Volume 44, Number 4, 2015

Pages 1039-1062
Roger M. Lane, Taher Darreh-Shori
Understanding the Beneficial and Detrimental Effects of Donepezil and Rivastigmine to Improve their Therapeutic Value
Abstract: Cholinesterase enzymes metabolize acetylcholine (ACh). Inhibition of acetylcholinesterase (AChE) in damaged but functional cholinergic synapses in the brains of dementia patients increases intrasynaptic ACh. This enhances cholinergic neurotransmission and improves cognition. There is a window of opportunity for this symptomatic treatment effect that opens and closes during the course of dementia depending on when significant synaptic damage occurs. Cholinesterases also metabolize extrasynaptic ACh with butyrylcholinesterase (BuChE) apparently playing the major dynamic role in extracellular ACh homeostasis. Extracellular ACh plays a key regulatory role in controlling the reactivity and functional states of non-excitable cells, such as neuroglia. Current inhibitors of cholinesterases (ChEIs) have similar effects on intrasynaptic ACh, but differ markedly in abilities to upregulate extracellular AChE, inhibit BuChE, and influence the fibrilization of amyloid-β peptides. Importantly, ChEIs can have detrimental disease modifying effects in particular individuals characterized by age, gender, and genotype. In contrast, preliminary evidence suggests that the right dose of the right ChEI in the right patient might significantly slow the progression of neurodegenerative processes. For a particular patient, understanding the condition of cholinergic synapses and the reactivity and functional status of neuroglia could allow administration of appropriate ChEI therapy for symptomatic and disease modifying benefits.

Pages 1063-1068
Short Communication
Lara Ordóñez-Gutiérrez, Marta Antón, Francisco Wandosell (Handling Associate Editor: Ricardo Allegri)
Peripheral Amyloid Levels Present Gender Differences Associated with Aging in AβPP/PS1 Mice
Abstract: The accumulation of amyloid-β (Aβ) peptide is one of the major neuropathological hallmarks of Alzheimer’s disease (AD). We have analyzed whether the progression of amyloidosis differentially affects males and females along aging in AβPP/PS1 transgenic mice. The levels of peripheral amyloid, Aβ40 and Aβ42, are not modified in either sex until 9 months of age. After that, however, there is an increase in amyloid levels in plasma among females and a decrease among males. These findings could be essential to design gender-specific strategies in other in vivo experiments or even in AD treatments.

Pages 1069-1074
Short Communication
Natalie S. Ryan, Tammaryn Lashley, Tamas Revesz, Kiran Dantu, Nick C. Fox, Huw R. Morris
Spontaneous ARIA (Amyloid-Related Imaging Abnormalities) and Cerebral Amyloid Angiopathy Related Inflammation in Presenilin 1-Associated Familial Alzheimer’s Disease
Abstract: Amyloid-related imaging abnormalities (ARIA), thought to reflect immune responses to vascular amyloid, have been detected in several amyloid-modifying therapy trials for Alzheimer’s disease (AD). We report a case of ARIA developing spontaneously during the course of Presenilin 1 (PSEN1)-associated familial AD (FAD), in an APOE4 homozygous patient. Severe cerebral amyloid angiopathy with associated inflammation was subsequently found at autopsy. Recognition that ARIA may arise spontaneously during FAD and of the potential risk factors for its development are important observations given the recent launch of amyloid-modifying therapy trials for FAD.

Pages 1075-1085
Yeo Jin Kim, Hanna Cho, Yun Joong Kim, Chang-Seok Ki, Sun Ju Chung, Byoung Seok Ye, Hee Jin Kim, Jung-Hyun Kim, Sung Tae Kim, Kyung Han Lee, Seun Jeon, Jong-Min Lee, Juhee Chin, Jeong-Hun Kim, Duk L. Na, Joon-Kyung Seong, Sang Won Seo (Handling Associate Editor: YoungSoo Shim)
Apolipoprotein E4 Affects Topographical Changes in Hippocampal and Cortical Atrophy in Alzheimer’s Disease Dementia: A Five-Year Longitudinal Study
Abstract: Apolipoprotein E4 (APOE4) is a genetic risk factor for developing Alzheimer’s disease (AD). Once AD manifests clinically, however, the effects of APOE4 are less clear. Therefore, we investigated the longitudinal effects of APOE4 on topographical changes in AD patient brain atrophy. We prospectively recruited 35 patients with AD (19 APOE4 carriers and 16 non-carriers), and 14 normal controls, then followed them for five years. We measured hippocampal deformities and cortical thickness. Hippocampal comparison between APOE4 carriers and non-carriers with AD showed carriers had rapid changes in the head and body, while non-carriers had rapid changes in a small portion of the body. Cortical thickness comparison between APOE4 carriers and non-carriers with AD dementia showed carriers had rapid thinning in the lateral frontal, temporal, and parietal regions, while no region showed more rapid cortical thinning in non-carriers than in carriers. These findings underlined the importance of the APOE4 allele for designing and interpreting future treatment trials in patients with AD dementia.

Pages 1087-1098
Shea Andrews, Debjani Das, Kaarin J. Anstey, Simon Easteal (Handling Associate Editor: Corinne Engelman)
Interactive Effect of APOE Genotype and Blood Pressure on Cognitive Decline: The PATH Through Life Study
Abstract: The apolipoprotein E (APOE) *ε4 allele and hypertension are two of the most prevalent risk factors for cognitive decline in later life. Here we investigate whether cognitive decline is affected by interaction between these two risk factors. Specifically, we examine whether APOE*ε4 moderates the association between high blood pressure and cognition in later life. Cognitive function was assessed at three time points over a period of 8 years in 1,474 cognitively normal, community-dwelling adults aged 60-64 years at baseline. Blood pressure and APOE genotype were assessed at baseline. Blood pressure was measured categorically as ‘Hypertension’ and continuously as ‘Mean Arterial Pressure’ (MAP). Multilevel models were used to investigate main and interactive effects of APOE genotype and both hypertension and MAP on the rate of change of episodic memory, working memory, verbal ability, perceptual speed, and global cognition. The APOE–hypertension interaction was associated with a small but statistically significant increase in the rate of decline of episodic memory, verbal ability, and global cognition. However, its inclusion in the model did not increase the amount of outcome variation explained beyond that already explained by the effect of time. In contrast, the APOE-MAP interaction had no effect on the rate of decline in any of these domains of cognitive performance. These results provide tentative evidence that APOE genotype moderates the association between high blood pressure and cognitive decline in later life.

