Volume 72, Number 2, 2019

Pages 361-362

Suzanne M. de la Monte, John E. Donahue, Bassam I. Aswad
Edward G. Stopa, MD

Pages 363-372
Pierre Krolak-Salmon, Audrey Maillet, Nicola Vanacore, Geir Selbaek, Konrad Rejdak, Latchezar Traykov, Antonios Politis, Jean Georges, Soo Borson, Armelle Leperre-Desplanques
Toward a Sequential Strategy for Diagnosing Neurocognitive Disorders: A Consensus from the “Act On Dementia” European Joint Action
Abstract: Neurocognitive disorders causing progressive cognitive, functional, and behavioral impairment remain underdiagnosed. The needs for a timely diagnosis are now widely acknowledged since person-centered care helps to preserve life quality and prevent crises. One powerful barrier to detection in primary care is the lack of an easy-to-follow stepwise approach, grounded in evidence and consistent with high-quality specialty practice. To help fill this gap, the current European Joint Action proposes a graduated diagnosis strategy tailored to the patients’ needs and wills, clarifying appropriate components for primary and specialty care. This strategy considers a first evaluation in primary care that may detect a neurocognitive disorder, that would lead to a second evaluation step allowing etiological diagnosis hypotheses performed mostly by the specialist. A third evaluation stage considering some biological, electrophysiological, or neuroimaging complementary techniques would be proposed to atypical cases or patients willing to consider access to research.

Pages 373-388
Paolo Maria Rossini, Stefano F. Cappa, Fabrizia Lattanzio, Daniela Perani, Patrizia Spadin, Fabrizio Tagliavini, Nicola Vanacore
The Italian INTERCEPTOR Project: From the Early Identification of Patients Eligible for Prescription of Antidementia Drugs to a Nationwide Organizational Model for Early Alzheimer’s Disease Diagnosis
Abstract: Alzheimer’s disease is the most common age-related neurodegenerative disorder and its burden on patients, families, and society grows significantly with lifespan. Early modifications of risk-enhancing lifestyles and treatment initiation expand personal autonomy and reduce management costs. Many clinical trials with potentially disease-modifying drugs are devoted to mild cognitive impairment (MCI) prodromal-to-Alzheimer’s disease. The identification of biomarkers for early diagnosis may thus be crucial for early intervention and identification of high-risk subjects, the most appropriate target of new drugs as soon as they will be discovered. INTERCEPTOR is a strategic project by the Italian Ministry of Health and the Italian Medicines Agency (AIFA), aiming to validate the best combination (highly accurate, non-invasive, available on the whole national territory and financially sustainable) of biomarkers and organizational model for early diagnosis. 500 MCI subjects will be enrolled at baseline and followed-up for 3 years for at least 400 of them in order to define a “hub & spoke” nationwide model with recruiting (spokes) centers for MCI identification and expert (hubs) centers for risk diagnosis.

Pages 389-399
Juanjuan Jiang*, Zhuangzhi Yan, Can Sheng*, Min Wang, Qinglan Guan, Zhihua Yu, Ying Han, Jiehui Jiang *These authors have contributed equally to this work.
A Novel Detection Tool for Mild Cognitive Impairment Patients Based on Eye Movement and Electroencephalogram
Abstract: Background: Detecting subtle changes in visual attention from electroencephalography (EEG) and the perspective of eye movement in mild cognitive impairment (MCI) patients can be of great significance in screening early Alzheimer’s disease (AD) in a large population at primary care. Objective: We proposed an automatic, non-invasive, and quick MCI detection approach based on multimodal physiological signals for clinical decision-marking. Methods: The proposed model recruited 152 patients with MCI and 184 healthy elderly controls (HC) who underwent EEG and eye movement signal recording under a visual stimuli task, as well as other neuropsychological assessments. Forty features were extracted from EEG and eye movement signals by linear and nonlinear analysis. The features related to MCI were selected by logistic regression analysis. To evaluate the efficacy of this MCI detection approach, we applied the same procedures to achieve the Clinical model, EEG model, Eye movement model, EEG+ Clinical model, Eye movement+ Clinical model, and Combined model, and compared the classification accuracy between the MCI and HC groups with the above six models. Results: After the penalization of logistic regression analysis, five features from EEG and eye movement features exhibited significant differences (p < 0.05). In the classification experiment, the combined model resulted in the best accuracy. The average accuracy for the Clinical/EEG/Eye movement/EEG+ Clinical/Eye movement+ Clinical/Combined model was 68.69%, 61.79%, 73.13%, 69.46%, 75.61%, and 81.51%, respectively. Conclusion: These results suggest that the proposed MCI detection tool has the potential to screen MCI patients from HCs and may be a powerful tool for personalized precision MCI screening in the large-scale population under primary care condition.

