Volume 25, Number 4, August 2011

Pages 571-581
Simon D’Alton, Daniel R. George (Handling Associate Editor: D. Allan Butterfield)
Changing Perspectives on Alzheimer’s Disease: Thinking Outside the Amyloid Box
Abstract: The past two decades have seen an explosion in funding and research for Alzheimer’s disease (AD), which has resulted in a wealth of data exploring the potential underlying processes, particularly with regard to amyloid-β (Aβ).   However, to date, therapies based on this knowledge have not been forthcoming.  In seeking an explanation for our current pharmacological failures, it has become clear that amyloid is only one part of a multi-factorial disease process incorporating a wealth of deleterious factors.  Additionally, there is strong evidence that the initial production of Aβ is part of the evolutionarily conserved stress response, triggered by a host of upstream factors highly altered in aging.  Taken together, these observations place Aβ in a drastically different context, with toxicity occurring secondarily to upstream deleterious factors and rendering current therapeutic strategies oversimplified.  This re-conceptualization necessitates a paradigm shift in our scientific and social response to AD, placing a greater emphasis on upstream interventions and public health awareness of the measures that can be taken by most individuals to reduce the risk of AD.  With the increasing prevalence of AD and the realization that disease-modifying drugs may not be available in the near future, it is the responsibility of science to better communicate the worth of preventative healthcare measures to the public.

Pages 583-594
Toni T. Seppälä, Anne M. Koivisto, Päivi Hartikainen, Seppo Helisalmi, Hilkka Soininen, Sanna-Kaisa Herukka (Handling Associate Editor: Henrik Zetterberg)
Longitudinal Changes of CSF Biomarkers in Alzheimer’s Disease
Abstract: Longitudinal changes of cerebrospinal fluid (CSF) biomarkers in Alzheimer’s disease (AD) have been studied, but there are few consistent conclusions and even less is known about their variation during the different stages of the disease. We hypothesized that changes in CSF biomarker values would correlate with the progression of the cognitive decline in AD. One hundred and thirty-one memory clinic patients [56 AD, 57 mild cognitive impairment (MCI), 10 other neurological disorders, eight unimpaired subjects] underwent a clinical follow-up with repeated Mini-Mental Status Examination (MMSE) tests and two lumbar punctures with a median interval of 3 years. Levels of CSF amyloid-β (Aβ) 42, tau, and p-tau-181 were measured using commercially available ELISA. Twenty-one of the MCI subjects progressed to AD, whereas 26 subjects remained stable and 56 subjects had AD already at the baseline. The subjects displaying the most rapid MMSE decline rate had the lowest baseline Aβ42, highest tau, and highest p-tau-181 CSF concentrations. An annual decrease of 2.20 pg/ml/year in the CSF p-tau-181 concentration was seen in AD-AD patients (p=0.001). The difference was significant compared to stable MCI-MCI (increase of 1.24 pg/ml/year, p=0.001) and cognitively healthy (increase of 0.84 pg/ml/year, p=0.013) subjects (p for group difference 0.004). The decrease rate of p-tau-181 correlated with the MMSE decrease rate in AD subjects (r=0.579, p<0.001). The CSF Aβ42 level decreased in the AD-AD group (decrease 11.9 pg/ml/year, p<0.001). Concentrations of hyperphosphorylated tau decline in the late stages of the AD process. The decrease of p-tau-181 appears to correlate with cognitive functioning and probably reflects neuronal loss. More longitudinal studies of CSF biomarker dynamics are needed, especially in patients during the preclinical stage of the disease.

