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2.  αCaMKII Autophosphorylation Controls the Establishment of Alcohol Drinking Behavior 
Neuropsychopharmacology  2013;38(9):1636-1647.
The α-Ca2+/calmodulin-dependent protein kinase II (αCaMKII) is a crucial enzyme controlling plasticity in the brain. The autophosphorylation of αCaMKII works as a ‘molecular memory' for a transient calcium activation, thereby accelerating learning. We investigated the role of αCaMKII autophosphorylation in the establishment of alcohol drinking as an addiction-related behavior in mice. We found that alcohol drinking was initially diminished in αCaMKII autophosphorylation-deficient αCaMKIIT286A mice, but could be established at wild-type level after repeated withdrawals. The locomotor activating effects of a low-dose alcohol (2 g/kg) were absent in αCaMKIIT286A mice, whereas the sedating effects of high-dose (3.5 g/kg) were preserved after acute and subchronic administration. The in vivo microdialysis revealed that αCaMKIIT286A mice showed no dopamine (DA) response in the nucleus accumbens to acute or subchronic alcohol administration, but enhanced serotonin (5-HT) responses in the prefrontal cortex. The attenuated DA response in αCaMKIIT286A mice was in line with altered c-Fos activation in the ventral tegmental area after acute and subchronic alcohol administration. In order to compare findings in mice with the human condition, we tested 23 single-nucleotide polymorphisms (SNPs) in the CAMK2A gene for their association with alcohol dependence in a population of 1333 male patients with severe alcohol dependence and 939 controls. We found seven significant associations between CAMK2A SNPs and alcohol dependence, one of which in an autophosphorylation-related area of the gene. Together, our data suggest αCaMKII autophosphorylation as a facilitating mechanism in the establishment of alcohol drinking behavior with changing the DA–5-HT balance as a putative mechanism.
PMCID: PMC3717547  PMID: 23459588
Alcohol & Alcoholism; alphaCaMKII; Animal models; autophosphorylation; c-Fos; Dopamine; microdialysis; Serotonin; αCaMKII; autophosphorylation; alcohol; reinforcement; dopamine; serotonin
3.  CSF biomarker variability in the Alzheimer’s Association quality control program 
The cerebrospinal fluid (CSF) biomarkers amyloid beta 1–42, total tau, and phosphorylated tau are used increasingly for Alzheimer’s disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories.
Data from the first nine rounds of the Alzheimer’s Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Mölndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection.
A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1–42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP).
The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.
PMCID: PMC3707386  PMID: 23622690
Alzheimer’s disease; Cerebrospinal fluid; Biomarkers; External assurance; Quality control; Proficiency testing
4.  The Alzheimer’s Association external quality control program for cerebrospinal fluid biomarkers 
Mattsson, Niklas | Andreasson, Ulf | Persson, Staffan | Arai, Hiroyuki | Batish, Sat Dev | Bernardini, Sergio | Bocchio-Chiavetto, Luisella | Blankenstein, Marinus A. | Carrillo, Maria C. | Chalbot, Sonia | Coart, Els | Chiasserini, Davide | Cutler, Neal | Dahlfors, Gunilla | Duller, Stefan | Fagan, Anne M. | Forlenza, Orestes | Frisoni, Giovanni B. | Galasko, Douglas | Galimberti, Daniela | Hampel, Harald | Handberg, Aase | Heneka, Michael T. | Herskovits, Adrianna Z. | Herukka, Sanna-Kaisa | Holtzman, David M. | Humpel, Christian | Hyman, Bradley T. | Iqbal, Khalid | Jucker, Mathias | Kaeser, Stephan A. | Kaiser, Elmar | Kapaki, Elisabeth | Kidd, Daniel | Klivenyi, Peter | Knudsen, Cindy S. | Kummer, Markus P. | Lui, James | Lladó, Albert | Lewczuk, Piotr | Li, Qiao-Xin | Martins, Ralph | Masters, Colin | McAuliffe, John | Mercken, Marc | Moghekar, Abhay | Molinuevo, José Luis | Montine, Thomas J. | Nowatzke, William | O’Brien, Richard | Otto, Markus | Paraskevas, George P. | Parnetti, Lucilla | Petersen, Ronald C. | Prvulovic, David | de Reus, Herman P. M. | Rissman, Robert A. | Scarpini, Elio | Stefani, Alessandro | Soininen, Hilkka | Schröder, Johannes | Shaw, Leslie M. | Skinningsrud, Anders | Skrogstad, Brith | Spreer, Annette | Talib, Leda | Teunissen, Charlotte | Trojanowski, John Q. | Tumani, Hayrettin | Umek, Robert M. | Van Broeck, Bianca | Vanderstichele, Hugo | Vecsei, Laszlo | Verbeek, Marcel M. | Windisch, Manfred | Zhang, Jing | Zetterberg, Henrik | Blennow, Kaj
The cerebrospinal fluid (CSF) biomarkers amyloid β (Aβ)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer’s disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer’s Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program.