Pages 401-412
Francesca La Rosa, Marina Saresella, Ivana Marventano, Federica Piancone, Enrico Ripamonti, Nasser Al-Daghri, Chiara Bazzini, Chiara Paola Zoia, Elisa Conti, Carlo Ferrarese, Mario Clerici (Handling Associate Editor: Hongxing Lei)
Stavudine Reduces NLRP3 Inflammasome Activation and Modulates Amyloid-β Autophagy
Abstract: Background: Alzheimer’s disease (AD) is associated with the accumulation of amyloid-β (Aβ) within senile plaques in the brain and neuroinflammation, possibly driven by the activation of the NLRP3 inflammasome. Nucleoside reverse transcriptase inhibitors (NRTI) hamper the NLRP3 inflammasome assembly. Objective: We utilized an in vitro model reproducing the Aβ-driven inflammation seen in AD to analyze whether stavudine (D4T), a prototypical NRTI, modulates Aβ-mediated inflammasome activation and the ability of macrophages to eliminate Aβ via phagocytosis and autophagy. Methods: THP-1-derived macrophages were stimulated in vitro with Aβ42 or with Aβ42 after LPS-priming in the presence/absence of D4T. NLRP3 and TREM2 expression was analyzed by RT-PCR; phagocytosis, as well as ASC-Speck formation, was analyzed by Amnis FlowSight Imaging; NLRP3-produced cytokines were quantified by ELISA and, finally, autophagy was analyzed by measuring p-ERK1/2, p-AKT, beclin, p70-S6Kinase, and Lamp by ELISA and western blot. Results: IL-1β, IL-18, and caspase-1 were increased whereas Aβ phagocytosis and TREM2 were reduced in LPS+Aβ42-stimulated cells. D4T reduced NLRP3 assembly as well as IL-18 and caspase-1 production, but did not affect IL-1β production and TREM2 expression. Notably, whereas D4T reduced Aβ phagocytosis, Aβ autophagy by macrophages was stimulated by D4T, as witnessed by the down-modulation of ERK1/2 and AKT phosphorylation and the upregulation of beclin, LAMP, and p70-S6K, their downstream targets. Conclusion: In this in vitro model of AD, D4T reduces NLRP3 inflammasome-associated inflammation and stimulates Aβ autophagy by macrophages. It will be interesting to verify the possibly beneficial effects of D4T in the clinical scenario.

Pages 413-423
Shengzhen Zou, Jie Zhang, for Alzheimer’s Disease Neuroimaging Initiative, Wei Chen
Subtypes Based on Six Apolipoproteins in Non-Demented Elderly Are Associated with Cognitive Decline and Subsequent Tau Accumulation in Cerebrospinal Fluid
Abstract: Apolipoproteins (APOs) have been implicated in the pathogenesis of Alzheimer’s disease (AD). In the present study, we aimed to investigate if patterns of cerebrospinal fluid (CSF) APOs (APOA-I, APOC-III, APOD, APOE, APOH, and APOJ) levels are associated with changes over time in cognition, memory performance, neuroimaging markers, and AD-related pathologies (CSF Aβ42, t-tau, and p-tau) in non-demented older adults. At baseline, a total of 241 non-demented older adults with CSF APOs data was included in the present analysis. Hierarchical agglomerative cluster analysis including the six CSF APOs was carried out. Among non-demented older adults, we identified two clusters. Compare with the first cluster, the second cluster had higher levels of APOs in CSF. Additionally, the second cluster showed a more benign disease course, including slower cognitive decline and slower p-tau accumulation in CSF. Our data highlight the importance of APOs in the pathogenesis of AD.