Pages 595-603
Hans Wouters, Jos Van Campen, Bregje Appels, Robert Lindeboom, Maarten Buiter, Rob J. de Haan, Aeilko H. Zwinderman, Willem A. van Gool, Ben Schmand
Does Adaptive Cognitive Testing Combine Efficiency with Precision? Prospective Findings
Abstract: Longer cognitive tests, such as the Alzheimer’s disease assessment scale (ADAS-cog) or the Cambridge cognitive examination (CAMCOG), are more precise but less efficient than briefer tests, such as the Mini Mental State Examination (MMSE). We examined if computerized adaptive testing (CAT) of cognitive impairment can combine brevity with precision by tailoring a precise test to each individual patient. We conducted a prospective study of 84 participants [normal aging, n = 41; mild cognitive impairment (MCI), n = 21; dementia, n = 22]. CAT estimated a participant’s ability during testing by selecting only items of appropriate difficulty from either the CAMCOG or the CAMCOG supplemented with ADAS-cog items and neuropsychological tests (the CAMCOG-Plus). After tailored testing with CAT, the remaining CAMCOG and CAMCOG-Plus items not selected by CAT were administered. The time needed to complete the CAT was compared to that needed for the whole CAMCOG and CAMCOG-Plus. Results showed that testing time reductions achieved with CAT were 37% or more compared to the whole CAMCOG and 55% or more compared to the whole CAMCOG-Plus. Estimated ability levels with CAT were in excellent agreement with those based on the whole CAMCOG and CAMCOG-Plus (intraclass correlations 0.99 and 0.98, respectively). Diagnostic accuracy of detecting mild dementia and MCI seemed better for the CAT administered tests than for the MMSE, but the differences were not significant. We conclude that adaptive testing combines brevity with precision, especially in grading the severity of cognitive impairment.

Supplementary Data for Wouters et al. article (PDF)

Pages 605-622
Robert G. Nagele, Peter M. Clifford, Gilbert Siu, Eli C. Levin, Nimish K. Acharya, Min Han, Mary C. Kosciuk, Venkat Venkataraman, Semah Zavareh, Shabnam Zarrabi, Kristin Kinsler, Nikhil Patel, Eric P. Nagele, Jacqueline Dash, Hoau Y. Wang, Andrew Levitas (Handling Associate Editor: Thomas Shea)
Brain-Reactive Autoantibodies Prevalent in Human Sera Increase Intraneuronal Amyloid-β1-42 Deposition
Abstract: Previous studies have reported immunoglobulin-positive neurons in Alzheimer’s disease (AD) brains, an observation indicative of blood-brain barrier (BBB) breakdown.  Recently, we demonstrated the nearly ubiquitous presence of brain-reactive autoantibodies in human sera.  The significance of these observations to AD pathology is unknown.  Here, we show that IgG-immunopositive neurons are abundant in brain regions exhibiting AD pathology, including intraneuronal amyloid-β42 (Ab42) and amyloid plaques, and confirm by western analysis that brain-reactive autoantibodies are nearly ubiquitous in human serum.  To investigate a possible interrelationship between neuronal antibody binding and Aβ pathology, we tested the effects of human serum autoantibodies on the intraneuronal deposition of soluble Aβ42 peptide in adult mouse neurons in vitro (organotypic brain slice cultures).  Binding of human autoantibodies to mouse neurons dramatically increased the rate and extent of intraneuronal Aβ42 accumulation in the mouse cerebral cortex and hippocampus.  Additionally, individual sera exhibited variable potency related to their capacity to enhance intraneuronal Aβ42 peptide accumulation and immunolabel neurons in AD brain sections.  Replacement of human sera with antibodies targeting abundant neuronal surface proteins resulted in a comparable enhancement of Aβ42 accumulation in mouse neurons.  Overall, results suggest that brain-reactive autoantibodies are ubiquitous in the blood and that a defective BBB allows these antibodies to access the brain interstitium, bind to neuronal surfaces and enhance intraneuronal deposition of Aβ42 in AD brains.  Thus, in the context of BBB compromise, brain-reactive autoantibodies may be an important risk factor for the initiation and/or progression of AD as well as other neurodegenerative diseases.