The program is open for laboratories using commercially available kits for Aβ, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Molndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples.
Forty laboratories participated. Twenty-six used INNOTESTenzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aβ-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aβ triplex (AβN-42, AβN-40, and AβN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories.
Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.
PMCID: PMC3710290  PMID: 21784349
Alzheimer’s disease; Cerebrospinal fluid; Biomarkers; External assurance; External control; Proficiency testing
6.  Influence of brain-derived neurotrophic factor and apolipoprotein E genetic variants on hemispheric and lateral ventricular volume of young healthy adults 
Brain-derived neurotrophic factor (BDNF) and apolipoprotein E (ApoE) are thought to be implicated in a variety of neuronal processes, including cell growth, resilience to noxious stimuli and synaptic plasticity. A Val to Met substitution at codon 66 in the BDNF protein has been associated with a variety of neuropsychiatric conditions. The ApoE4 allele is considered a risk factor for late-onset Alzheimer’s disease, but its effects on young adults are less clear. We sought to investigate the effects of those two polymorphisms on hemispheric and lateral ventricular volumes of young healthy adults.
Hemispheric and lateral ventricular volumes of 144 healthy individuals, aged 19–35 years, were measured using high resolution magnetic resonance imaging and data were correlated with BDNF and ApoE genotypes.
There were no correlations between BDNF or ApoE genotype and hemispheric or lateral ventricular volumes.
These findings indicate that it is unlikely that either the BDNF Val66Met or ApoE polymorphisms exert any significant effect on hemispheric or lateral ventricular volume. However, confounding epistatic genetic effects as well as relative insensitivity of the volumetric methods used cannot be ruled out. Further imaging analyses are warranted to better define any genetic influence of the BDNF Val6Met and ApoE polymorphism on brain structure of young healthy adults.
PMCID: PMC3119566  PMID: 21701702
apolipoprotein E; brain-derived neurotrophic factor; magnetic resonance imaging; polymorphism; volumetry
7.  Qualification of the analytical and clinical performance of CSF biomarker analyses in ADNI 
Acta neuropathologica  2011;121(5):597-609.
The close correlation between abnormally low pre-mortem cerebrospinal fluid (CSF) concentrations of amyloid-β1-42 (Aβ1–42) and plaque burden measured by amyloid imaging as well as between pathologically increased levels of CSF tau and the extent of neurode-generation measured by MRI has led to growing interest in using these biomarkers to predict the presence of AD plaque and tangle pathology. A challenge for the wide-spread use of these CSF biomarkers is the high variability in the assays used to measure these analytes which has been ascribed to multiple pre-analytical and analytical test performance factors. To address this challenge, we conducted a seven-center inter-laboratory standardization study for CSF total tau (t-tau), phospho-tau (p-tau181) and Aβ1–42 as part of the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Aliquots prepared from five CSF pools assembled from multiple elderly controls (n = 3) and AD patients (n = 2) were the primary test samples analyzed in each of three analytical runs by the participating laboratories using a common batch of research use only immunoassay reagents (INNO-BIA AlzBio3, xMAP technology, from Innogenetics) on the Luminex analytical platform. To account for the combined effects on overall precision of CSF samples (fixed effect), different laboratories and analytical runs (random effects), these data were analyzed by mixed-effects modeling with the following results: within center %CV 95% CI values (mean) of 4.0–6.0% (5.3%) for CSF Aβ1–42; 6.4–6.8% (6.7%) for t-tau and 5.5–18.0% (10.8%) for p-tau181 and inter-center %CV 95% CI range of 15.9–19.8% (17.9%) for Aβ1–42, 9.6–15.2% (13.1%) for t-tau and 11.3–18.2% (14.6%) for p-tau181. Long-term experience by the ADNI biomarker core laboratory replicated this degree of within-center precision. Diagnostic threshold CSF concentrations for Aβ1–42 and for the ratio t-tau/Aβ1–42 were determined in an ADNI independent, autopsy-confirmed AD cohort from whom ante-mortem CSF was obtained, and a clinically defined group of cognitively normal controls (NCs) provides statistically significant separation of those who progressed from MCI to AD in the ADNI study. These data suggest that interrogation of ante-mortem CSF in cognitively impaired individuals to determine levels of t-tau, p-tau181 and Aβ1–42, together with MRI and amyloid imaging biomarkers, could replace autopsy confirmation of AD plaque and tangle pathology as the “gold standard” for the diagnosis of definite AD in the near future.