Pages 1099-1106
Yu-Tzu Wu, William B. Grant, A. Matthew Prina, Hsin-yi Lee, Carol Brayne
Nutrition and the Prevalence of Dementia in Mainland China, Hong Kong, and Taiwan: An Ecological Study
Abstract: Background: Western diets are associated with obesity, vascular diseases, and metabolic syndrome and might increase dementia risk in later life. If these associations are causal, those low- and middle-income countries experiencing major changes in diet might also see an increasing prevalence of dementia. Objective: To investigate the relationship of dietary supply and the prevalence of dementia in mainland China, Hong Kong, and Taiwan over time using existing data and taking diagnostic criteria into account. Methods: Estimated total energy supply and animal fat from the United Nations was linked to the 70 prevalence studies in mainland China, Hong Kong, and Taiwan from 1980 to 2012 according to the current, 10 years, and 20 years before starting year of investigation. Studies using newer and older diagnostic criteria were separated into two groups. Spearman’s rank correlation was calculated to investigate whether trends in total energy, animal fat supply, and prevalence of dementia were monotonically related. Results: The supply of total energy and animal fat per capita per day in China increased considerably over the last 50 years. The original positive relationship of dietary supply and dementia prevalence disappeared after stratifying by newer and older diagnostic criteria and there was no clear time lag effect. Conclusion: Taking diagnostic criteria into account, there is no cross-sectional or time lag relationship between the dietary trends and changes in dementia prevalence. It may be too early to detect any such changes because current cohorts of older people did not experience these dietary changes in their early to mid-life.

Pages 1107-1120
Fabio Di Domenico, Gilda Pupo, Cesare Mancuso, Eugenio Barone, Francesca Paolini, Andrea Arena, Carla Blarzino, Frederick A. Schmitt, Elizabeth Head, D. Allan Butterfield, Marzia Perluigi (Handling Associate Editor: Tommaso Cassano)
Bach1 Overexpression in Down Syndrome Correlates with the Alteration of the HO-1/BVR-A System: Insights for Transition to Alzheimer’s Disease
Abstract: Bach1, among the genes encoded on chromosome 21, is a transcription repressor, which binds to antioxidant response elements of DNA thus inhibiting the transcription of specific genes involved in the cell stress response including heme oxygenase-1 (HO-1). HO-1 and its partner, biliverdin reductase-A (BVR-A), are upregulated in response to oxidative stress in order to protect cells against further damage. Since oxidative stress is an early event in Down syndrome (DS) and might contribute to the development of multiple deleterious DS phenotypes, including Alzheimer’s disease (AD) pathology, we investigated the status of the Bach1/HO-1/BVR-A axis in DS and its possible implications for the development of AD. In the present study, we showed increased total Bach1 protein levels in the brain of all DS cases coupled with reduced induction of brain HO-1. Furthermore, increased oxidative stress could, on one hand, overcome the inhibitory effects of Bach1 and, on the other hand, promote BVR-A impairment. Our data show that the development of AD in DS subjects is characterized by (i) increased Bach1 total and poly-ubiquitination; (ii) increased HO-1 protein levels; and (iii) increased nitration of BVR-A followed by reduced activity. To corroborate our findings, we analyzed Bach1, HO-1, and BVR-A status in the Ts65Dn mouse model at 3 (young) and 15 (old) months of age. The above data support the hypothesis that the dysregulation of HO-1/BVR-A system contributes to the early increase of oxidative stress in DS and provide potential mechanistic paths involved in the neurodegenerative process and AD development.

Pages 1121-1129
Paul T. Williams
Lower Risk of Alzheimer’s Disease Mortality with Exercise, Statin, and Fruit Intake
Abstract: Background: Whether lifestyle affects Alzheimer’s disease (AD) risk remains controversial. Objective: Test whether exercise, diet, or statins affect AD mortality in 153,536 participants of the National Runners’ and Walkers’ Health Studies. Methods: Hazard ratios (HR) and 95% confidence intervals (95%CI) were obtained from Cox proportional hazard analyses for AD mortality versus baseline metabolic equivalent (MET) hours/d of exercise energy expenditure (1 MET equals approximately 1 km run), statin use, and fruit intake when adjusted for age, race, gender, education, and exercise mode. Results: The National Death Index identified 175 subjects who died with AD listed as an underlying (n=116) or contributing (n=59) cause of death during 11.6-year average mortality surveillance. Relative to exercising <1.07 MET-hours/d, AD mortality was 6.0% lower for 1.07 to 1.8 MET-hours/d (HR: 0.94, 95%CI: 0.59 to 1.46, p=0.79), 24.8% lower for 1.8 to 3.6 MET-hours/d (HR: 0.75, 95%CI: 0.50 to 1.13, p=0.17), and 40.1% lower for ≥3.6 MET-hours/d (HR: 0.60, 95%CI: 0.37 to 0.97, p=0.04). Relative to non-use, statin use was associated with 61% lower AD mortality (HR: 0.39, 95%CI: 0.15 to 0.82, p=0.01), whereas use of other cholesterol-lowering medications was not (HR: 0.78, 95%CI: 0.40 to 1.38, p=0.42). Relative to < 1 piece of fruit/day, consuming 2 to 3 pieces daily was associated with 39.7% lower AD mortality (HR: 0.60, 95%CI: 0.39 to 0.91, p=0.02) and ≥3 pieces/day with 60.7% lower AD mortality (HR: 0.39, 95%CI: 0.22 to 0.67, p=0.0004). Conclusions: Exercise, statin, and fruit intake were associated with lower risk for AD mortality.