Pages 425-441
Xiaoguang Liu, Michaeline L. Hebron, Sanjana Mulki, Chen Wang, Elizabeth Lekah, Dalila Ferrante, Wangke Shi, Bahjat Kurd-Misto, Charbel Moussa
Ubiquitin Specific Protease 13 Regulates Tau Accumulation and Clearance in Models of Alzheimer’s Disease
Abstract: Ubiquitin Specific Protease-13 (USP13) is a de-ubiquinating enzyme that regulates protein ubiquitination and clearance. The role of USP13 is largely unknown in neurodegeneration. In this study we aim to demonstrate whether tau accumulation and/or clearance depends on ubiquitination/de-ubiquitination via USP-13. We used transgenic animal models of human amyloid precursor protein (APP) or P301L tau mutations and genetically knocked-down USP13 expression via shRNA to determine USP13 effects on tau ubiquitination and levels. We found a two-fold increase of USP13 levels in postmortem Alzheimer’s disease (AD) brains. USP13 knockdown significantly increased the activity of the 20S proteasome and reduced the levels of hyper-phosphorylated tau (p-tau) in primary cortical neurons. USP13 knockdown also reduced the levels of amyloid and increased p-tau ubiquitination and clearance in transgenic animal models that overexpress murine tau as a result of the expression of familial APP mutations (TgAPP) and the human mutant P301L tau (rTg4510), respectively. Clearance of p-tau appears to be mediated by autophagy in these animal models. Taken together, these data suggest that USP13 knockdown reduces p-tau accumulation via regulation of ubiquitination/de-ubiquitination and mediates its clearance via autophagy and/or the proteasome. These results suggest that USP13 inhibition may be a therapeutic strategy to reduce accumulation of plaques and toxic p-tau in AD and human tauopathies.

Pages 443-454
Guanqun Chen, Kun Yang, Wenying Du, Xiaochen Hu, Ying Han
Clinical Characteristics in Subjective Cognitive Decline with and without Worry: Baseline Investigation of the SILCODE Study
Abstract: Background: Subjective cognitive decline (SCD) refers to the self-perception of cognitive decline among cognitively normal elderly individuals. SCD related worry confers a higher risk of developing cognitive decline. However, the clinical characteristics of SCD patients with worry are not clear. Objective: To explore the clinical characteristics of SCD patients with worry. Methods: A cross-sectional study was carried out, with 270 consecutive participants of the Sino Longitudinal Study on Cognitive Decline (SILCODE) study. Participants were classified as normal controls (n=36), SCD patients without worry (n=91), or SCD patients with worry (n=143) and were comprehensively compared on 1) their self-perception of cognitive decline, 2) multiple cognitive domains, 3) neuropsychiatric symptoms, and 4) sleep status. Results: SCD patients with worry had significantly more self-perception of cognitive decline (p<0.001); increased depression (p<0.001) and anxiety (p<0.001); decreased sleep quality (p<0.001), sleep latency (p<0.05), sleep time (p<0.01), and sleep efficiency (p<0.05); more sleep disorders (p<0.05) and daytime dysfunction (p<0.05); and a higher global score on the Pittsburgh Sleep Quality Index (p<0.001) than normal controls. Although there was a significant increase only in self-perception of cognitive decline (p<0.001), anxiety (p<0.001), and Pittsburgh Sleep Quality Index scores (p<0.05), the severity of the increase in those without worry was between that in SCD patients with worry and normal controls. Conclusion: Our findings show that participants who had SCD with worry showed distinct clinical characteristics compared with normal controls and SCD patients without worry, which could be useful for understanding the higher risk in SCD patients with worry of subsequently developing Alzheimer’s disease.

