iba1) were quantified both in the whole visual‐field and specifically within the NVU, and the sample set was additionally analyzed using anti‐tau (AT8) and three different anti‐Aβ (clones G2‐10, G2‐11, 4G8) antibodies. Analyses of lectin labeled sections showed an altered vascular distribution in AD patients as revealed by a reduced nearest distance between capillaries. Within the NVU, a Braak‐stage dependent reduction in pericyte coverage was identified as the earliest structural alteration during AD progression. In comparison to non‐demented elderly controls, AD patients showed a significantly higher astrocyte coverage within the NVU, which was paralleled by a reduced microglial coverage around capillaries. Assessment of perivascular macrophages moreover demonstrated a relocation of these cells from leptomeningeal arteries to penetrating parenchymal vessels in AD patients. Collectively, the results of our study represent a comprehensive first in‐depth analysis of AD‐related structural changes in the NVU and suggest distinct alterations in all components of the NVU during AD progression.

RG6102 is a bispecific 2+1 monoclonal antibody (mAb) under development for the treatment of Alz- heimer’s disease (AD). It combines the anti-amyloid beta antibody gantenerumab with a transferrin receptor 1-binding “Brain Shuttle” module, enabling active receptor-mediated transport across the blood–brain barrier.In preclinical studies, RG6102 has shown superior distribution, target engagement and amyloid plaque clearance compared with gantenerumab. In a human Phase Ia study, there was a markedly increased cerebrospinal fluid (CSF)/plasma ratio for RG6102 compared with typical mAbs.Brainshuttle AD is a 28-week, randomised, global, multicentre, double-blind, placebo-controlled, parallel- group Phase Ib/IIa study evaluating the safety, tolerability, immunogenicity and pharmacokinetics/phar- macodynamics (PK/PD) of RG6102. Multiple-ascending intravenous doses of RG6102 are administered every 4 weeks to patients with prodromal or mild-to-moderate AD. The study consists of a screening period, a double-blind treatment period and a safety follow-up period.The primary objective is to evaluate the safety and tolerability of RG6102. Secondary outcome measures include the change from baseline in brain amyloid load, plasma and CSF concentration of RG6102 and incidence of anti-drug antibodies to RG6102. Exploratory endpoints include the clinical effect of multiple doses of RG6102 on clinical outcome measures and on various PD biomarkers.Recruitment for Brainshuttle AD is currently ongoing.

The beta-site APP cleaving enzyme 1 (BACE1) is known primarily for its initial cleavage of the amyloid precursor protein (APP), which ultimately leads to the generation of Aβ peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aβ40 and Aβ42 into a common Aβ34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aβ34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aβ34 levels correlate with the overall Aβ clearance rates in amyloid positive individuals. Using CSF samples from the PREVENT-AD cohort (cognitively normal individuals at risk for Alzheimer’s disease), we further demonstrate that the Aβ34/Aβ42 ratio, representing Aβ degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aβ34 represents a marker of amyloid clearance and may be helpful for the characterization of Aβ turnover in clinical samples., Aβ34 is generated from degradation of Aβ40 and Aβ42 by β-secretase. Here, the authors show that Aβ34 is a marker for amyloid clearance and is elevated in the CSF of patients that go on to convert from mild cognitive impairment to Alzheimer’s disease, suggesting it may be a useful biomarker.

Fast and minimally invasive approaches for early, preclinical diagnosis of neurodegenerative Alzheimer’s disease (AD) are highly anticipated. Evidence of adaptive immune cells responding to cerebral β-amyloidosis, one of the pathological hallmarks of AD, has raised the question of whether immune markers could be used as proxies for β-amyloid accumulation in the brain. Here, we deploy multidimensional mass cytometry combined with unbiased machine learning techniques to immunophenotype peripheral blood mononuclear cells from study participants in cross-sectional and longitudinal cohorts. We show that increases in antigen-experienced T and B cell subpopulations in blood are associated with early accumulation of β-amyloid in still cognitively healthy human brains. Our results suggest that preclinical AD pathology stages are associated with systemic alterations of the adaptive immune system. These β-amyloid-induced immunophenotype changes may be exploited in the future to identify and develop novel diagnostic tools for early AD detection and prediction of clinical outcomes. Funding Information: This work was supported by grants from the Synapsis Foundation – Alzheimer Research Switzerland ARS (No. 2019-PI06 to R.M.N., C.G., and A.G.), the Swiss National Science Foundation (SNF 33CM30-124111, SNF 320030-125387/1 to C.H.), the Maxi Foundation (to C.H.) and the Velux Foundation (to L.K.). Declaration of Interests: T.K. is currently an employee of Biogen, Switzerland; L.K. and V.Tosevski are employees of Roche, Switzerland; C.H. and R.M.N. are members of the board of directors and shareholders of Neurimmune AG, Switzerland; M.T.F. is co-founder and CSO of the Women’s Brain Project. Ethics Approval Statement: All participants gave written informed consent. The studies were conducted in concordance with the guidelines of the local ethics committee (Kantonale Ethikkommission Zurich) and the Declaration of Helsinki (World Medical Association, 2013). The current analyses were conducted with permission of the ethics committee for further use of data and samples.