Pages 1131-1143
Andrew D. Watt, Keyla A. Perez, Ching-Seng Ang, Paul O’Donnell, Alan Rembach, Kelly K. Pertile, Rebecca L. Rumble, Brett O. Trounson, Christopher J. Fowler, Noel G. Faux, Colin L. Masters, Victor. L. Villemagne, Kevin J. Barnham
Peripheral α-Defensins 1 and 2 are Elevated in Alzheimer’s Disease
Abstract: Biomarkers enabling the preclinical identification of Alzheimer’s disease (AD) remain one of the major unmet challenges in the field. The blood cellular fractions offer a viable alternative to current cerebrospinal fluid and neuroimaging modalities. The current study aimed to replicate our earlier reports of altered binding within the AD-affected blood cellular fraction to copper-loaded immobilized metal affinity capture (IMAC) arrays. IMAC and anti-amyloid-β (Aβ) antibody arrays coupled with mass spectrometry were used to analyze blood samples collected from 218 participants from within the AIBL Study of Aging. Peripheral Aβ was fragile and prone to degradation in the AIBL samples, even when stored at -80°C. IMAC analysis of the AIBL samples lead to the isolation and identification of alpha-defensins 1 and 2 at elevated levels in the AD periphery, validating earlier findings. Alpha-defensins 1 and 2 were elevated in AD patients indicating that an inflammatory phenotype is present in the AD periphery; however, peripheral Aβ levels are required to supplement their prognostic power.

Pages 1145-1156
Christelle Frederick*, Kunie Ando*, Karelle Leroy, Céline Héraud, Valérie Suain, Luc Buée, Jean-Pierre Brion *These authors contributed equally to this work.
Rapamycin Ester Analog CCI-779/Temsirolimus Alleviates Tau Pathology and Improves Motor Deficit in Mutant Tau Transgenic Mice
Abstract: Neurofibrillary tangles are intracellular inclusions made of tau protein that accumulates in neurons in Alzheimer’s disease (AD) and in other tauopathies. We have investigated the ability of the rapamycin ester CCI-779/Temsilorimus, a mTOR inhibitor with better stability and pharmacological properties compared to rapamycin, to interfere with the development of a motor phenotype and tau pathology in a mutant tau mouse model developing neurofibrillary tangles, by stimulation of mTOR dependent macroautophagy. Mutant tau mice (Tg30) were treated with CCI-779 before onset of motor signs for 7 months (from 5 to 12 months of age) or after the onset of motor signs for 2 months (from 10 to 12 months of age). End-point motor deficits were 50% lower in the group of Tg30 mice treated for 7 months. Inhibition of mTOR signaling and stimulation of macroautophagy in the brain of CCI-779 treated Tg30 mice was suggested by decreased phosphorylation of mTOR downstream signaling molecules p70S6 kinase and Akt and increased level of the autophagy markers Rab7 and LC3-II. CCI-779 treatment decreased the brain levels of Sarkosyl-insoluble tau and phosphotau inTg30 mice both after 2 months or 7 months of treatment. The density of neurofibrillary tangles was significantly decreased when treatment was started prior onset of motor signs. These results indicate that stimulation of mTOR dependent autophagy by CCI-779 compound is efficient to counteract the accumulation of abnormal tau when administered early or late in a tauopathy model and to improve a motor deficit when started before onset of motor signs.

Pages 1157-1169
John M. Nolan, Ekaterina Loskutova, Alan Howard, Riona Mulcahy, Rachel Moran, Jim Stack, Maggie Bolger, Robert F. Coen, Jessica Dennison, Kwadwo Owusu Akuffo, Niamh Owens, Rebecca Power, David Thurnham, Stephen Beatty
The Impact of Supplemental Macular Carotenoids in Alzheimer’s Disease: A Randomized Clinical Trial
Abstract: Background: Patients with Alzheimer’s disease (AD) exhibit significantly less macular pigment (MP) and poorer vision when compared to control subjects. Objective: To investigate supplementation with the macular carotenoids on MP, vision, and cognitive function in patients with AD versus controls. Methods: A randomized, double-blind clinical trial with placebo and active arms. 31 AD patients and 31 age-similar control subjects were supplemented for six months with either Macushield (10 mg meso-zeaxanthin [MZ]; 10 mg lutein [L]; 2 mg zeaxanthin [Z]) or placebo (sunflower oil). MP was measured using dual-wavelength autofluorescence (Heidelberg Spectralis®). Serum L, Z, and MZ were quantified by high performance liquid chromatography. Visual function was assessed by best corrected visual acuity and contrast sensitivity (CS). Cognitive function was assessed using a battery of cognition tests, including the Cambridge Neuropsychological Test Automated Battery (CANTAB)). Results: Subjects on the active supplement (for both AD and non-AD controls) exhibited statistically significant improvement in serum concentrations of L, Z, MZ, and MP (p<0.001, for all) and also CS at 1.2 cpd (p<0.039). Also, for subjects on the active supplement, paired samples t-tests exhibited four significant results (from five spatial frequencies tested) in the AD group, and two for the non-AD group, and all indicating improvements in CS. We found no significant changes in any of the cognitive function outcome variables measured (p>0.05, for all). Conclusion: Supplementation with the macular carotenoids (MZ, Z, and L) benefits patients with AD, in terms of clinically meaningful improvements in visual function and in terms of MP augmentation.

Pages 1171-1180
Pamela M. Rist, Jessica R. Marden, Benjamin D. Capistrant, Qiong Wu, M. Maria Glymour (Handling Associate Editor: Barbara Caracciolo)
Physical Activity, Smoking, Drinking, or Depression Modify Transitions from Cognitive Impairment to Functional Disability?
Abstract: Background: Individual-level modifiers can delay onset of limitations in basic activities of daily living (ADLs) among cognitively impaired individuals. We assessed whether these modifiers also delayed onset of limitations in instrumental ADLs (IADLs) among individuals at elevated dementia risk. Objectives: To determine whether modifiable individual-level factors delay incident IADL limitations among adults stratified by dementia risk. Methods: Health and Retirement Study participants aged 65+ without activity limitations in 1998 or 2000 (n=5,219) were interviewed biennially through 2010. Dementia probability, categorized in quartiles, was used to predict incident IADL limitations with Poisson regression. We estimated relative (risk ratio) and absolute (number of limitations) effects from models including dementia, individual-level modifiers (physical inactivity, smoking, no alcohol consumption, and depression) and interaction terms between dementia and individual-level modifiers. Results: Dementia probability quartile predicted incident IADL limitations (relative risk for highest versus lowest quartile=0.44; 95% CI: 0.28-0.70). Most modifiers did not significantly increase risk of IADL limitations among the cognitively impaired. Physical inactivity (RR=1.60; 95% CI: 1.16, 2.19) increased the risk of IADL limitations among the cognitively impaired. The interaction between physical inactivity and low dementia probability was statistically significant (p=0.009) indicating that physical inactivity had significantly larger effects on incident IADLs among cognitively normal than among those with high dementia probability. Conclusion: Physical activity may protect against IADL limitations while smoking, alcohol consumption, and depression do not afford substantial protection among the cognitively impaired. Results highlight the need for extra support for IADLs among individuals with cognitive losses.

