Early ß-amyloid accumulation in the brain is associated with peripheral T cell alterations.

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 ß-amyloidosis has raised the question of whether immune markers could be used as proxies for ß-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 ß-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.

Mechanisms of amyloid-ß34 generation indicate a pivotal role for BACE1 in amyloid homeostasis.

The beta­site amyloid precursor protein (APP) cleaving enzyme (BACE1) was discovered due to its "amyloidogenic" activity which contributes to the production of amyloid-beta (Aß) peptides. However, BACE1 also possesses an "amyloidolytic" activity, whereby it degrades longer Aß peptides into a non­toxic Aß34 intermediate. Here, we examine conditions that shift the equilibrium between BACE1 amyloidogenic and amyloidolytic activities by altering BACE1/APP ratios. In Alzheimer disease brain tissue, we found an association between elevated levels of BACE1 and Aß34. In mice, the deletion of one BACE1 gene copy reduced BACE1 amyloidolytic activity by ~ 50%. In cells, a stepwise increase of BACE1 but not APP expression promoted amyloidolytic cleavage resulting in dose-dependently increased Aß34 levels. At the cellular level, a mislocalization of surplus BACE1 caused a reduction in Aß34 levels. To align the role of γ-secretase in this pathway, we silenced Presenilin (PS) expression and identified PS2-γ-secretase as the main γ-secretase that generates Aß40 and Aß42 peptides serving as substrates for BACE1's amyloidolytic cleavage to generate Aß34.

Evidence for Effects of Extracellular Vesicles on Physical, Inflammatory, Transcriptome and Reward Behaviour Status in Mice.

Immune-inflammatory activation impacts extracellular vesicles (EVs), including their miRNA cargo. There is evidence for changes in the EV miRNome in inflammation-associated neuropsychiatric disorders. This mouse study investigated: (1) effects of systemic lipopolysaccharide (LPS) and chronic social stress (CSS) on plasma EV miRNome; and (2) physiological, transcriptional, and behavioural effects of peripheral or central delivered LPS-activated EVs in recipient mice. LPS or CSS effects on the plasma EV miRNome were assessed by using microRNA sequencing. Recipient mice received plasma EVs isolated from LPS-treated or SAL-treated donor mice or vehicle only, either intravenously or into the nucleus accumbens (NAc), on three consecutive days. Bodyweight, spleen or NAc transcriptome and reward (sucrose) motivation were assessed. LPS and CSS increased the expression of 122 and decreased expression of 20 plasma EV miRNAs, respectively. Peripheral LPS-EVs reduced bodyweight, and both LPS-EVs and SAL-EVs increased spleen expression of immune-relevant genes. NAc-infused LPS-EVs increased the expression of 10 immune-inflammatory genes. Whereas motivation increased similarly across test days in all groups, the effect of test days was more pronounced in mice that received peripheral or central LPS-EVs compared with other groups. This study provides causal evidence that increased EV levels impact physiological and behavioural processes and are of potential relevance to neuropsychiatric disorders.

Differential Expression of Serum Extracellular Vesicle miRNAs in Multiple Sclerosis: Disease-Stage Specificity and Relevance to Pathophysiology.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). Its first clinical presentation (clinically isolated syndrome, CIS) is often followed by the development of relapsing-remitting MS (RRMS). The periphery-to-CNS transmission of inflammatory molecules is a major pathophysiological pathway in MS. This could include signalling via extracellular vesicle (EV) microRNAs (miRNAs). In this study, we investigated the serum EV miRNome in CIS and RRMS patients and matched controls, with the aims to identify MS stage-specific differentially expressed miRNAs and investigate their biomarker potential and pathophysiological relevance. miRNA sequencing was conducted on serum EVs from CIS-remission, RRMS-relapse, and viral inflammatory CNS disorder patients, as well as from healthy and hospitalized controls. Differential expression analysis was conducted, followed by predictive power and target-pathway analysis. A moderate number of dysregulated serum EV miRNAs were identified in CIS-remission and RRMS-relapse patients, especially relative to healthy controls. Some of these miRNAs were also differentially expressed between the two MS stages and had biomarker potential for patient-control and CIS-RRMS separations. For the mRNA targets of the RRMS-relapse-specific EV miRNAs, biological processes inherent to MS pathophysiology were identified using in silico analysis. Study findings demonstrate that specific serum EV miRNAs have MS stage-specific biomarker potential and contribute to the identification of potential targets for novel, efficacious therapies.