Pages 623-633
Eugenio Barone*, Fabio Di Domenico*, Giovanna Cenini, Rukhsana Sultana, Raffaella Coccia, Paolo Preziosi, Marzia Perluigi, Cesare Mancuso, D. Allan Butterfield *Both the authors contributed equally.
Oxidative and Nitrosative Modifications of Biliverdin Reductase-A in the Brain of Subjects with Alzheimer’s Disease and Amnestic Mild Cognitive Impairment
Abstract: Biliverdin reductase-A (BVR-A) is a pleiotropic enzyme and plays pivotal role in the antioxidant defense against free radicals as well as in cell homeostasis. Together with heme oxygenase, BVR-A forms a powerful system involved in the cell stress response during neurodegenerative disorders including Alzheimer’s disease (AD), whereas due to the serine/threonine/tyrosine kinase activity the enzyme regulates glucose metabolism and cell proliferation. In this paper, we report results that demonstrate BVR-A undergoes post-translational oxidative and nitrosative modifications in the hippocampus, but not cerebellum, of subjects with AD and amnestic mild cognitive impairment (MCI). A significant increase of nitrated BVR-A was demonstrated only in AD and MCI hippocampi, whereas no significant modifications were found in cerebellar tissue. In addition, a significant reduction in protein carbonyl-derivatives of BVR-A was found in both AD and MCI hippocampi (15% and 18%, respectively). Biliverdin reductase-bound 4-hydroxynonenals were not modified in hippocampi and cerebella from AD and MCI subjects. These results supported the hypothesis of a prevalence of nitrosative stress-induced modifications on BVR-A structure, and this evidence was confirmed by a significant upregulation of inducible nitric oxide synthase in hippocampal tissue of subjects with AD and MCI that was not present in cerebellum. In conclusion, nitrosative stress-induced modifications on hippocampal BVR-A are an early event in the pathogenesis of AD since they appear also in MCI subjects and could contribute to the antioxidant and metabolic derangement characteristic of these neurodegenerative disorders.

Pages 635-644
James C. Turton*, James Bullock*, Christopher Medway, Hui Shi, Kristelle Brown, Olivia Belbin, Noor Kalsheker, Minerva M. Carrasquillo, Dennis W. Dickson, Neill R. Graff-Radford, Ronald C. Petersen, Steven G. Younkin, Kevin Morgan (Handling Associate Editor: Francesco Panza) * These authors contributed equally to this work.
Investigating Statistical Epistasis in Complex Disorders
Abstract: The missing heritability exhibited by late-onset Alzheimer’s disease is unexplained and has been partly attributed to epistatic interaction. Methods available to explore this are often based on logistic regression and allow for determination of deviation from an expected outcome as a result of statistical epistasis. Three such methodologies including Synergy Factor and the PLINK modules, --epistasis and --fast-epistasis, were applied to study an epistatic interaction between interleukin-6 and interleukin-10. The models analyzed consisted of two synergistic interactions (SF ≈ 4.2 and 1.6) and two antagonistic interactions (SF ≈ 0.9 and 0.6). As with any statistical test, power to detect association is paramount; and most studies will be underpowered for the task. However, the availability of large sample sizes through genome-wide association studies make it feasible to examine approaches for determining epistatic interactions. This study documents the sample sizes needed to achieve a statistically significant outcome from each of the methods examined and discusses the limitations/advantages of the chosen approaches.

Pages 645-653
Elisa Canepa, Roberta Borghi, Jose Viña, Nicola Traverso, Juan Gambini, Cinzia Domenicotti, Umberto M. Marinari, Giuseppe Poli, Maria A. Pronzato, Roberta Ricciarelli (Handling Associate Editor: Luigi Iuliano)
Cholesterol and Amyloid-β: Evidence for a Cross-Talk between Astrocytes and Neuronal Cells
Abstract: Accumulating data supports the concept that alterations of cholesterol metabolism might influence the development of Alzheimer’s disease (AD), a neurodegenerative disorder characterized by progressive accumulation of amyloid-β (Aβ) peptides in the brain. Changes in the neuronal production of Aβ have been described as a function of cholesterol levels, thus suggesting a causal link between cholesterol homeostasis dysregulation and AD pathogenesis. Under physiological conditions, cholesterol uptake in the brain is efficiently prevented by the blood-brain barrier, and mature neurons are thought to rely on glial cells for their cholesterol supply. In the present study, we tested the hypothesis that Aβ may serve as a signaling molecule capable of informing the astroglial network about the neuronal need for cholesterol. Collectively, our data bolster this hypothesis and demonstrate, for the first time, that Aβ42 exerts an inhibitory effect on the expression of the cholesterol transporter ABCA1 in cultured astrocytes. Accordingly, we also show that ABCA1 expression is reduced in the brain of AβPP/PS1 transgenic mice. These results provide a biological function for Aβ peptides and may help to define the pathogenic relationship between cholesterol metabolism in brain and AD.