Objective Brain calcifications are associated with several neurodegenerative diseases. Here, we describe the occurrence of intracranial calcifications as a new phenotype in transgenic P301L mice overexpressing four repeat tau, a model of human tauopathy. Materials and methods Thirty-six P301L mice (Thy1.2) and ten age-matched non-transgenic littermates of different ages were assessed. Gradient echo data were acquired in vivo and ex vivo at 7 T and 9.4 T for susceptibility-weighted imaging (SWI) and phase imaging. In addition, ex vivo micro-computed tomography (μCT) was performed. Histochemistry and immunohistochemistry were used to investigate the nature of the imaging lesions. Results SW images revealed regional hypointensities in the hippocampus, cortex, caudate nucleus, and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, µCT detected hyperdense lesions, though fewer lesions were observed compared to MRI. Diamagnetic susceptibility lesions in the hippocampus increased with age. The immunochemical staining of brain sections revealed osteocalcin-positive deposits. Furthermore, intra-neuronal and vessel-associated osteocalcin-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus, while vascular osteocalcin-containing nodules were detected in the thalamus in the absence of phosphorylated-tau deposition. Discussion SWI and phase imaging sensitively detected intracranial calcifications in the P301L mouse model of human tauopathy.

Familial forms of Alzheimer’s disease (AD) are caused by mutations in the presenilin genes or in the gene encoding for the amyloid precursor protein (APP). Proteolytic cleavage of APP generates the β-amyloid peptide (Aβ), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aβ sequence, so-called intra-Aβ mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aβ sequence. One of these mutations is the Osaka mutation, E693Δ, which has unique aggregation properties with patients showing unusually low brain amyloid levels on amyloid PET scans. Despite intense research on the pathomechanisms of different intra-Aβ mutants, our knowledge is limited due to controversial findings in various studies. Here, we investigated in an ex vivo experimental system the neuro- and synaptotoxic properties of two intra-Aβ mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aβ. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aβ40 or Aβ42 containing the respective intra-Aβ mutations. Our analyses revealed that wt Aβ and E22G Aβ, both recombinant and transgenic, caused a loss of dendritic spines along with an increase in tau phosphorylation and tau-dependent neurodegeneration. In all experiments, the 42-residue variants of wt and E22G Aβ showed stronger effects than the respective Aβ40 isoforms. In contrast, E22Δ Aβ neither reduced dendritic spine density nor resulted in increased tau phosphorylation or neuronal cell death in our ex vivo system. Our findings suggest that the previously reported major differences in the aggregation kinetics between E22G and E22Δ Aβ are likely reflected in different disease pathomechanisms., PLoS ONE, 15 (9), ISSN:1932-6203

An impairment of amyloid β-peptide (Aβ) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer’s disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aβ40 and Aβ42 results in the formation of a common Aβ34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aβ34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aβ34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aβ34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aβ34 immunoreactivity was largely lost. Aβ34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aβ40, but not with Aβ42 levels. Moreover, a significantly decreased Aβ34/Aβ40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aβ40 to Aβ34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aβ34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aβ34 levels upon treatment with recombinant Aβ40 peptides while Aβ34 production was impaired when Aβ40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aβ34 is generated by a novel BACE1-mediated Aβ clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is implicated in cardiovascular disease by modulating apoptosis and oxidative stress. We hypothesized that LOX-1 may be involved in pathophysiology of stroke by mediating ischaemia/reperfusion (I/R)-dependent cell death. Transient middle cerebral artery occlusion (tMCAO) was performed in wild-type (WT) mice, endothelial-specific LOX-1 transgenic mice (eLOX-1TG) and WT animals treated with LOX-1 silencing RNA (siRNA). In WT mice exposed to tMCAO, LOX-1 expression and function were increased in the MCA. Compared to WT animals, eLOX-1TG mice displayed increased stroke volumes and worsened outcome after I/R. Conversely, LOX-1-silencing decreased both stroke volume and neurological impairment. Similarly, in HBMVECs, hypoxia/reoxygenation increased LOX-1 expression, while LOX-1 overexpressing cells showed increased death following hypoxia reoxygenation. Increased caspase-3 activation was observed following LOX-1 overexpression both in vivo and in vitro, thus representing a likely mediator. Finally, monocytes from ischaemic stroke patients exhibited increased LOX-1 expression which also correlated with disease severity. Our data unequivocally demonstrate a key role for LOX-1 in determining outcome following I/R brain damage. Our findings could be corroborated in human brain endothelial cells and monocytes from patients, underscoring their translational relevance and suggesting siRNA-mediated LOX-1 knockdown as a novel therapeutic strategy for stroke patients.

Brain calcification is associated with several neurodegenerative proteinopathies. Here, we report a new phenotype of intracranial calcification in transgenic P301L mice overexpressing 4 repeat tau. P301L mice (Thy1.2) of 3, 5, 9 and 18-25 months-of-age and age-matched non-transgenic littermates were assessed using in vivo/ex vivo magnetic resonance imaging (MRI) with a gradient recalled echo sequence and micro computed tomography (μCT). Susceptibility weighted images computed from the gradient recalled echo data revealed regional hypointensities in the hippocampus, cortex, caudate nucleus and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, µCT detected hyperdense lesions. Occurrence of diamagnetic susceptibility lesions in the hippocampus, increased with age. Immunochemical staining of brain sections revealed bone protein-positive deposits. Furthermore, intra-neuronal and vessel-associated protein-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus. Protein-containing nodules were detected also in the thalamus in the absence of phosphorylated-tau deposition. In contrast, osteocalcin-containing nodules were vessel-associated, indicating ossified vessels, in the thalamus in absence of phosphorylated-tau. In summary, MRI and µCT demonstrated imaging pattern of intracranial calcification, concomitant with immunohistochemical evidence of formation of protein deposits containing bone proteins along with phosphorylated-tau in the P301L mouse model of human tauopathy. The P301L mouse model may thus serve as a future model to study the pathogenesis of brain calcifications in tauopathies.