Pages 1181-1191
Keri Diamond, Loren Mowszowski, Nicole Cockayne, Louisa Norrie, Matthew Paradise, Daniel F. Hermens, Simon J.G. Lewis, Ian B. Hickie, Sharon L. Naismith (Handling Associate Editor: Michael Hornberger)
Randomized Controlled Trial of a Healthy Brain Aging Cognitive Training Program: Effects on Memory, Mood, and Sleep
Abstract: Background: With the rise in the aging population and absence of a cure for dementia, cost-effective prevention strategies for those ‘at risk’ of dementia including those with depression and/or mild cognitive impairment are urgently required. Objective: This study evaluated the efficacy of a multifaceted Healthy Brain Ageing Cognitive Training (HBA-CT) program for older adults ‘at risk’ of dementia. Methods: Using a single-blinded design, 64 participants (mean age = 66.5 years, SD = 8.6) were randomized to an immediate treatment (HBA-CT) or treatment-as-usual control arm. The HBA-CT intervention was conducted twice-weekly for seven weeks and comprised group-based psychoeducation about cognitive strategies and modifiable lifestyle factors pertaining to healthy brain aging, and computerized cognitive training. Results: In comparison to the treatment-as-usual control arm, the HBA-CT program was associated with improvements in verbal memory (p = 0.03), self-reported memory (p = 0.03), mood (p = 0.01), and sleep (p = 0.01). While the improvements in memory (p = 0.03) and sleep (p = 0.02) remained after controlling for improvements in mood, only a trend in verbal memory improvement was apparent after controlling for sleep. Conclusion: The HBA-CT program improves cognitive, mood, and sleep functions in older adults ‘at risk’ of dementia, and therefore offers promise as a secondary prevention strategy.

Pages 1193-1201
Therese Koal, Kristaps Klavins, Daniele Seppi, Georg Kemmler, Christian Humpel
Sphingomyelin SM(d18:1/18:0) is Significantly Enhanced in Cerebrospinal Fluid Samples Dichotomized by Pathological Amyloid-β42, Tau, and Phospho-Tau-181 Levels
Abstract: Alzheimer’s disease (AD) is a severe and chronic neurodegenerative disorder of the brain. The laboratory diagnosis is limited to the analysis of three biomarkers in cerebrospinal fluid (CSF): amyloid-β42 (Aβ42), total tau, and phospho-tau-181 (P-tau-181). However, there is a need to find more biomarkers in CSF that can improve the sensitivity and specificity. The aim of the present study was to analyze endogenous small metabolites (metabolome) in the CSF, which may provide potentially new insights into biochemical processes involved in AD. One hundred CSF samples were dichotomized by normal (n=50) and pathological decreased Aβ42 and increased tau and P-tau-181 levels (n=50; correlating to an AD-like pathology). These CSF samples were analyzed using the AbsoluteIDQ® p180 Kit (BIOCRATES Life Sciences), which included 40 acyl carnitines metabolites, 21 amino acids, 19 proteinogenic aminoacids, 15 sphingolipids, and 90 glycerophospholipids. Our data show that two sphingomyelins (SM (d18:1/18:0) and SM (d18:1/18:1)), 5 glycerophospholipids (PC aa C32:0, PC aa C34:1, PC aa C36:1, PC aa C38:4 and PC aa C38:6), and 1 acyl carnitine (C3-DC-M/C5-OH) were significantly altered in the CSF with pathological "AD-like pathology". Sphingomyelin SM (d18:1/18:0) proved to be a specific (76%) and sensitive (66%) biomarker with a defined cut-off of 546 nM. Correct diagnoses for 21 out of 32 unknown samples could be achieved using this SM (d18:1/18:0) cut-off value. In conclusion, the sphingolipid SM (d18:1/18:0) is significantly increased in CSF of patients displaying pathological levels of Aβ42, tau, and P-tau-181.

Pages 1203-1212
Alfredo Ramirez*, Steffen Wolfsgruber*, Carolin Lange, Hanna Kaduszkiewicz, Siegfried Weyerer, Jochen Werle, Michael Pentzek, Angela Fuchs, Steffi G. Riedel-Heller, Tobias Luck, Edelgard Mösch, Horst Bickel, Birgitt Wiese, Jana Prokein, Hans-Helmut König, Christian Brettschneider, Monique M. Breteler, Wolfgang Maier, Frank Jessen**, and Martin Scherer** for the AgeCoDe Study Group (Handling Associate Editor: Patrick Kehoe) *These authors contributed equally to this work. **Shared last authors
Elevated HbA1c is Associated with Increased Risk of Incident Dementia in Primary Care Patients
Abstract: Type 2 diabetes mellitus (T2DM) is a risk factor of dementia. The effect of T2DM treatment quality on dementia risk, however, is unclear. 1,342 elderly individuals recruited via general practitioner registries (AgeCoDe cohort) were analyzed. This study analyzed the association between HbA1c level and the incidence of all-cause dementia (ACD) and of Alzheimer’s disease dementia (referred to here as AD). HbA1c levels ≥6.5% were associated with 2.8-fold increased risk of incident ACD (p=0.027) and for AD (p=0.047). HbA1c levels ≥7% were associated with a five-fold increased risk of incident ACD (p=0.001) and 4.7-fold increased risk of incident AD (p=0.004). The T2DM diagnosis per se did not increase the risk of either ACD or AD. Higher levels of HbA1c are associated with increased risk of ACD and AD in an elderly population. T2DM diagnosis was not associated with increased risk if HbA1c levels were below 7%.