Pages 655-669
Wei-Qin Zhao*, Dawn Toolan*, Robert W. Hepler, Abigail L. Wolfe, Yuanjiang Yu, Eric Price, Victor N. Uebele, Joel B. Schachter, Ian J. Reynolds, John J. Renger, Alexander McCampbell, William J. Ray * Authors contributed equally to the study.
High Throughput Monitoring of Amyloid-β42 Assembly into Soluble Oligomers Achieved by Sensitive Conformation State-Dependent Immunoassays
Abstract: Accumulation of small soluble assemblies of amyloid-β (Aβ)42 in the brain is thought to play a key role in the pathogenesis of Alzheimer's disease. As a result, there has been much interest in finding small molecules that inhibit the formation of synaptotoxic Aβ42 oligomers that necessitates sensitive methods for detecting the initial steps in the oligomerization of Aβ42. Modeling suggests that oligomerized Aβ42 adopts a conformation in which the C-terminus is embedded in the center, whereas the N-terminus is exposed at the periphery of the oligomer.  Here we report that an inverse change in Aβ42 C-terminal and N-terminal epitope accessibility provides the basis of a sensitive method for assessing early steps in Aβ42 oligomerization. Using ELISA and AlphaLISA, we found that Aβ42 C-terminal immunoreactivity decreased in a time- and concentration-dependent manner under conditions favoring oligomerization. This reduction was accompanied by an increase in the N-terminal immunoreactivity, suggesting that assemblies with multiple exposed N-terminal epitopes were detected. Importantly the assay generates a robust window between monomers and oligomers at as low as 1 nM Aβ42. Using this assay, known oligomerization inhibitors produced a dose-dependent unmasking of the Aβ42 C-terminal epitope.  After automation, the assay proved to be highly reproducible and effective for high throughput screening of small molecules that inhibit Aβ42 oligomerization.

Pages 671-677
Elisabeth M.C. Schrijvers, Nese Direk, Peter J. Koudstaal, Clemens Kirschbaum, Albert Hofman, Henning Tiemeier, Monique M.B. Breteler
Associations of Serum Cortisol with Cognitive Function and Dementia: The Rotterdam Study
Abstract: Higher levels of cortisol have been observed in persons with cognitive decline and dementia. It is unknown whether these higher levels are a cause or a consequence of disease. We investigated whether morning levels of serum cortisol were associated with cognitive function, cognitive decline, and the risk of dementia and Alzheimer’s disease in the Rotterdam Study, a large prospective population based cohort study. Cortisol levels were assessed in fasting blood serum in 3341 participants, who were free of dementia at baseline (1997-1999). Cognitive function was assessed with a dedicated neuropsychological test battery at baseline and at follow-up examination (2002-2004). In addition, the cohort was continuously monitored for incident dementia until January 1, 2007. After a mean follow-up of 7.1 years, 243 participants had developed dementia, of whom 210 were diagnosed with Alzheimer’s disease. Morning serum levels of cortisol were neither related to cognitive function at baseline, nor to annual cognitive decline. There was no relation between serum levels of cortisol and the risk of developing dementia or Alzheimer’s disease. These results suggest that that morning serum cortisol is not a causal factor in the development of dementia.