Résumé. Le diagnostic précoce des atteintes cognitives, ressenties subjectivement ou rapportées par un tiers, est essentiel pour détecter des maladies neurodégénératives ou exclure des causes traitables telles que des pathologies de médecine interne, neurologiques ou psychiatriques. C’est la seule façon de garantir un traitement anticipé. Dans le cadre du projet 3.1 de la stratégie nationale en matière de démences 2014–2019 («Mise en place et extension d’un réseau de centres de compétences régionaux pour le diagnostic»), l’association Swiss Memory Clinics (SMC) s’est fixé pour objectif d’améliorer les normes de qualité en matière de diagnostic des démences et de soins de proximité dans ce domaine. Ces recommandations contiennent des directives d’ordre général sur le diagnostic et les différentes possibilités d’examens, et proposent des normes pour les procédures à appliquer. Elles expliquent en détail les différents éléments du diagnostic standard, tels que l’anamnèse, l’examen clinique, l’analyse de laboratoire, les tests neuropsychologiques et les procédures neuroradiologiques, et présentent des examens complémentaires pouvant alimenter les réflexions sur le diagnostic différentiel. Les principaux objectifs des recommandations SMC pour le diagnostic des démences sont les suivants: assurer l’accès à un diagnostic de haute qualité à un maximum de personnes atteintes, améliorer le diagnostic précoce de la démence, ainsi que proposer aux médecins de premier recours et aux collaborateurs de Memory Clinics un outil d’investigations diagnostiques utile.

PURPOSE OF REVIEW: The concept of preclinical Alzheimer's disease has emerged to describe the long 'silent' phase of the disease when significant pathophysiological changes occur in the brain but clinical symptoms are not yet manifest. In this review, a summary of the recent advances in cerebrospinal fluid (CSF) biomarker-based diagnostics of preclinical Alzheimer's disease will be presented. RECENT FINDINGS: The association between core CSF biomarkers of Alzheimer's disease and between CSF and neuroimaging markers has been a major focus of various recently published studies in cognitively healthy individuals. Longitudinal results from several research groups suggest that CSF Aβ42 is altered early in preclinical Alzheimer's disease, even preceding changes on amyloid PET imaging. In line with the proposed NIA-AA criteria, elevated tau levels and/or Aβ/tau interactions appear to be a prerequisite for neurodegeneration and future cognitive decline. Novel candidate CSF markers, including markers of neuronal and synaptic injury as well as neuroinflammation, may complement CSF-based diagnostics in preclinical Alzheimer's disease. SUMMARY: Further longitudinal research is necessary to delineate the temporal changes of core and candidate CSF biomarkers in preclinical Alzheimer's disease and to investigate their association with established and emerging neuroimaging markers as well as with comorbidities and other risk factors for age-related cognitive decline.

Mutations in the gene encoding superoxide dismutase 1 (SOD1) lead to misfolding and aggregation of SOD1 and cause familial amyotrophic lateral sclerosis (FALS). However, the implications of wild-type SOD1 misfolding in sporadic forms of ALS (SALS) remain unclear. By screening human memory B cells from a large cohort of healthy elderly subjects, we generated a recombinant human monoclonal antibody (α-miSOD1) that selectively bound to misfolded SOD1, but not to physiological SOD1 dimers. On postmortem spinal cord sections from 121 patients with ALS, α-miSOD1 antibody identified misfolded SOD1 in a majority of cases, regardless of their SOD1 genotype. In contrast, the α-miSOD1 antibody did not bind to its epitope in most of the 41 postmortem spinal cord sections from non-neurological control (NNC) patients. In transgenic mice overexpressing disease-causing human SOD1G37R or SOD1G93A mutations, treatment with the α-miSOD1 antibody delayed the onset of motor symptoms, extended survival by up to 2 months, and reduced aggregation of misfolded SOD1 and motor neuron degeneration. These effects were obtained whether α-miSOD1 antibody treatment was administered by direct brain infusion or peripheral administration. These results support the further development of α-miSOD1 antibody as a candidate treatment for ALS involving misfolding of SOD1.

Neurodegenerative diseases of the central nervous system frequently affect the locomotor system resulting in impaired movement and gait. In this study we performed a whole-body high-speed video gait analysis in three different mouse lines of neurodegenerative movement disorders to investigate the motor phenotype. Based on precise computerized motion tracking of all relevant joints and the tail, a custom-developed algorithm generated individual and comprehensive locomotor profiles consisting of 164 spatial and temporal parameters. Gait changes observed in the three models corresponded closely to the classical clinical symptoms described in these disorders: Muscle atrophy due to motor neuron loss in SOD1 G93A transgenic mice led to gait characterized by changes in hind-limb movement and positioning. In contrast, locomotion in huntingtin N171-82Q mice modeling Huntington's disease with basal ganglia damage was defined by hyperkinetic limb movements and rigidity of the trunk. Harlequin mutant mice modeling cerebellar degeneration showed gait instability and extensive changes in limb positioning. Moreover, model specific gait parameters were identified and were shown to be more sensitive than conventional motor tests. Altogether, this technique provides new opportunities to decipher underlying disease mechanisms and test novel therapeutic approaches.