Distinct changes in all major components of the neurovascular unit across different neuropathological stages of Alzheimer's disease.

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-ß (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-Aß (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.

Familial Alzheimer's disease mutations at position 22 of the amyloid ß-peptide sequence differentially affect synaptic loss, tau phosphorylation and neuronal cell death in an ex vivo system.

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 ß-amyloid peptide (Aß), which aggregates into amyloid plaques, one of the major hallmarks of AD. APP mutations within the Aß sequence, so-called intra-Aß mutations, cluster around position E693 of APP, which corresponds to position E22 in the Aß 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-Aß 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-Aß mutants with different intrinsic aggregation propensities, the Osaka mutation E22Δ and the Arctic mutation E22G, and compared them to wild-type (wt) Aß. Experiments in hippocampal slice cultures from transgenic mice were complemented by treating wild-type slices with recombinantly produced Aß40 or Aß42 containing the respective intra-Aß mutations. Our analyses revealed that wt Aß and E22G Aß, 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 Aß showed stronger effects than the respective Aß40 isoforms. In contrast, E22Δ Aß 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 Aß are likely reflected in different disease pathomechanisms.

SWI and phase imaging reveal intracranial calcifications in the P301L mouse model of human tauopathy.

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.

A Practical Guide to the Automated Analysis of Vascular Growth, Maturation and Injury in the Brain.

The distinct organization of the brain's vasculature ensures the adequate delivery of oxygen and nutrients during development and adulthood. Acute and chronic pathological changes of the vascular system have been implicated in many neurological disorders including stroke and dementia. Here, we describe a fast, automated method that allows the highly reproducible, quantitative assessment of distinct vascular parameters and their changes based on the open source software Fiji (ImageJ). In particular, we developed a practical guide to reliably measure aspects of growth, repair and maturation of the brain's vasculature during development and neurovascular disease in mice and humans. The script can be used to assess the effects of different external factors including pharmacological treatments or disease states. Moreover, the procedure is expandable to blood vessels of other organs and vascular in vitro models.

The amyloid-ß degradation intermediate Aß34 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer's disease.

An impairment of amyloid ß-peptide (Aß) 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 ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aß40 and Aß42 results in the formation of a common Aß34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aß34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aß34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aß34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aß34 immunoreactivity was largely lost. Aß34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aß40, but not with Aß42 levels. Moreover, a significantly decreased Aß34/Aß40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aß40 to Aß34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aß34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aß34 levels upon treatment with recombinant Aß40 peptides while Aß34 production was impaired when Aß40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aß34 is generated by a novel BACE1-mediated Aß 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.

Aß34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer's disease progression.

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 Aß peptides. Here, we provide evidence that altered BACE1 levels and activity impact the degradation of Aß40 and Aß42 into a common Aß34 intermediate. Using human cerebrospinal fluid (CSF) samples from the Amsterdam Dementia Cohort, we show that Aß34 is elevated in individuals with mild cognitive impairment who later progressed to dementia. Furthermore, Aß34 levels correlate with the overall Aß 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 Aß34/Aß42 ratio, representing Aß degradation and cortical deposition, associates with pre-clinical markers of neurodegeneration. We propose that Aß34 represents a marker of amyloid clearance and may be helpful for the characterization of Aß turnover in clinical samples.

fMRI Reveals Mitigation of Cerebrovascular Dysfunction by Bradykinin Receptors 1 and 2 Inhibitor Noscapine in a Mouse Model of Cerebral Amyloidosis.