Pages 679-694
Verena C. Buschert, Uwe Friese, Stefan J. Teipel, Philine Schneider, Wibke Merensky, Dan Rujescu, Hans-Jürgen Möller, Harald Hampel, Katharina Buerger
Effects of a Newly Developed Cognitive Intervention in Amnestic Mild Cognitive Impairment and mild Alzheimer’s disease: A Pilot Study
Abstract: Recent studies have shown that patients with Alzheimer’s disease (AD) and its possible prodromal stage mild cognitive impairment benefit from cognitive interventions. Few studies so far have used an active control condition and determined effects in different stages of disease. We evaluated a newly developed 6-month group-based multicomponent cognitive intervention in a randomized controlled pilot study on subjects with amnestic mild cognitive impairment (aMCI) and mild AD patients. Forty-three subjects with aMCI and mild AD were recruited. Primary outcome measures were change in global cognitive function as determined by the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog) and the Mini Mental Status Examination (MMSE). Secondary outcomes were specific cognitive and psychopathological ratings. Thirty-nine patients were randomized to intervention groups (IGs: 12 aMCI, 8 AD) and active control groups (CGs: 12 aMCI, 7 AD). At the end of the study, we found significant improvements in the IGMCI compared to the CGMCI in the ADAS-cog (p=0.02) and for the secondary endpoint Montgomery Asberg Depression Rating Scale (MADRS) (p<0.01) Effects on the MMSE score showed a non-significant trend (p=0.07). In AD patients, we found no significant effect of intervention on the primary outcome measures. In conclusion, these results suggest that participation in a 6-month cognitive intervention can improve cognitive and non-cognitive functions in aMCI subjects. In contrast, AD patients showed no significant benefit from intervention. The findings in this small sample support the use of the intervention in larger scales studies with an extended follow-up period to determine long-term effects.

Pages 695-706
Stefan Förster*, Verena C. Buschert*, Hans-Georg Buchholz, Stefan J. Teipel, Uwe Friese, Christian Zach, Christian la Fougere, Axel Rominger, Alexander Drzezga, Harald Hampel, Peter Bartenstein, Katharina Buerger *Both authors contributed equally
Effects of a 6-Month Cognitive Intervention Program on Brain Metabolism in Amnestic Mild Cognitive Impairment and Mild Alzheimer’s Disease
Abstract: The effect of cognitive intervention on brain metabolism in Alzheimer’s disease (AD) is largely unexplored. Therefore, we aimed to investigate clinical cognitive parameters and 18FDG PET to test for effects of a cognitive intervention in patients with amnestic mild cognitive impairment (aMCI) or mild AD. Patients with aMCI (n=24) or mild AD (n=15) were randomly assigned either to cognitive intervention groups (IGs), receiving weekly sessions of group-based multicomponent cognitive intervention, or active control groups (CGs), receiving pencil-paper exercises for self-study. We obtained resting-state FDG-PET scans and neuropsychological testing at baseline and after six-months.  Normalized FDG-PET images were analyzed using voxel-based SPM5 approaches to determine longitudinal changes, group-by-time interactions, and correlations with neuropsychological outcome parameters. Primary global cognitive outcome was determined by analyses of covariance with MMSE and ADAS-cog scores as dependent measures. Both, aMCI and AD subgroups of CGs showed widespread bilateral cortical declines in FDG uptake, while the AD subgroup of IGs showed discrete decline or rather no decline in case of the aMCI subgroup. Group by time analyses revealed strongest attenuation of metabolic decline in the aMCI subgroup of the IGs, involving left superior temporal- and anterior cingulate gyrus. However, correlation analyses revealed only weak non-significant associations between increased FDG uptake and improvement in primary or secondary outcome parameters. Concurrently, there was significant improvement in global cognitive status in the aMCI subgroup of the IGs. A six-month cognitive intervention imparted cognitive benefits in patients with aMCI, which were concurrent with an attenuated decline of glucose metabolism in cortical regions affected by neurodegenerative AD.

Pages 707-717
Kathryn P. Riley, Gregory A. Jicha, Daron Davis, Erin L. Abner, Gregory E. Cooper, Nancy Stiles,  Charles D. Smith, Richard J. Kryscio, Peter T. Nelson, Linda J. Van Eldik, Frederick A. Schmitt
Prediction of Preclinical Alzheimer’s Disease: Longitudinal Rates of Change in Cognition
Abstract: Preclinical Alzheimer’s disease (pAD) reflects neuropathological findings of AD in cognitively normal subjects. The present study represents an effort to determine if differences could be identified in the longitudinal patterns of cognitive performance in persons classified as pAD compared to those who did not meet criteria for AD at autopsy. We included 121 subjects who were cognitively normal from baseline through their last assessment before death and who underwent autopsy. Participants were classified into two groups: pathologically normal (PN; NIA-Reagan low or no-likelihood of AD, n=89) and preclinicalAD (pAD; NIA-Reagan criteria of intermediate or high-likelihood of AD in the absence of clinical dementia symptoms, n=32) followed for a mean 7.5 years prior to death.  Longitudinal rates and patterns of change in scores on a standard cognitive battery were compared between these two groups. While cognitive results at baseline and last evaluations revealed no clear cross sectional group differences after adjustment for age, ApoE status, education, and gender, statistically significant differences between the pAD and PN groups in slope of decline were seen on a composite score of cognitive function. Further analyses showed three components of this score reached significance: constructional praxis, delayed recall of a word list, and category verbal fluency. Despite being clinically viewed as normal at enrollment and at the final exam, there are significant differences in rates of cognitive decline in participants classified as pAD compared to those without this pathology.  Longitudinal changes in slope of decline in specific cognitive test measures can serve as non-invasive methods for the detection of pAD.