Pages 1213-1229
Xinhua Zhan, Glen C. Jickling, Bradley P. Ander1, Boryana Stamova, DaZhi Liu, Patricia F. Kao, Mariko A Zelin, Lee-Way Jin, Charles DeCarli, Frank R. Sharp
Myelin Basic Protein Associates with AβPP, Aβ1-42, and Amyloid Plaques in Cortex of Alzheimer’s Disease Brain
Abstract: The goal of this study was to show that myelin and axons in cortical gray matter are damaged in Alzheimer’s disease (AD) brain. Superior temporal gyrus gray matter of AD patients (9 male, 14 female) was compared to cognitively normal controls (8 male, 7 female). Myelin basic protein (MBP) and a degraded myelin basic protein complex (dMBP) were quantified by western blot. Brain sections were immunostained for MBP, dMBP, axonal neurofilament protein (NF), autophagy marker microtubule-associated proteins 1A/B light chain 3B precursor (LC3B), amyloid-β protein precursor (AβPP), and amyloid markers amyloid β1-42 (Aβ1-42) and FSB. Co-immunoprecipitation and mass spectroscopy evaluated interaction of AβPP/Aβ1-42 with MBP/dMBP. Evidence of axonal injury in AD cortex included appearance of AβPP in NF stained axons, and NF at margins of amyloid plaques. Evidence of myelin injury in AD cortex included (1) increased dMBP in AD gray matter compared to control (p<0.001); (2) dMBP in AD neurons; and (3) increased LC3B that co-localized with MBP. Evidence of interaction of AβPP/Aβ1-42 with myelin or axonal components included (1) greater binding of dMBP with AβPP in AD brain; (2) MBP at the margins of amyloid plaques; (3) dMBP co-localized with Aβ1-42 in the core of amyloid plaques in AD brains; and (4) interactions between Aβ1-42 and MBP/dMBP by co-immunoprecipitation and mass spectrometry. We conclude that damaged axons may be a source of AβPP. dMBP, MBP, and NF associate with amyloid plaques and dMBP associates with AβPP and Aβ1-42. These molecules could be involved in formation of amyloid plaques.

Pages 1231-1239
Thomas J. Littlejohns, Katarina Kos, William E. Henley, Antonio Cherubini, Luigi Ferrucci, Iain A. Lang, Kenneth M. Langa, David Melzer, David J. Llewellyn
Serum Leptin and Risk of Cognitive Decline in Elderly Italians
Abstract: Background: US studies suggest that leptin, a fat-derived hormone, may be protective against the development of dementia. Objective: To investigate the complex relationship between leptin levels and cognitive decline in elderly Italians. Methods: We studied circulating fasting leptin levels in 809 elderly adults free from dementia who participated in the prospective Italian population-based InCHIANTI study between 1998 and 2009 (mean follow-up of 8.0 years). Global cognitive decline was defined as a reduction of ≥5 points on the Mini-Mental State Examination (MMSE). Trail-Making Tests A and B were also incorporated, with cognitive decline defined as discontinued testing or the worst 10% of change from baseline. We also investigated whether any association could be explained by midlife weight and whether cognitive decline was associated with changing leptin levels. Results: The multivariate adjusted relative risk ([RR]; 95% confidence interval [CI]) of cognitive decline on the MMSE was 0.84 (95% CI 0.73-0.97) in relation to baseline sex-standardized log-leptin levels. High leptin levels showed a non-significant trend toward a reduced risk of decline on the Trail-Making Tests A (RR=0.85, 95% CI 0.71-1.02) and B (RR=0.90, 0.79-1.02). Adjusting for midlife weight or change in weight did not alter the pattern of results, and cognitive decline was not associated with changing leptin levels. Conclusions: High leptin levels were independently associated with a reduced risk of cognitive decline in elderly Italians.

Pages 1241-1251
Matteo Bulati*, Silvio Buffa*, Adriana Martorana, Francesco Gervasi, Cecilia Camarda, Delia Maria Azzarello, Roberto Monastero, Calogero Caruso, Giuseppina Colonna-Romano *These authors contributed equally to this work.
Double Negative (IgG+IgD-CD27-) B Cells are Increased in a Cohort of Moderate-Severe Alzheimer’s Disease Patients and Show a Pro-Inflammatory Trafficking Receptor Phenotype
Abstract: Alzheimer’s disease (AD) is a progressive, irreversible, and debilitating disease for which no effective preventive or disease modifying therapies or treatments have so far been detected. The crucial step in AD pathogenesis is the production of amyloid-β42 peptide, which causes chronic inflammation. Activated cells in the central nervous system (CNS) produce pro-inflammatory mediators that lead to the recruitment of myeloid or lymphocytic cells. As a consequence, the communication between the CNS and peripheral blood of AD subjects could influence the lymphocyte distribution and/or the expression of phenotypic markers. In the present paper, we show a significant decrease in total CD19+ B lymphocytes and a remodeling of the B cell subpopulations in moderate-severe AD patients, compared with their coeval healthy controls and mild AD subjects. In particular, we report a significant reduction in naïve B cells (IgD+CD27-) and a simultaneous increase in double negative (DN, IgD-CD27-) memory B lymphocytes. We have also evaluated the expression of the pro-inflammatory chemokine receptors CCR6 and CCR7 in total and naïve/memory B cells from mild and moderate-severe AD patients, with the aim to detect a possible relationship between the trafficking profile and the stage of the disease. Our results demonstrate that both the amount and the trafficking profile of B cells are related to the severity of AD. The results discussed in this paper suggest a well-selected antibody panel should be used as an additional test for the identification of early AD.