The early diagnosis of subjectively perceived or externally anamnestically observed cognitive impairments is essential for proving neurodegenerative diseases or excluding treatable causes such as internal, neurological or psychiatric disorders. Only in this way is early treatment made possible. As part of the project 3.1 of the National Dementia Strategy 2014–2019 («Development and expansion of regional and networked centres of competence for diagnostics»), the association Swiss Memory Clinics (SMC) set itself the goal of developing quality standards for dementia clarification and improving the community-based care in this field. In these recommendations, general guidelines of diagnostics and individual examination possibilities are presented, and standards for the related processes are suggested. Individual areas such as anamnesis, clinical examination, laboratory examination, neuropsychological testing and neuroradiological procedures are discussed in detail as part of standard diagnostics, and supplementary examination methods for differential diagnosis considerations are portrayed. The most important goals of the SMC recommendations for the diagnosis of dementia are to give all those affected access to high-quality diagnostics, if possible, to improve early diagnosis of dementia and to offer the basic service providers and the employees of Memory Clinics a useful instrument for the clarification.

Zusammenfassung. Die Frühdiagnostik subjektiv wahrgenommener oder fremdanamnestisch beobachteter kognitiver Beeinträchtigungen ist essenziell, um neurodegenerative Erkrankungen nachzuweisen oder behandelbare Ursachen wie internistische, neurologische oder psychiatrische Störungen auszuschliessen. Nur dadurch wird eine frühzeitige Behandlung ermöglicht. Im Rahmen des Projekts 3.1 der Nationalen Demenzstrategie 2014–2019 («Auf- und Ausbau regionaler und vernetzter Kompetenzzentren für die Diagnostik») hat sich der Verein Swiss Memory Clinics (SMC) zum Ziel gesetzt, Qualitätsstandards für die Demenzabklärung zu entwickeln und die wohnortsnahe Versorgung in diesem Bereich zu verbessern. In den vorliegenden Empfehlungen werden allgemeine Richtlinien der Diagnostik und einzelne Untersuchungsmöglichkeiten vorgestellt, sowie Standards für die diesbezüglichen Abläufe vorgeschlagen. Einzelne Bereiche wie Anamneseerhebung, klinische Untersuchung, Laborparameter, neuropsychologische Testung und neuroradiologische Verfahren werden als Teil der Standarddiagnostik ausführlich diskutiert, ergänzende Untersuchungsmethoden für differenzialdiagnostische Überlegungen abgebildet. Die wichtigsten Ziele der SMC-Empfehlungen zur Diagnostik der Demenzerkrankungen sind, möglichst allen Betroffenen Zugang zu einer qualitativ hochstehenden Diagnostik zu ermöglichen, die Frühdiagnostik der Demenz zu verbessern und den Grundversorgern sowie den Mitarbeitenden der Memory Clinics ein nützliches Instrument für die Abklärung anzubieten.

European Heart Journal, 36, ISSN:1522-9645, ISSN:0195-668X

Background: Strong genetic and epidemiological evidence points to a crucial role of the immune system in the development of Alzheimer disease (AD). CD3+ T lymphocytes have been described in brains of postmortem AD patients and in transgenic models of AD-like cerebral amyloidosis and tau pathology. However, the occurrence of T cells in AD brains is still controversial; furthermore, the relationship between T cells and hallmarks of AD pathology (amyloid plaques and neurofibrillary tangles) remains to be established. Objectives: We have studied the occurrence of T cells in postmortem hippocampi and mid frontal gyrus (MFG) samples of AD patients (Braak stage V-VI) and nondemented control subjects and correlated it with amyloid and tau pathology burden. Methods: Confocal microscopy and bright-field immunohistochemistry were used to identify brain-associated T cells. Extravascular CD3+ T cells were quantified and compared to nondemented controls. In addition, numbers of extravascular CD3+ T cells were correlated with amyloid (6E10 staining) and tau pathology (AT8 staining) in the same sections. Results: Several CD3+, extravascular T cells were observed in the brains of AD patients, mostly of the CD8+ subtype. AD hippocampi harbored significantly increased numbers of extravascular CD3+ T cells compared to nondemented controls. CD3+ T cells significantly correlated with tau pathology but not with amyloid plaques in AD samples. Conclusions: Our data support the notion of T-cell occurrence in AD brains and suggest that, in advanced stages of AD, T-cell extravasation is driven by tau-related neurodegenerative changes rather than by cerebral amyloidosis. T cells could be crucial for driving the amyloid-independent phase of the AD pathology.

The mammalian ShcA adaptor protein p66Shc is a key regulator of mitochondrial reactive oxygen species (ROS) production and has previously been shown to mediate amyloid β (Aβ)-peptide-induced cytotoxicity in vitro. Moreover, p66Shc is involved in mammalian longevity and lifespan determination as revealed in the p66Shc knockout mice, which are characterized by a 30% prolonged lifespan, lower ROS levels and protection from age-related impairment of physical and cognitive performance. In this study, we hypothesized a role for p66Shc in Aβ-induced toxicity in vivo and investigated the effects of genetic p66Shc deletion in the PSAPP transgenic mice, an established Alzheimer's disease mouse model of β-amyloidosis. p66Shc-ablated PSAPP mice were characterized by an improved survival and a complete rescue of Aβ-induced cognitive deficits at the age of 15 months. Importantly, these beneficial effects on survival and cognitive performance were independent of Aβ levels and amyloid plaque deposition, but were associated with improved brain mitochondrial respiration, a reversal of mitochondrial complex I dysfunction, restored adenosine triphosphate production and reduced ROS levels. The results of this study support a role for p66Shc in Aβ-related mitochondrial dysfunction and oxidative damage in vivo, and suggest that p66Shc ablation may be a promising novel therapeutic strategy against Aβ-induced toxicity and cognitive impairment.