Pages 719-726
Mauro Silvestrini, Giovanna Viticchi, Lorenzo Falsetti, Clotilde Balucani, Fabrizio Vernieri, Raffaella Cerqua, Simona Luzzi, Marco Bartolini, Leandro Provinciali (Handling Associate Editor: Jack de la Torre)
The Role of Carotid Atherosclerosis in Alzheimer’s Disease Progression
Abstract: The aim of this 12-month prospective study was to establish whether severe internal carotid artery stenosis is associated with faster progression of the cognitive impairment in patients with Alzheimer’s disease (AD). Four hundred and eleven patients with AD underwent extracranial carotid Doppler ultrasound evaluation. Cerebrovascular reactivity to hypercapnia was measured by means of the breath-holding index (BHI) in those with severe carotid artery stenosis using transcranial Doppler ultrasonography. Cognitive status was quantified with the Mini Mental State Evaluation (MMSE). Ninety-eight patients had severe carotid artery stenosis, 41 right (group 1), and 57 left (group 2), while 313 had no significant stenosis (group 3). Group 1 and 2 patients showed an increased probability compared with group 3 patients to develop severe dementia (MMSE scores < 21) during the 12-month follow-up period: OR 2.36 (95% CI: 1.14-4.87) and OR 4.90 (95% CI: 2.65-9.04), respectively (p < 0.05, multiple logistic regression analysis). A BHI value ipsilateral to the stenosis < 0.69 predicted a worse MMSE score at 12 months irrespective of the side of the stenosis. These findings suggest that severe internal carotid artery stenosis can be considered as a marker of a faster rate of progression of the cognitive decline in AD. They also indicate that cerebral hemodynamic evaluation could be applied to identify patients at higher risk of rapid cognitive decline, who may benefit from aggressive treatment, and warrant investigation of the advantages of carotid revascularization procedures in these patients.

Pages 727-737
Lorena Arranz, Nuria M. De Castro, Isabel Baeza, Lydia Giménez-Llort, Mónica De la Fuente
Effect of Environmental Enrichment on the Immunoendocrine Aging of Male and Female Triple-Transgenic 3xTg-AD Mice for Alzheimer’s Disease
Abstract: We have previously shown that 3xTgAD mice (triple-transgenic mice for Alzheimer’s disease, harboring PS1M146V, AβPPSwe, tauP301L transgenes) suffer detrimental changes in some key lymphocyte functions, described as health and longevity markers, with males being more affected than females and showing higher mortality rates. In the present work, 3xTgAD and wild type 129/C57BL6 male and female non- and environmentally enriched mice were used. The enriched environment (EE) began in the adulthood (6 months) and lasted for 5.5 months. The animals were sacrificed at advanced stages of the disease (15 month-old), and spleen, thymus, and plasma were obtained. The results indicate that 3xTg-AD males are especially benefitted from EE exposure, as shown by the improvement in lymphocyte functional activities such as chemotaxis and natural killer cytotoxicity, as well as in plasma corticosterone levels. By contrast, wild type females seem to be highly sensitive to EE removal, as regards the proliferation capacity of lymphocytes and their intracellular glutathione content. These results support the relevance of gender differences in AD when screening for new strategies for the control of the disease, and suggest that active life, by means of EE, should be maintained until natural death in order to preserve all the positive effects that this strategy exerts on the immune system.