PMCID: PMC3175107  PMID: 21311900
Alzheimer’s Disease Neuroimaging Initiative; Cerebrospinal fluid; Amyloid-β1-42; Total tau; p-tau181; Interlaboratory study; Mixed-effects modeling
8.  Update on the Biomarker Core of the Alzheimer's Disease Neuroimaging Initiative Subjects 
Here we review progress by the Penn Biomarker Core in the Alzheimer's disease Neuroimaging Initiative (ADNI) towards developing a pathological cerebrospinal fluid (CSF) and plasma biomarker signature for mild Alzheimer's disease (AD) as well as a biomarker profile that predicts conversion of mild cognitive impairment (MCI) and/or normal control (NC) subjects to AD. The Penn Biomarker Core also collaborated with other ADNI Cores to integrate data across ADNI to temporally order changes in clinical measures, imaging data and chemical biomarkers that serve as mileposts and predictors of the conversion of NC to MCI as well as MCI to AD, and the progression of AD.
Initial CSF studies by the ADNI Biomarker Core revealed a pathological CSF biomarker signature of AD defined by the combination of Aβ1-42 and total tau (T-tau) that effectively delineates mild AD in the large multisite prospective clinical investigation conducted in ADNI. This signature appears to predict conversion from MCI to AD. Data fusion efforts across ADNI Cores generated a model for the temporal ordering of AD biomarkers which suggests that Aβ amyloid biomarkers become abnormal first, followed by changes in neurodegenerative biomarkers (CSF tau, FDG-PET, MRI) and the onset of clinical symptoms. The timing of these changes varies in individual patients due to genetic and environmental factors that increase or decrease an individual's resilience in response to progressive accumulations of AD pathologies. Further studies in ADNI will refine this model and render the biomarkers studied in ADNI more applicable to routine diagnosis and to clinical trials of disease modifying therapies.
PMCID: PMC2867838  PMID: 20451871
Alzheimer's disease; cerebrospinal fluid; plasma; biomarkers; mild cognitive impairment
9.  Perfusion Imaging with SPECT in the Era of Pathophysiology-Based Biomarkers for Alzheimer's Disease 
SPECT allows registration of regional cerebral blood flow (rCBF) which is altered in a characteristic temporoparietal pattern in Alzheimer's Dementia. Numerous studies have shown the diagnostic value of reduced cerebral blood flow and metabolic changes using perfusion SPECT and FDG-PEPT in AD diagnosis as well as in differential diagnosis against frontotemporal dementia, dementia with Lewy bodies and vascular disease. Recently more pathophysiology-based biomarkers in CSF and Amyloid-PET tracers have been developed that probably have a higher diagnostic accuracy than the more indirect rCBF changes seen in perfusion SPECT. In the paper review, we describe recent advances in AD biomarkers as well as improvements in the SPECT technique.
PMCID: PMC3010620  PMID: 21197480
10.  Phagocytosis and LPS alter the maturation state of β-amyloid precursor protein and induce different Aβ peptide release signatures in human mononuclear phagocytes 
The classic neuritic β-amyloid plaque of Alzheimer's disease (AD) is typically associated with activated microglia and neuroinflammation. Similarly, cerebrovascular β-amyloid (Aβ) deposits are surrounded by perivascular macrophages. Both observations indicate a contribution of the mononuclear phagocyte system to the development of β-amyloid.
Human CD14-positive mononuclear phagocytes were isolated from EDTA-anticoagulated blood by magnetic activated cell sorting. After a cultivation period of 72 hours in serum-free medium we assessed the protein levels of amyloid precursor protein (APP) as well as the patterns and the amounts of released Aβ peptides by ELISA or one-dimensional and two-dimensional urea-based SDS-PAGE followed by western immunoblotting.
We observed strong and significant increases in Aβ peptide release upon phagocytosis of acetylated low density lipoprotein (acLDL) or polystyrene beads and also after activation of the CD14/TLR4 pathway by stimulation with LPS. The proportion of released N-terminally truncated Aβ variants was increased after stimulation with polystyrene beads and acLDL but not after stimulation with LPS. Furthermore, strong shifts in the proportions of single Aβ1-40 and Aβ2-40 variants were detected resulting in a stimulus-specific Aβ signature. The increased release of Aβ peptides was accompanied by elevated levels of full length APP in the cells. The maturation state of APP was correlated with the release of N-terminally truncated Aβ peptides.