Passive immunization with anti-amyloid-β peptide (Aβ) antibodies is effective in animal models of Alzheimer disease. With the advent of efficient in vitro selection technologies, the novel class of designed ankyrin repeat proteins (DARPins) presents an attractive alternative to the immunoglobulin scaffold. DARPins are small and highly stable proteins with a compact modular architecture ideal for high affinity protein-protein interactions. In this report, we describe the selection, binding profile, and epitope analysis of Aβ-specific DARPins. We further showed their ability to delay Aβ aggregation and prevent Aβ-mediated neurotoxicity in vitro. To demonstrate their therapeutic potential in vivo, mono- and trivalent Aβ-specific DARPins (D23 and 3×D23) were infused intracerebroventricularly into the brains of 11-month-old Tg2576 mice over 4 weeks. Both D23 and 3×D23 treatments were shown to result in improved cognitive performance and reduced soluble Aβ levels. These findings demonstrate the therapeutic potential of Aβ-specific DARPins for the treatment of Alzheimer disease.

In Alzheimer’s disease (AD), cerebral arteries, in contrast to cerebral microvessels, show both cerebral amyloid angiopathy (CAA) -dependent and -independent vessel wall pathology. However, it remains unclear whether CAA-independent vessel wall pathology affects arterial function, thereby chronically reducing cerebral perfusion, and, if so, which mechanisms mediate this effect. To this end, we assessed the ex vivo vascular function of the basilar artery and a similar-sized peripheral artery (femoral artery) in the Swedish-Arctic (SweArc) transgenic AD mouse model at different disease stages. Furthermore, we used quantitative immunohistochemistry to analyze CAA, endothelial morphology, and molecular pathways pertinent to vascular relaxation. We found that endothelium-dependent, but not smooth muscle-dependent, vasorelaxation was significantly impaired in basilar and femoral arteries of 15-mo-old SweArc mice compared with that of age-matched wild-type and 6-mo-old SweArc mice. This impairment was accompanied by significantly reduced levels of cyclic GMP, indicating a reduced nitric oxide (NO) bioavailability. However, no age- and genotype-related differences in oxidative stress as measured by lipid peroxidation were observed. Although parenchymal capillaries, arterioles, and arteries showed abundant CAA in the 15-mo-old SweArc mice, no CAA or changes in endothelial morphology were detected histologically in the basilar and femoral artery. Thus our results suggest that, in this AD mouse model, dysfunction of large intracranial, extracerebral arteries important for brain perfusion is mediated by reduced NO bioavailability rather than by CAA. This finding supports the growing body of evidence highlighting the therapeutic importance of targeting the cerebrovasculature in AD. NEW & NOTEWORTHY We show that vasorelaxation of the basilar artery, a large intracranial, extracerebral artery important for cerebral perfusion, is impaired independent of cerebral amyloid angiopathy in a transgenic mouse model of Alzheimer’s disease. Interestingly, this dysfunction is specifically endothelium related and is mediated by impaired nitric oxide–cyclic GMP bioavailability. Listen to this article’s corresponding podcast at http://ajpheart.podbean.com/e/cerebroarterial-dysfunction-in-swedish-arctic-ad-mice/ .

Cerebral beta-amyloidosis, one of the pathological hallmarks of Alzheimer’s disease (AD), elicits a well- characterised, microglia-mediated local innate immune response. In contrast, it is not clear whether cells of the adaptive immune system, in particular T-cells, react to cerebral amyloidosis in AD. Even though parenchymal T-cells have been described in post-mortem brains of AD patients, it is not known whether infiltrating T-cells are specifically recruited to the extracellular deposits of beta-amyloid, and whether they are locally activated into proliferating, effector cells upon interaction with antigen-presenting cells (APCs). To address these issues we have analysed by confocal microscopy and flow-cytometry the local- isation and activation status of both T-cells and APCs in transgenic (tg) mice models of AD-like cerebral amyloidosis. Increased numbers of infiltrating T-cells were found in amyloid-burdened brain regions of tg mice, with concomitant up-regulation of endothelial adhesion molecules ICAM-1 and VCAM-1, compared to non-tg littermates. The infiltrating T-cells in tg brains did not co-localise with amyloid plaques, pro- duced less interferon-gamma than those in controls and did not proliferate locally. Bona-fide dendritic cells were virtually absent from the brain parenchyma of both non-tg and tg mice, and APCs from tg brains showed an immature phenotype, with accumulation of MHC-II in intracellular compartments. These results indicate that cerebral amyloidosis promotes T-cell infiltration but interferes with local anti- gen presentation and T-cell activation. The inability of the brain immune surveillance to orchestrate a protective immune response to amyloid-beta peptide might contribute to the accumulation of amyloid in the progression of the disease. 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