Pages 1253-1261
Romain Salza, Jean-Baptiste Oudart, Laurent Ramont, François-Xavier Maquart, Serge Bakchine, Henri Thoannès, Sylvie Ricard-Blum
Endostatin Level in Cerebrospinal Fluid of Patients with Alzheimer’s Disease
Abstract: The aim of this study was to measure the level of endostatin, a fragment of collagen XVIII that accumulates in the brain of patients with Alzheimer’s disease (AD), in the cerebrospinal fluids (CSF) of patients with neurodegenerative diseases. The concentrations of total protein, endostatin, amyloid-β1-42 peptide, tau, and hyperphosphorylated tau proteins were measured by enzyme-linked immunosorbent assay in CSF of patients with AD (n=57), behavioral frontotemporal dementia (bvFTD, n=22), non AD and non FTD dementia (nAD/nFTD, n=84), and 45 subjects without neurodegenerative diseases. The statistical significance of the results was assessed by Mann-Whitney and Kruskal and Wallis tests, and by ROC analysis. The concentration of endostatin in CSF was higher than the levels of the three markers of AD both in control subjects and in patients with neurodegenerative diseases. The endostatin/amyloid-β1-42 ratio was significantly increased in patients with AD (257%, p<0.0001) and nAD/nFTD (140%, p<0.0001) compared to controls. The endostatin/tau protein ratio was significantly decreased in patients with AD (-49%, p<0.0001) but was increased in bvFTD patients (89%, p<0.0001) compared to controls. In the same way, the endostatin/hyperphosphorylated tau protein ratio was decreased in patients with AD (-21%, p=0.0002) but increased in patients with bvFTD (81%, p=0.0026), compared to controls. The measurement of endostatin in CSF and the calculation of its ratio relative to well-established AD markers improve the diagnosis of bvFTD patients and the discrimination of patients with AD from those with bvFTD and nAD/nFTD.

Pages 1263-1278
Kendra L. Puig, Brianna M. Lutz, Siri A. Urquhart, Andrew A. Rebel, Xudong Zhou, Gunjan D. Manocha, MaryAnn Sens, Ashok K. Tuteja, Norman L. Foster, Colin K. Combs
Overexpression of Mutant Amyloid-β Protein Precursor and Presenilin 1 Modulates Enteric Nervous System
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder histologically characterized by amyloid-β (Aβ) protein accumulation and activation of associated microglia. Although these features are well described in the central nervous system, the process and consequences of Aβ accumulation in the enteric nervous system have not been extensively studied. We hypothesized that Aβ also may accumulate in the enteric nervous system and lead to immune cell activation and neuronal dysfunction in the digestive tract not unlike that observed in diseased brain. To test this hypothesis, ileums of the small intestine of thirteen month old AβPP/PS1 and C57BL/6 (wild type) mice were collected and analyzed using immunohistochemistry, western blot analysis, cytokine arrays, and ELISA. AβPP/PS1 mice demonstrated no differences in intestinal motility or water absorption but elevated luminal IgA levels compared to wild type mice. They also had increased protein levels of AβPP and the proteolytic enzyme, BACE, corresponding to an increase in Aβ1-40 in the intestinal lysate as well as an increase in both Aβ1-40 and Aβ1-42 in the stool. This correlated with increased protein markers of proinflammatory and immune cell activation. Histologic analysis localized AβPP within enteric neurons but also intestinal epithelial cells with elevated Aβ immunoreactivity in the AβPP/PS1 mice. The presence of AβPP, Aβ, and CD68 immunoreactivity in the intestines of some patients with neuropathologically-confirmed AD are consistent with the findings in this mouse model. These data support the hypothesis that in AD the intestine, much like the brain, may develop proinflammatory and immune changes related to AβPP and Aβ.

Pages 1279-1290
Emma L. Elcombe, Jim Lagopoulos, Shantel Duffy, Simon J.G. Lewis, Louisa Norrie Ian B. Hickie, Sharon L. Naismith
Hippocampal Volume in Older Adults at Risk of Cognitive Decline: The Role of Sleep, Vascular Risk, and Depression
Abstract: Background and Objectives: Decreased hippocampal volume in older adults is associated with neurodegenerative and psychiatric diseases. Several modifiable risk factors have been associated with the size of this structure, however the relative contribution of these factors to hippocampal atrophy is unclear. This study aimed to examine the relationship between modifiable risk factors and hippocampal volume in older adults at risk of cognitive decline. Methods: Two hundred and eighteen participants (mean age=67.3 years, MMSE=28.6) with mood and/or memory complaints underwent clinical and neuropsychological assessment, and magnetic resonance imaging. Measures of depression, global cognitive functioning, exercise, vascular health, cognitive reserve, sleep, and memory were collected. Hippocampal volumes were derived using image segmentation as implemented by FMRIB Software Library. Results: Smaller hippocampal volumes were strongly associated with poorer verbal learning and memory as well as diagnoses of either multiple or amnestic mild cognitive impairment. Based on univariate correlations, multivariable regressions were performed (controlling for age and total intracranial volume) to determine which modifiable risk factors were associated with hippocampal volume. For the left hippocampus, poor sleep efficiency and greater than five years untreated depressive illness remained a significant predictors. For the right hippocampus, diabetes and low diastolic blood pressure were significant predictors. Conclusions: Although their contribution is small, lower sleep efficiency, low blood pressure, diabetes, and untreated depression are associated with reduced hippocampal volumes. Studies exploring the impact of early intervention for these risk factors on hippocampal integrity are warranted.