Pages 455-465
Stefan J. Teipel, Jan O. Kuper-Smith, Claudia Bartels, Frederic Brosseron, Martina Buchmann, Katharina Buerger, Cihan Catak, Daniel Janowitz, Peter Dechent, Laura Dobisch, Birgit Ertl-Wagner, Klaus Fließbach, John-Dylan Haynes, Michael T. Heneka, Ingo Kilimann, Christoph Laske, Siyao Li, Felix Menne, Coraline D. Metzger, Josef Priller, Verena Pross, Alfredo Ramirez, Klaus Scheffler, Anja Schneider, Annika Spottke, Eike J. Spruth, Michael Wagner, Jens Wiltfang, Steffen Wolfsgruber5, Emrah Düzel, Frank Jessen, Martin Dyrba, and the DELCODE study group (Handling Associate Editor: Juan Helen Zhou)
Multicenter Tract-Based Analysis of Microstructural Lesions within the Alzheimer’s Disease Spectrum: Association with Amyloid Pathology and Diagnostic Usefulness
Abstract: Diffusion changes as determined by diffusion tensor imaging are potential indicators of microstructural lesions in people with mild cognitive impairment (MCI), prodromal Alzheimer’s disease (AD), and AD dementia. Here we extended the scope of analysis toward subjective cognitive complaints as a pre-MCI at risk stage of AD. In a cohort of 271 participants of the prospective DELCODE study, including 93 healthy controls and 98 subjective cognitive decline (SCD), 45 MCI, and 35 AD dementia cases, we found reductions of fiber tract integrity in limbic and association fiber tracts in MCI and AD dementia compared with controls in a tract-based analysis (p < 0.05, family wise error corrected). In contrast, people with SCD showed spatially restricted white matter alterations only for the mode of anisotropy and only at an uncorrected level of significance. DTI parameters yielded a high cross-validated diagnostic accuracy of almost 80% for the clinical diagnosis of MCI and the discrimination of Aβ positive MCI cases from Aβ negative controls. In contrast, DTI parameters reached only random level accuracy for the discrimination between Aβ positive SCD and control cases from Aβ negative controls. These findings suggest that in prodromal stages of AD, such as in Aβ positive MCI, multicenter DTI with prospectively harmonized acquisition parameters yields diagnostic accuracy meeting the criteria for a useful biomarker. In contrast, automated tract-based analysis of DTI parameters is not useful for the identification of preclinical AD, including Aβ positive SCD and control cases.

Pages 467-478
Jing Tian, Tienju Wang, Qi Wang, Lan Guo, Heng Du (Handling Associate Editor: P. Hemachandra Reddy)
MK0677, a Ghrelin Mimetic Improves Neurogenesis but Fails to Prevent Hippocampal Lesions in a Mouse Model of Alzheimer’s Disease Pathology
Abstract: Hippocampal lesions including synaptic injury, neuroinflammation, and impaired neurogenesis are featured pathology closely associated with neuronal stress and cognitive impairment in Alzheimer’s disease (AD). Previous studies suggest that ghrelin and its receptor, growth hormone secretagogue receptor 1α (GHSR1α), promote hippocampal synaptic function and neurogenesis. GHSR1α activation thus holds the potential to be a therapeutic avenue for the treatment of hippocampal pathology in AD; however, a comprehensive study on the preventive effect of MK0677 on hippocampal lesions in AD-related conditions is still lacking. In this study, we treated a transgenic mouse model of AD-like amyloidosis (5xFAD mice) at the asymptomatic stage with MK0677, a potent ghrelin mimetic. We found that MK0677 fostered hippocampal neurogenesis in 5xFAD mice but observed little preventive function with regards to the development of hippocampal amyloid-β (Aβ) deposition, synaptic loss, microglial activation, or cognitive impairment. Furthermore, MK0677 at a dose of 3 mg/kg significantly increased 5xFAD mouse mortality. Despite enhanced hippocampal neurogenesis, MK0677 treatment has little beneficial effect to prevent hippocampal lesions or cognitive deficits against Aβ toxicity. This study, together with a failed large-scale clinical trial, suggests the ineffectiveness of MK0677 alone for AD prevention and treatment.