Alzheimer's disease is a fatal neurodegenerative disorder affecting the aging population. Neuroimaging methods, in particular magnetic resonance imaging (MRI), have helped reveal alterations in the brain structure, metabolism, and function of patients and in groups at risk of developing AD, yet the nature of these alterations is poorly understood. Neuroimaging in mice is attractive for investigating mechanisms underlying functional and structural changes associated with AD pathology. Several preclinical murine models of AD have been generated based on transgenic insertion of human mutated APP genes. Depending on the specific mutations, mouse strains express different aspects of amyloid pathology, e.g. intracellular amyloid-β (Aβ) aggregates, parenchymal plaques, or cerebral amyloid angiopathy. We have applied multi-parametric MRI in three transgenic mouse lines to compare changes in brain function with resting-state fMRI and structure with diffusion tensor imaging and high resolution anatomical imaging. E22ΔAβ developing intracellular Aβ aggregates did not present functional or structural alterations compared to their wild-type littermates. PSAPP mice displaying parenchymal amyloid plaques displayed mild functional changes within the supplementary and barrel field cortices, and increased isocortical volume relative to controls. Extensive reduction in functional connectivity in the sensory-motor cortices and within the default mode network, as well as local volume increase in the midbrain relative to wild-type have been observed in ArcAβ mice bearing intracellular Aβ aggregates as well as parenchymal and vascular amyloid deposits. Patterns of functional and structural changes appear to be strain-specific and not directly related to amyloid deposition.

Introduction In Alzheimer’s disease, accumulation and pathological aggregation of amyloid β-peptide is accompanied by the induction of complex immune responses, which have been attributed both beneficial and detrimental properties. Such responses implicate various cell types of the innate and adaptive arm of the immunesystem, both inside the central nervous system, and in the periphery. To investigate the role of the adaptive immune system in brain β-amyloidosis, PSAPP transgenic mice, an established mouse model of Alzheimer’s disease, were crossbred with the recombination activating gene-2 knockout (Rag2 ko) mice lacking functional B and T cells. In a second experimental paradigm, aged PSAPP mice were reconstituted with bone marrow cells from either Rag2 ko or wildtype control mice. Results Analyses from both experimental approaches revealed reduced β-amyloid pathology and decreased brain amyloid β-peptide levels in PSAPP mice lacking functional adaptive immune cells. The decrease in brain β-amyloid pathology was associated with enhanced microgliosis and increased phagocytosis of amyloid β-peptide aggregates. Conclusion The results of this study demonstrate an impact of the adaptive immunity on cerebral β-amyloid pathology in vivo and suggest an influence on microglia-mediated amyloid β-peptide clearance as a possible underlying mechanism. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0251-x) contains supplementary material, which is available to authorized users.

Background: Increased plasma homocysteine levels have been described as an independent risk factor for Alzheimer’s disease (AD), but the underlying pathophysiology is unclear. Objective: This single-center, cross-sectional, correlational study analyzed homocysteine metabolism in 60 AD patients and 60 control subjects. Methods: Fasting plasma levels of vitamin B12, folate and homocysteine as well as cerebrospinal fluid (CSF) levels of folate derivates, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and homocysteine were measured. In addition, the apolipoprotein E (APOE) genotype was determined. Results: As expected, the APOE4 allele was significantly overrepresented in AD patients compared with controls (p < 0.001). Homocysteine plasma levels in the highest quartile were more frequent in the AD patients than in the controls (p = 0.008). In addition, AD patients had significantly lower CSF levels of the methyl group donor SAM (193 ± 31 vs. 207 ± 37 nmol/l; p = 0.032). Accordingly, the SAM/SAH ratio, which represents the methylation capacity, was significantly lower in the CSF of the AD patients (7.6 ± 2.4 vs. 9.1 ± 2.8; p = 0.003). Further, explorative analysis of all subjects showed that CSF SAM levels were lower in carriers of the APOE4 allele compared with noncarriers (189 ± 30 vs. 207 ± 36 nmol/l; p = 0.010). Of the individuals with CSF SAM levels in the lowest quartile, 63% carried the APOE4 allele compared with 17% of the individuals with CSF SAM levels in the highest quartile (Pearson: χ2 = 9.9; p = 0.002; odds ratio 0.126, 95% confidence interval 0.32–0.49). Conclusion: These data suggest that AD is associated with lower CSF SAM levels and that this is at least partly due to an association of the APOE4 allele with reduced SAM levels in the CSF.

Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure. Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity, decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions in the DHCR24−/− mouse. Electronic supplementary material The online version of this article (doi:10.1007/s11064-008-9893-4) contains supplementary material, which is available to authorized users.