Pages 1291-1302
Melanie Hüttenrauch, Sandra Baches, Janina Gerth, Thomas A. Bayer, Sascha Weggen, Oliver Wirths
Neprilysin Deficiency Alters the Neuropathological and Behavioral Phenotype in the 5XFAD Mouse Model of Alzheimer’s Disease
Abstract: The deposition of amyloid-β (Aβ) is one of the major neuropathological hallmarks of Alzheimer’s disease (AD). In the case of sporadic AD, an imbalance in Aβ in production and clearance seems to be the reason for an enhanced Aβ accumulation. Besides a systematic clearance through the blood-brain barrier, Aβ is cleared from the brain by Aβ-degrading enzymes. The metalloprotease neprilysin (NEP) is an important Aβ-degrading enzyme as shown by numerous in vitro, in vivo, and reverse genetics studies. 5XFAD mice represent an early-onset AD mouse model which develops plaque pathology starting with 2 months of age in addition to robust behavioral deficits at later time points. By crossing 5XFAD mice with homozygous NEP-knock-out mice (NEP-/-), we show that hemizygous NEP deficiency aggravates the behavioral and neuropathological phenotype of 5XFAD mice. We found that 5XFAD mice per se showed strongly decreased NEP expression levels compared to wildtype mice, which was aggravated by NEP reduction. 5XFAD/NEP+/- mice demonstrated impairment in spatial working memory and increased astrocytosis in all studied brain areas, in addition to an overall increased level of soluble Aβ42 as well as region-specific increases in extracellular Aβ deposition. Surprisingly, in young mice, a more abundant cortical Aβ plaque pathology was observed in 5XFAD compared to 5XFAD/NEP+/- mice. Additionally, young 5XFAD/NEP+/- as well as hemi and homozygous NEP knockout mice showed elevated levels of endothelin-converting enzyme 1 (ECE1), suggesting a mutual regulation of ECE1 and NEP at young ages. The present data indicate that NEP mainly degrades soluble Aβ peptides, which confirms previous observations. Increased ECE1 levels correlated well with the strongly reduced extracellular plaque load in young 5XFAD/NEP+/- mice and might suggest a reciprocal effect between ECE and NEP activities in Aβ degradation.

Pages 1303-1312
Zita Oláh, János Kálmán, Melinda E. Tóth, Ágnes Zvara, Miklós Sántha, Eszter Ivitz, Zoltán Janka, Magdolna Pákáski (Handling Associate Editor: Inga Zerr)
Proteomic Analysis of Cerebrospinal Fluid in Alzheimer’s Disease: Wanted Dead or Alive
Abstract: Clinical diagnosis of Alzheimer’s disease (AD) relying on symptomatic features has a low specificity, emphasizing the importance of the pragmatic use of neurochemical biomarkers. The most advanced and reliable markers are amyloid-β (Aβ42), total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) with relatively high levels of sensitivity, specificity, and diagnostic accuracy. Recent advances within the field of proteomics offer the potential to search for novel biomarkers in CSF by using modern methods, such as microarrays. The purpose of this study was to identify pathognostic proteins in CSF obtained from patients whose clinical AD diagnosis was confirmed by the ‘‘core’’ biomarkers. CSF samples were obtained from 25 AD patients and 25 control individuals. The levels of Aβ42, t-tau, and p-tau were measured by ELISA. In the microarray experiments, ultrasensitive slides representing of 653 antigens were used. Apolipoprotein E genotyping was also determined. A decrease of seven CSF proteins in AD were found, four of them (POLG, MGMT, parkin, and ApoD) have a protective function against neuronal death, while the remaining three proteins (PAR-4, granzyme B, Cdk5) trigger multiple pathways facilitating neuronal cell death. Since these proteins from CSF samples could not be identified by western blot, their decreased levels in AD patients were not verified. Our results provide new information of pathognostic importance of POLG and granzyme B in AD. Although the function of MGMT, parkin, ApoD, PAR-4, and Cdk5 was previously known in AD, the findings presented here provide novel evidence of the significance of CSF analysis in the mapping of the AD pathomechanism.

Pages 1313-1322
Petronella Kettunen*, Susanna Larsson, Sandra Holmgren, Sandra Olsson, Lennart Minthon, Henrik Zetterberg, Kaj Blennow, Staffan Nilsson, Annica Sjölander* (Handling Associate Editor: Daniela Galimberti) *These authors contributed equally this work.
Genetic Variants of GSK3B are Associated with Biomarkers for Alzheimer’s Disease and Cognitive Function
Abstract: Background: Glycogen synthase kinase 3 beta (GSK3B) is the major kinase phosphorylating tau protein. Hyperphosphorylated tau is one of the hallmarks of Alzheimer’s disease (AD). Despite extensive research, the role of GSK3B in AD pathogenesis is not fully understood. Objective: To evaluate possible associations between gene variants of GSK3B and risk of AD. Methods: Twelve GSK3B tag single-nucleotide polymorphisms (SNPs), together with the previously AD-associated rs334558, were analyzed in 583 AD patients and 673 controls. Analyses on single marker and haplotype levels were done to relate to risk of AD, Mini-Mental State Examination (MMSE) scores, and cerebrospinal fluid (CSF) biomarker levels of total tau (T-tau), hyperphosphorylated tau (P-tau181), and amyloid-β (Aβ42). Results: After correction for multiple testing, we found a number of associations of gene variants with CSF biomarker levels and cognitive function in the AD patients. Firstly, rs334558 was associated with elevated T-tau levels (pc=0.04). Next, rs1154597 showed association with reduced Aβ42 levels (pc=0.007). Lastly, rs3107669 was associated with lower MMSE scores (pc=0.03). In addition, one more SNP was nominally significantly associated with reduced Aβ42 levels and another was associated with reduced MMSE. Conclusion: We found GSK3B gene variants associated with cognitive function and CSF biomarkers T-tau and Aβ42. To our knowledge, this is the first time GSK3B has been associated with cognitive function or CSF biomarkers reflecting neuronal degeneration (T-tau) and brain amyloid load (Aβ42). The regulation of GSK3B needs to be investigated further, to fully understand how these GSK3B gene variants are involved in AD pathogenesis.

Pages 1323-1331
Silvia Persichilli, Jacopo Gervasoni, Alessandra Di Napoli, Andrea Fuso, Vincenzina Nicolia, Bruno Giardina, Sigfrido Scarpa, Claudia Desiderio, Rosaria A. Cavallaro
Plasma Thiols Levels in Alzheimer’s Disease Mice under Diet-Induced Hyperhomocysteinemia: Effect of S-Adenosylmethionine and Superoxide-Dismutase Aupplementation
Abstract: Widely confirmed reports were published on association between hyperhomocysteinemia, B vitamin deficiency, oxidative stress, and amyloid-β in Alzheimer’s disease (AD). Homocysteine, cysteine, cysteinylglycine, and glutathione are metabolically interrelated thiols that may be potential indicators of health status and disease risk; they all participate in the metabolic pathway of homocysteine. Previous data obtained in one of our laboratories showed that B vitamin deficiency induced exacerbation of AD-like features in TgCRND8 AD mice; these effects were counteracted by S-adenosylmethionine (SAM) supplementation, through the modulation of DNA methylation and antioxidant pathways. Since the cellular response to oxidative stress typically involves alteration in thiols content, a rapid and sensitive HPLC method with fluorescence detection was here used to evaluate the effect of SAM and superoxide-dismutase (SOD) supplementation on thiols level in plasma, in TgCRND8 mice. The quantitative data obtained from HPLC analysis of mice plasma samples showed significant decrease of thiols level when the B vitamin deficient diet was supplemented with SAM+SOD and SOD alone, the latter showing the greatest effect. All these considerations point out the measurement of plasma thiols concentration as a powerful tool of relevance for all clinical purposes involving the evaluation of oxidative stress. The coupling of HPLC with fluorimetric detection, here used, provided a strong method sensitivity allowing thiols determination at very low levels.