These findings indicate that mononuclear phagocytes potentially contribute to the various N-truncated Aβ variants found in AD β-amyloid plaques, especially under neuroinflammatory conditions.
PMCID: PMC2958903  PMID: 20929546
11.  cNEUPRO: Novel Biomarkers for Neurodegenerative Diseases 
“clinical NEUroPROteomics of neurodegenerative diseases” (cNEUPRO) is a Specific Targeted Research Project (STREP) within the sixth framework program of the European Commission dedicated to the search for novel biomarker candidates for Alzheimer's disease and other neurodegenerative diseases. The ultimate goal of cNEUPRO is to identify one or more valid biomarker(s) in blood and CSF applicable to support the early and differential diagnosis of dementia disorders. The consortium covers all steps required for the discovery of novel biomarker candidates such as acquisition of high quality CSF and blood samples from relevant patient groups and controls, analysis of body fluids by various methods, and finally assay development and assay validation. Here we report the standardized procedures for diagnosis and preanalytical sample-handling within the project, as well as the status of the ongoing research activities and some first results.
PMCID: PMC2945639  PMID: 20886057
12.  Combined Analysis of CSF Tau, Aβ42, Aβ1–42% and Aβ1–40ox% in Alzheimer's Disease, Dementia with Lewy Bodies and Parkinson's Disease Dementia 
We studied the diagnostic value of CSF Aβ42/tau versus low Aβ1–42% and high Aβ1–40ox% levels for differential diagnosis of Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), respectively. CSF of 45 patients with AD, 15 with DLB, 21 with Parkinson's disease dementia (PDD), and 40 nondemented disease controls (NDC) was analyzed by Aβ-SDS-PAGE/immunoblot and ELISAs (Aβ42 and tau). Aβ42/tau lacked specificity in discriminating AD from DLB and PDD. Best discriminating biomarkers were Aβ1–42% and Aβ1–40ox% for AD and DLB, respectively. AD and DLB could be differentiated by both Aβ1–42% and Aβ1–40ox% with an accuracy of 80% at minimum. Thus, we consider Aβ1–42% and Aβ1–40ox% to be useful biomarkers for AD and DLB, respectively. We propose further studies on the integration of Aβ1–42% and Aβ1–40ox% into conventional assay formats. Moreover, future studies should investigate the combination of Aβ1–40ox% and CSF alpha-synuclein for the diagnosis of DLB.
PMCID: PMC2938459  PMID: 20862375
13.  Cerebrospinal Fluid Biomarker Signature in Alzheimer’s Disease Neuroimaging Initiative Subjects 
Annals of neurology  2009;65(4):403-413.
Develop a cerebrospinal fluid biomarker signature for mild Alzheimer’s disease (AD) in Alzheimer’s Disease Neuroimaging Initiative (ADNI) subjects.
Amyloid-β 1 to 42 peptide (Aβ1-42), total tau (t-tau), and tau phosphorylated at the threonine 181 were measured in (1) cerebrospinal fluid (CSF) samples obtained during baseline evaluation of 100 mild AD, 196 mild cognitive impairment, and 114 elderly cognitively normal (NC) subjects in ADNI; and (2) independent 56 autopsy-confirmed AD cases and 52 age-matched elderly NCs using a multiplex immunoassay. Detection of an AD CSF profile for t-tau and Aβ1-42 in ADNI subjects was achieved using receiver operating characteristic cut points and logistic regression models derived from the autopsy-confirmed CSF data.
CSF Aβ1-42 was the most sensitive biomarker for AD in the autopsy cohort of CSF samples: receiver operating characteristic area under the curve of 0.913 and sensitivity for AD detection of 96.4%. In the ADNI cohort, a logistic regression model for Aβ1-42, t-tau, and APOε4 allele count provided the best assessment delineation of mild AD. An AD-like baseline CSF profile for t-tau/Aβ1-42 was detected in 33 of 37 ADNI mild cognitive impairment subjects who converted to probable AD during the first year of the study.
The CSF biomarker signature of AD defined by Aβ1-42 and t-tau in the autopsy-confirmed AD cohort and confirmed in the cohort followed in ADNI for 12 months detects mild AD in a large, multisite, prospective clinical investigation, and this signature appears to predict conversion from mild cognitive impairment to AD.
PMCID: PMC2696350  PMID: 19296504
14.  Erythropoietin therapy for acute stroke is both safe and beneficial. 
Molecular Medicine  2002;8(8):495-505.
BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.
PMCID: PMC2040012  PMID: 12435860

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