Pages 479-494
Ran Nie, Zhou Wu, Junjun Ni, Fan Zeng, Weixian Yu, Yufeng Zhang, Tomoko Kadowaki, Haruhiko Kashiwazaki, Jessica L. Teeling, Yanmin Zhou (Handling Associate Editor: Sim Singhrao)
Porphyromonas gingivalis Infection Induces Amyloid-β Accumulation in Monocytes/Macrophages
Abstract: Abnormal accumulation of amyloid-β (Aβ) in the brain is the most significant pathological hallmark of Alzheimer’s disease (AD). We have found that chronic systemic exposure to lipopolysaccharide of Porphyromonas gingivalis (P. gingivalis) induces the accumulation of Aβ in the brain of middle-aged mice. On the other hand, recent research has shown that circulating Aβ is transferred into the brain; however, the involvement of chronic systemic P. gingivalis infection in the peripheral Aβ metabolism is unknown. We hypothesized that chronic P. gingivalis infection expands Aβ pools in peripheral inflammatory tissues and thereby contributes to the accumulation of Aβ in the brain of patients with periodontitis. We showed that the increased expression of IL-1β, AβPP770, CatB, Aβ1-42, and Aβ3-42 was mainly co-localized with macrophages in the liver of P. gingivalis infected mice. Blocking CatB and NF-κB significantly inhibited the P. gingivalis-induced expression of IL-1β, AβPP770, Aβ1-42, and Aβ3-42 in RAW264.7 cells. Aβ3-42, but not Aβ1-42, induced the significant death of macrophages, and the reduction of phagocytic abilities induced by Aβ3-42 tended to be higher than that induced by Aβ1-42. Additionally, the expression of AβPP770, CatB, Aβ1-42, and Aβ3-42 was determined in the macrophages of gingival tissues from periodontitis patients. These findings indicate that chronic systemic P. gingivalis infection induces the Aβ accumulation in inflammatory monocytes/macrophages via the activation of CatB/NF-κB signaling, thus suggesting monocytes/macrophages serve as a circulating pool of Aβ in patients with periodontitis. Taken together, CatB may be a novel therapeutic target for preventing the periodontitis-related AD initiation and pathological progression.

Pages 495-506
Jet M.J. Vonk, Miguel Arce Rentería, Valerie M. Medina, Margaret A. Pericak-Vance, Goldie S. Byrd, Jonathan Haines, Adam M. Brickman, Jennifer J. Manly (Handling Associate Editor: Maheen Adamson)
Education Moderates the Relation Between APOE ε4 and Memory in Nondemented Non-Hispanic Black Older Adults
Abstract: Background: The APOE ε4 allele is a well-known risk factor for Alzheimer’s disease (AD). Previous research argues that higher education helps to preserve cognition in older adults with AD pathology because of its key role in cognitive reserve and resilience. Objective: To test if higher educational level buffers the effect of APOE ε4 on cognition among older non-Hispanic Blacks. Methods: Participants were 849 non-demented older non-Hispanic Blacks (38.3% APOE ε4+), who underwent a comprehensive neuropsychological evaluation. Multiple linear regression models tested the relationship between APOE ε4 status and twelve cognitive measures with education (up to high school and beyond high school) as a moderator. Results: Education buffered the effects of the APOE ε4 allele, such that there was no impact of APOE ε4 status on word-list memory retention and working memory among participants with more than a high school degree. This pattern was not observed for ten other cognitive measures of verbal and visual episodic memory, semantic memory, executive function, and processing speed—although a similar trend was observed for switching ability in executive functioning. The buffering effect of education was stronger among women than men. Conclusion: Our findings suggest that genetic effects on late-life cognition may be modified by environmental factors such as educational attainment. These results are consistent with the framework of cognitive reserve such that engaging in cognitively enriching activities and acquiring skills and knowledge with more years of education may increase the capacity to maintain cognitive function despite high genetic risk for impairment.

Pages 507-514
Felicia C. Goldstein, David W. Loring, Tiffany Thomas, Sabria Saleh, Ihab Hajjar
Recognition Memory Performance as a Cognitive Marker of Prodromal Alzheimer’s Disease
Abstract: Background: The utility of recognition memory for identifying persons with biomarker evidence of Alzheimer’s disease (AD) is unclear since prior studies of mild cognitive impairment (MCI) relied only on clinical diagnosis and did not include simultaneous measures of central amyloidosis and tauopathy. Objective: We evaluated whether recognition memory and associated indices, including discriminability and response bias from signal detection theory, differentiate persons with amnestic MCI (aMCI) due to prodromal AD from non-prodromal AD. Method: Sixty older adults with aMCI were classified as prodromal AD (n=28) or non-prodromal AD (n=32) based upon cerebrospinal fluid levels of amyloid-β and tau. Memory was assessed using the Hopkins Verbal Learning Test-Revised which includes free recall and recognition. Results: ANCOVAs adjusting for age indicated comparable (all p>0.05) performances between prodromal and non-prodromal MCI groups respectively on traditional HVLT-R recognition measures of hits (mean±SD: 9.5±3.0 versus 10.9±1.7), false alarms (1.8±1.8 versus 1.5±1.5), and hits minus false alarms (7.7±3.0 versus 9.2;±2.6). In contrast, discriminability (d’), which reflects how easily targets and distractors are distinguished, was significantly (p=0.009) poorer in the prodromal versus non-prodromal groups (3.1±1.9 versus 4.8±2.0, effect size=0.87). In addition, only d’ significantly predicted group membership (OR=0.66, CI=0.48-0.92, p=0.04). Response bias, the tendency to report that a target did or did not appear, was comparable between groups (0.08±1.1 versus -0.04±1.3). Conclusion: Recognition discriminability is significantly poorer in aMCI with biomarker evidence of prodromal AD. In contrast to traditional recognition indices, discriminability from signal detection theory may be superior in identifying aMCI due to AD versus non-AD etiologies.