Functional magnetic resonance imaging (fMRI) techniques can be used to assess cerebrovascular dysfunction in Alzheimer's disease, an important and early contributor to pathology. We hypothesized that bradykinin receptor inhibition alleviates the vascular dysfunction in a transgenic arcAß mouse model of cerebral amyloidosis and that fMRI techniques can be used to monitor the treatment response. Transgenic arcAß mice, and non-transgenic littermates of 14 months-of-age were either treated with the bradykinin receptors 1 and 2 blocker noscapine or received normal drinking water as control over 3 months (n = 8-11/group) and all mice were assessed using fMRI at the end of the treatment period. Perfusion MRI using an arterial spin labeling technique showed regional hypoperfusion in arcAß compared to non-transgenic controls, which was alleviated by noscapine treatment. Similarly, measuring cerebral blood volume changes upon pharmacological stimulation using vessel dilator acetazolamide revealed recovery of regional impairment of cerebral vascular reactivity in arcAß mice upon noscapine treatment. In addition, we assessed with immunohistochemistry beta-amyloid (Aß) and inflammation levels in brain sections. Immunohistological stainings for Aß deposition (6E10) and related microgliosis (Iba1) in the cortex and hippocampus were found comparable between noscapine-treated and untreated arcAß mice. In addition, levels of soluble and insoluble Aß38, Aß40, Aß42 were found to be similar in brain tissue homogenates of noscapine-treated and untreated arcAß mice using electro-chemiluminescent based immunoassay. In summary, bradykinin receptors blockade recovered cerebral vascular dysfunction in a mouse model of cerebral amyloidosis. fMRI methods revealed the functional deficit in disease condition and were useful tools to monitor the treatment response.

Deleterious role of endothelial lectin-like oxidized low-density lipoprotein receptor-1 in ischaemia/reperfusion cerebral injury.

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.

A human-derived antibody targets misfolded SOD1 and ameliorates motor symptoms in mouse models of amyotrophic lateral sclerosis.

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.

Recommendations of Swiss Memory Clinics for the Diagnosis of Dementia / Die Empfehlungen der Swiss Memory Clinics für die Diagnostik der Demenzerkrankungen.

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 2014­2019 («Development and expansion of regional and networked centres of competence for diagnostics¼), 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.

Extravascular CD3+ T Cells in Brains of Alzheimer Disease Patients Correlate with Tau but Not with Amyloid Pathology: An Immunohistochemical Study.

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.

[Recommendations of Swiss Memory Clinics for the Diagnosis of Dementia]. / Recommandations de Swiss Memory Clinics pour le diagnostic des démences.

Recommendations of Swiss Memory Clinics for the Diagnosis of Dementia Abstract. 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.

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 2014­2019 («Mise en place et extension d'un réseau de centres de compétences régionaux pour le diagnostic¼), 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.

Multicenter Analytical Validation of Aß40 Immunoassays.

BACKGROUND: Before implementation in clinical practice, biomarker assays need to be thoroughly analytically validated. There is currently a strong interest in implementation of the ratio of amyloid-ß peptide 1-42 and 1-40 (Aß42/Aß40) in clinical routine. Therefore, in this study, we compared the analytical performance of six assays detecting Aß40 in cerebrospinal fluid (CSF) in six laboratories according to a recently standard operating procedure (SOP) developed for implementation of ELISA assays for clinical routine. METHODS: Aß40 assays of six vendors were validated in up to three centers per assay according to recently proposed international consensus validation protocols. The performance parameters included sensitivity, precision, dilutional linearity, recovery, and parallelism. Inter-laboratory variation was determined using a set of 20 CSF samples. In addition, test results were used to critically evaluate the SOPs that were used to validate the assays. RESULTS: Most performance parameters of the different Aß40 assays were similar between labs and within the predefined acceptance criteria. The only exceptions were the out-of-range results of recovery for the majority of experiments and of parallelism by three laboratories. Additionally, experiments to define the dilutional linearity and hook-effect were not executed correctly in part of the centers. The inter-laboratory variation showed acceptable low levels for all assays. Absolute concentrations measured by the assays varied by a factor up to 4.7 for the extremes. CONCLUSION: All validated Aß40 assays appeared to be of good technical quality and performed generally well according to predefined criteria. A novel version of the validation SOP is developed based on these findings, to further facilitate implementation of novel immunoassays in clinical practice.

Reduced nitric oxide bioavailability mediates cerebroarterial dysfunction independent of cerebral amyloid angiopathy in a mouse model of Alzheimer's 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.

Recent advances in cerebrospinal fluid biomarkers for the detection of preclinical Alzheimer's disease.

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 Aß42 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 Aß/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.

High-speed video gait analysis reveals early and characteristic locomotor phenotypes in mouse models of neurodegenerative movement disorders.

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.