The 3β-hydroxysterol-Δ24 reductase (DHCR24) is a broadly expressed oxidoreductase, sharing homologies with a family of flavin adenine dinucleotide-dependent reductases (26). The dhcr24 gene is the human orthologue of the diminuto/dwarf1, initially identified in plants, where it is required for the synthesis of brassinosteroids, a group of plant sterols that are essential for normal growth and development (4, 18, 23). In mammals, DHCR24 plays an indispensable role in cholesterol biosynthesis, catalyzing the conversion of desmosterol to cholesterol (6, 26, 27). However, DHCR24 was also described in a different context: dhcr24 expression was shown to be down-regulated in brain areas affected by Alzheimer's disease (11) and was therefore named seladin-1, the selective Alzheimer's disease indicator 1, suggesting an association of DHCR24/seladin-1 with the selective vulnerability of neurons in the affected brain areas. Conversely, high levels of DHCR24/seladin-1 exert protective functions, conferring resistance against oxidative stress and protecting cells from apoptotic cell death (2, 9, 11, 16). Endogenous DHCR24/seladin-1 levels are highly up-regulated upon acute oxidative stress (3, 28), while expression declines to very low levels upon chronic exposure (3), suggesting that DHCR24/seladin-1 plays a role in integrating cellular responses to oxidative stress. However, the precise molecular mechanism for this protective effect is not known. Intriguingly, recent findings identified an interaction between DHCR24/seladin-1 and the tumor suppressor protein p53. Following oncogenic and oxidative stress, seladin-1 binds to p53 in fibroblasts, thus displacing the E3 ubiquitin ligase Mdm2 from p53, which results in p53 accumulation (28). These data argue in favor of a potential tumor suppressor role of DHCR24/seladin-1, indicating that low protein levels enhance p53 degradation and thus prevent senescence in cellular response to Ras/p53-mediated oncogenic signaling (28). However, it was shown that DHCR24 activity of seladin-1, i.e., the oxidoreductase activity of the protein in cholesterol biosynthesis, is not required for p53 binding and the p53-dependent oxidative stress response (28). Naturally high transcription levels of DHCR24/seladin-1 and gene transfer of DHCR24/seladin-1 cDNA into various cell lines were associated with elevated cholesterol concentrations (3, 9), suggesting a possible role for cholesterol in the protection process. We recently showed that overexpression of DHCR24/seladin-1 in human neuroblastoma SH-SY5Y cells leads to elevated levels of cellular and membrane cholesterol, thereby enhancing the formation of lipid rafts in these cells (6). Oxidative stress leads to the production of reactive oxygen species which attack lipid membrane constituents such as unsaturated phospholipids, glycolipids, and cholesterol, resulting in cellular dysfunction and cell death (10). During apoptosis, sterol regulatory element-binding proteins (SREBPs), which regulate expression of genes involved in lipid and cholesterol homeostasis (30), were shown to be activated by caspase cleavage (21). These observations have led to the hypothesis that cholesterol may be required in the early stages of apoptosis to maintain plasma membrane integrity (25). Moreover, plasma membrane compartments rich in cholesterol may participate in signal transduction pathways activated upon oxidative stress, and thus enhance prosurvival pathways, while cholesterol depletion appears to increase apoptotic events triggered by hydrogen peroxide treatment (29). In addition, methyl-β-cyclodextrin-mediated cholesterol depletion causes apoptosis, which is associated with caspase-3 activation and Akt inactivation, while cholesterol reload replenishes rafts on the cell surface and restores Akt activation and cell viability (13, 29). In the present work, we analyzed the role of DHCR24/seladin-1 in integrating cellular responses to oxidative stress by employing primary neurons and neuroblastoma SH-SY5Y cells. We found that DHCR24/seladin-1 expression is up-regulated in an acute response and down-regulated in a long-term response to oxidative stress. In this context, protective effects of high DHCR24/seladin-1 levels were mediated by increased cellular cholesterol concentrations, which resulted from a generally enhanced cholesterol biosynthesis after exposure to oxidative stress. In contrast, protection against oxidative stress mediated by low levels of DHCR24/seladin-1 was associated with reduced levels of p53 and elevated p53 ubiquitination, while overexpression of DHCR24/seladin-1 stabilized p53, independent of DHCR24 activity and cholesterol concentrations. These findings reveal a dual capacity of DHCR24/seladin-1, which appears to be involved in two mechanistically different prosurvival effects, exerting an acute response as well as a late response to oxidative stress.

Biobanks are important resources for biomarker discovery and assay development. Biomarkers for Alzheimer's and Parkinson's disease (BIOMARKAPD) is a European multicenter study, funded by the EU Joint Programme-Neurodegenerative Disease Research, which aims to improve the clinical use of body fluid markers for the diagnosis and prognosis of Alzheimer's disease (AD) and Parkinson's disease (PD). The objective was to standardize the assessment of existing assays and to validate novel fluid biomarkers for AD and PD. To support the validation of novel biomarkers and assays, a central and a virtual biobank for body fluids and associated data from subjects with neurodegenerative diseases have been established. In the central biobank, cerebrospinal fluid (CSF) and blood samples were collected according to the BIOMARKAPD standardized pre-analytical procedures and stored at Integrated BioBank of Luxembourg. The virtual biobank provides an overview of available CSF, plasma, serum, and DNA samples at each site. Currently, at the central biobank of BIOMARKAPD samples are available from over 400 subjects with normal cognition, mild cognitive impairment (MCI), AD, frontotemporal dementia (FTD), vascular dementia, multiple system atrophy, progressive supranuclear palsy, PD, PD with dementia, and dementia with Lewy bodies. The virtual biobank contains information on over 8,600 subjects with varying diagnoses from 21 local biobanks. A website has been launched to enable sample requests from the central biobank and virtual biobank. © 2015 Reijs, Teunissen, Goncharenko, Betsou, Blennow, Baldeiras, Brosseron, Cavedo, Fladby, Froelich, Gabryelewicz, Gurvit, Kapaki, Koson, Kulic, Lehmann, Lewczuk, Lleó, Maetzler, de Mendonça, Miller, Molinuevo, Mollenhauer, Parnetti, Rot, Schneider, Simonsen, Tagliavini, Tsolaki, Verbeek, Verhey, Zboch, Winblad, Scheltens, Zetterberg and Visser.