Pages 1333-1347
Stelios Zygouris, Dimitrios Giakoumis, Konstantinos Votis, Stefanos Doumpoulakis, Ntovas Konstantinos, Sofia Segkouli, Karagiannidis Charalampos, Dimitrios Tzovaras, Magda Tsolaki
Can a Virtual Reality Cognitive Training Application Fulfill a Dual Role? Using the Virtual Supermarket Cognitive Training Application as a Screening Tool for Mild Cognitive Impairment
Abstract: Background: Recent research advocates the potential of virtual reality (VR) applications in assessing cognitive functions highlighting the possibility of using a VR application for mild cognitive impairment (MCI) screening. Objective: The aim of this study is to investigate whether a VR cognitive training application, the virtual supermarket (VSM), can be used as a screening tool for MCI. Methods: Two groups, one of healthy older adults (n=21) and one of MCI patients (n=34), were recruited from day centers for cognitive disorders and administered the VSM and a neuropsychological test battery. The performance of the two groups in the VSM was compared and correlated with performance in established neuropsychological tests. At the same time, the effectiveness of a combination of traditional neuropsychological tests and the VSM was examined. Results: VSM displayed a correct classification rate (CCR) of 87.30% when differentiating between MCI patients and healthy older adults, while it was unable to differentiate between MCI subtypes. At the same time, the VSM correlates with various established neuropsychological tests. A limited number of tests were able to improve the CCR of the VSM when combined with the VSM for screening purposes. Discussion: VSM appears to be a valid method of screening for MCI in an older adult population though it cannot be used for MCI subtype assessment. VSM’s concurrent validity is supported by the large number of correlations between the VSM and established tests. It is considered a robust test on its own as the inclusion of other tests failed to improve its CCR significantly.

Pages 1349-1359
Amira Zarrouk*, Jean-Marc Riedinger, Samia Hadj Ahmed, Sonia Hammami, Wafa Chaabane, Meryam Debbabi, Sofiene Ben Ammou, Olivier Rouaud, Mahbouba Frih, Gérard Lizard*, Mohamed Hammami *These authors contributed equally to this work.
Fatty Acid Profiles in Demented Patients: Identification of Hexacosanoic Acid (C26:0) as a Blood Lipid Biomarker of Dementia
Abstract: Background: Several lipid metabolism alterations have been described in the brain and plasma of Alzheimer’s disease (AD) patients, suggesting a relation between lipid metabolism alteration and dementia. Objective: We attempted to identify blood fatty acids as biomarkers of dementia. Methods: Fatty acid profiles were established using gas chromatography with or without mass spectrometry on matched plasma and red blood cells (RBCs) of demented patients diagnosed with AD, vascular dementia, or other dementia, and compared with a control group of elderly individuals. The severity of dementia was evaluated with the Mini-Mental State Examination test. Results: Fatty acid analysis showed significant variations of fatty acid levels in demented patients including AD patients. The highest plasma and RBC accumulation was found with hexacosanoic acid (C26:0). These data suggest that alterations of desaturase and elongase activity may contribute to cognitive dysfunction. Conclusion: The variations of fatty acid levels and the accumulation of C26:0 in the plasma and RBCs highlight an alteration of fatty acid metabolism in demented patients and point toward possible peroxisomal dysfunction. It is suggested that C26:0 may constitute a convenient blood biomarker of dementia that could be useful in routine medical practice.

Pages 1361-1373
Angela L. Jefferson, Timothy J. Hohman, Dandan Liu, Shereen Haj-Hassan, Katherine A. Gifford, Elleena M. Benson, Jeannine S. Skinner, Zengqi Lu, Jamie Sparling, Emily C. Sumner, Susan Bell, Frederick L. Ruberg
Adverse Vascular Risk is Related to Cognitive Decline in Older Adults
Abstract: Background: Cardiovascular disease (CVD) and related risk factors are associated with Alzheimer’s disease (AD). This association is less well-defined in normal cognition (NC) or prodromal AD (mild cognitive impairment, MCI). Objective: Cross-sectionally and longitudinally relate a vascular risk index to cognitive outcomes among elders free of clinical dementia. Methods: 3,117 MCI (74±8 years, 56% female) and 6,603 NC participants (72±8 years, 68% female) were drawn from the National Alzheimer’s Coordinating Center. A composite measure of vascular risk was defined using the Framingham Stroke Risk Profile (FSRP) score (i.e., age, systolic blood pressure, anti-hypertensive medication, diabetes, cigarette smoking, CVD history, atrial fibrillation). Ordinary linear regressions and generalized linear mixed models related baseline FSRP to cross-sectional and longitudinal cognitive outcomes, separately for NC and MCI, adjusting for age, gender, race, education, and follow-up time (in longitudinal models). Results: In NC participants, increasing FSRP was related to worse baseline global cognition, information processing speed, and sequencing abilities (p-values<0.0001) and a worse longitudinal trajectory on all cognitive measures (p-values<0.0001). In MCI, increasing FSRP correlated with worse longitudinal delayed memory (p=0.004). In secondary models using an age-excluded FSRP score, associations persisted in NC participants for global cognition, naming, information processing speed, and sequencing abilities. Conclusions: An adverse vascular risk profile is associated with worse cognitive trajectory, especially global cognition, naming, and information processing speed, among NC elders. Future studies are needed to understand how effective management of CVD and related risk factors can modify cognitive decline to identify the ideal timeframe for primary prevention implementation.