Pages 739-745
Rüdiger Zimmermann, Natalia Lelental, Oliver Ganslandt, Juan Manuel Maler, Johannes Kornhuber, Piotr Lewczuk (Handling Associate Editor: Julius Popp)
Preanalytical Sample Handling and Sample Stability Testing for the Neurochemical Dementia Diagnostics
Abstract: Preanalytical sample handling and storage procedures play an extremely important role in reliably measuring neurochemical dementia diagnostics (NDD) biomarkers: Aβ1-40, Aβ1-42, Tau, and pTau181. To test different handling and storage conditions, the following protocols were applied: (a) storage at room temperature for one week, (b) deep-freezing and thawing up to three cycles, (c) deep-freezing, thawing and keeping under +4°C for two days before the analysis, and (d) long-term stability of a deeply frozen sample. Between the first and the seventh day of the storage at room temperature, the percentage of the concentrations (compared to the starting concentrations) fluctuated: 104.3-105.3, 97.6-93.2, 100.6-96.8, and 97.9-90.2 for Aβ1-40, Aβ1-42, Tau, and pTau181, respectively. Re-freezing cycles resulted in the percentage fluctuations of the concentrations: 101.1-105.5, 95.4-99.7, 98.3-100.0, and 100.5-101.4 for Aβ1-40, Aβ1-42, Tau, and pTau181, respectively. Keeping previously frozen/thawed samples under +4°C for two days resulted in the percentage differences of the concentrations: +15.9, +2.2, -1.1, and -0.1 for Aβ1-40, Aβ1-42, Tau, and pTau181, respectively. During long-term stability, the coefficients of linear correlation (R2) were: Aβ1-40, 0.007; Aβ1-42, 0.02; Tau, 0.011; and pTau181, 0.02, and the corresponding inter-assay coefficients of variation: 13.9%, 13.9%, 11.0%, and 10.7% for Aβ1-40, Aβ1-42, Tau, and pTau181, respectively. We conclude that the NDD biomarkers are relatively stable when the cerebrospinal fluid sample is kept at room temperature for about four days; one or two thawing/refreezing cycles do not profoundly affect the biomarkers concentrations, however three cycles result in increased unsystematic variation. The four biomarkers seem to be stable in a sample stored deeply frozen for more than two years.

Pages 747-761
Lyudmyla Koval, Olena Lykhmus, Olena Kalashnyk, Nataliya Bachinskaya, Ganna Kravtsova, Mariya Soldatkina, Marios Zouridakis, Christos Stergiou, Socrates Tzartos, Victor Tsetlin, Sergiy Komisarenko, Maryna Skok
The Presence and Origin of Autoantibodies Against α4 and α7 Nicotinic Acetylcholine Receptors in the Human Blood: Possible Relevance to Alzheimer’s Pathology
Abstract: Alzheimer’s disease (AD) is characterized by a loss of α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) in the brain and severe memory impairments. Previously, we found that antibodies elicited against extracellular domain of α7 nAChR subunit decreased the number of α7 nAChRs in the brains of mice and impaired episodic memory. Here we show that antibodies capable to bind α7(1-208) are present in the blood of both healthy humans and AD patients.  In healthy individuals, their capacity to compete with [125-I]-α-bungarotoxin for the binding to α7(1-208) increased with age. The level of such antibodies was significantly elevated in  children with severe form of obstructive bronchitis and in mice injected with Lewis lung carcinoma cells expressing both α4β2 and α7 nAChRs. Elevated antibody levels were accompanied with decreased surface nAChRs on the blood lymphocytes of children and of mice immunized with α7(1-208). Among AD patients, the level of α7 nAChR-specific antibodies was significantly larger in people 62.5±1.5 years old with moderate or severe AD stages (15.2±1.3 MMSE scores) compared to those of 76±1.5 years old with the mild (22.7±0.1 MMSE scores) AD stage. We concluded that α7(1-208) nAChR-specific antibodies found in the human blood are formed as a result of common infections accompanied with the destruction of respiratory epithelium.  Elevated blood plasma levels of α7(1-208) nAChR-specific antibodies are characteristic for the early-onset AD and, therefore,  are suggested as one of the risk factors for the development of this form of the disease.