Indirect modulation of cholinergic activity by cholinesterase inhibition is currently a widely established symptomatic treatment for Alzheimer's disease (AD). Selective activation of certain muscarinic receptor subtypes has emerged as an alternative cholinergic-based amyloid-lowering strategy for AD, as selective muscarinic M1 receptor agonists can reduce amyloid-β (Aβ) production by shifting endoproteolytic amyloid-β protein precursor (AβPP) processing toward non-amyloidogenic pathways. In this study, we addressed the hypothesis that acute stimulation of muscarinic M1 receptors can inhibit Aβ production in awake and freely moving AβPP transgenic mice. By combining intracerebral microdialysis with retrodialysis, we determined hippocampal Aβ concentrations during simultaneous pharmacological modulation of brain M1 receptor function. Infusion with a M1 receptor agonist AF102B resulted in a rapid reduction of interstitial fluid (ISF) Aβ levels while treatment with the M1 antagonist dicyclomine increased ISF Aβ levels reaching significance within 120 minutes of treatment. The reduction in Aβ levels was associated with PKCα and ERK activation resulting in increased levels of the α-secretase ADAM17 and a shift in AβPP processing toward the non-amyloidogenic processing pathway. In contrast, treatment with the M1 receptor antagonist dicyclomine caused a decrease in levels of phosphorylated ERK that was independent of PKCα, and led to an elevation of β-secretase levels associated with increased amyloidogenic AβPP processing. The results of this study demonstrate rapid effects of in vivo M1 receptor modulation on the ISF pool of Aβ and suggest that intracerebral microdialysis with retrodialysis is a useful technical approach for monitoring acute treatment effects of muscarinic receptor modulators on AβPP/Aβ metabolism.

Amyloid beta-peptide (Abeta) containing plaques and neurofibrillary tangles (NFT) are the two major histopathological hallmarks of Alzheimer's disease (AD). According to the amyloid cascade hypothesis, deposition of Abeta is an initial and essential step in the pathogenesis of AD, and formation of NFT has been proposed to be caused by increased Abeta levels. Several previous studies revealed that Abeta plaque formation can be reduced or even prevented by active immunization with Abeta preparations or by administration of Abeta-specific antibodies. To assess the role of fibrillar preparations of Abeta42 in NFT formation, we previously performed intracerebral (i.c.) injections of Abeta42 into brains of NFT-forming P301L tau transgenic mice which caused significant increases in NFT numbers. To determine whether these increases in NFT can be blocked or reduced by active immunization, P301L tau mice were immunized with intraperitoneal injections of preaggregated Abeta42. Abeta42-specific titers were monitored and the mice injected i.c. with Abeta42. We found that i.c. injection of Abeta42 caused significant increases in NFT formation. However, this induction was not affected by active immunization despite high serum anti-Abeta42 titer levels and binding of anti-Abeta42 antibodies to the injected Abeta42 aggregates. We conclude that active immunization is not sufficient to prevent the effect of Abeta42 on tau aggregation in our model system. Further studies are required to determine whether modifications of our protocol could affect the Abeta42-mediated induction of NFT formation.

Most of the genetically inherited Alzheimer's disease cases are caused by mutations in the presenilin genes, PS1 and PS2. PS mutations result in the enhanced production of the highly amyloidogenic 42/43 amino acid variant of amyloid β-peptide (Aβ). We have introduced arbitrary mutations at position 286 of PS1, where a naturally occurring PS1 mutation has been described (L286V). Introduction of charged amino acids (L286E or L286R) resulted in an increase of Aβ42/43 production, which reached almost twice the level of the naturally occurring PS1 mutation. Although pathological Aβ production was increased, endoproteolysis of Notch and nuclear transport of its cytoplasmic domain was significantly inhibited. These results demonstrate that the biological function of PS proteins in the endoproteolysis of β-amyloid precursor protein and Notch can be separated.

Pathogenic amyloid-β peptide precursor (APP) mutations clustered around position 693 of APP-position 22 of the Aβ sequence--are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aβ APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aβ accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAβ) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAβ mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aβ oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aβ peptides form amyloid fibrils, aged E22ΔAβ mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aβ peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aβ42 on E22Δ Aβ40 fibrillogenesis. Moreover, E22Δ Aβ42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aβ aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

Serum antibodies against amyloid-β peptide (Aβ) in humans with or without diagnosis of Alzheimer's disease (AD) indicate the possibility of immune responses against brain antigens. In an unbiased screening for antibodies directed against brain proteins, we found in AD patients high serum levels of antibodies against the neuronal cytoskeletal protein ankyrin G (ankG); these correlated with slower rates of cognitive decline. Neuronal expression of ankG was higher in AD brains than in nondemented age-matched healthy control subjects. AnkG was present in exosomal vesicles, and it accumulated in β-amyloid plaques. Active immunization with ankG of arcAβ transgenic mice reduced brain β-amyloid pathology and increased brain levels of soluble Aβ(42). AnkG immunization induced a reduction in β-amyloid pathology, also in Swedish transgenic mice(.) Anti-ankG monoclonal antibodies reduced Aβ-induced loss of dendritic spines in hippocampal ArcAβ organotypic cultures. Together, these data established a role for ankG in the human adaptive immune response against resident brain proteins, and they show that ankG immunization reduces brain β-amyloid and its related neuropathology.