Pages 515-524
Heba Elshatoury, Egils Avots, Gholamreza Anbarjafari, for the Alzheimer’s Disease Neuroimaging Initiative
Volumetric Histogram-Based Alzheimer’s Disease Detection Using Support Vector Machine
Abstract: In this research work, machine learning techniques are used to classify magnetic resonance imaging brain scans of people with Alzheimer’s disease. This work deals with binary classification between Alzheimer’s disease and cognitively normal. Supervised learning algorithms were used to train classifiers in which the accuracies are being compared. The database used is from The Alzheimer’s Disease Neuroimaging Initiative (ADNI). Histogram is used for all slices of all images. Based on the highest performance, specific slices were selected for further examination. Majority voting and weighted voting is applied in which the accuracy is calculated and the best result is 69.5% for majority voting.

Pages 525-535
Patrick G. Kehoe, Noura Al Mulhim, Henrik Zetterberg, Kaj Blennow, James S. Miners
Cerebrospinal Fluid Changes in the Renin-Angiotensin System in Alzheimer’s Disease
Abstract: Observations in autopsied brain tissue indicate that overactivation of the classical renin-angiotensin system (cRAS) and underactivity within regulatory RAS pathways (rRAS) are associated with pathology in Alzheimer’s disease (AD). The primary aim of this study was to investigate whether cerebrospinal fluid (CSF) markers of RAS are altered in AD in relation to established CSF markers of disease pathology (lower Aβ42 and elevated tau) and CSF markers of capillary dysfunction. We studied 40 controls and 40 AD cases grouped according to a biomarker profile (i.e., AD cases t-tau >400 pg/mL, pTau >60 pg/mL, and Aβ42 <550 pg/mL). Angiotensin-II converting enyme-1 (ACE1) and ACE2 enzyme activity was measured using fluorogenic peptide substrates; sPDGFRβ and albumin level by sandwich ELISA; and angiotensin-I (Ang-I), Ang-II, and Ang-(1-7) by direct ELISA. CSF Aβ42, total, and phosphorylated tau levels were previously measured by INNOTEST sandwich ELISA. CSF ACE1 activity was significantly elevated in AD (p = 0.008) and positively correlated with ACE2 in AD (r = 0.420, p = 0.007). CSF ACE1 weakly correlated with t-tau (r = 0.294, p = 0.066) and p-tau (r = 0.329, p = 0.038) but not with Aβ42 in the controls but not in AD. ACE1 correlated positively with sPDGFRβ (r = 0.426, p = 0.007), a marker of pericyte injury, and ACE2 correlated positively with albumin (r = 0.422, p = 0.008), a marker of blood-brain barrier integrity. CSF Ang-I, Ang-II, and Ang-(1-7) levels were unchanged in AD. This cross-sectional CSF study indicates RAS dysfunction in relation to capillary damage in AD.

Pages 537-548
Lea S. van Husen, Sophia Schedin-Weiss, Minh Nguyen Trung, Manija Kazmi, Bengt Winblad, Thomas P. Sakmar, Simon J. Elsässer,Lars O. Tjernberg
Dual Bioorthogonal Labeling of the Amyloid-β Protein Precursor Facilitates Simultaneous Visualization of the Protein and Its Cleavage Products
Abstract: The amyloid-β protein precursor (AβPP) is critical in the pathophysiology of Alzheimer’s disease (AD), since two-step proteolytic processing of AβPP generates the neurotoxic amyloid-β peptide (Aβ). We developed a dual fluorescence labeling system to study the exact subcellular location of γ-secretase cleavage of AβPP. The C-terminal tail of AβPP was fluorescently labeled using a SNAP-tag, while the Aβ region of AβPP was fluorescently tagged with a dye at a genetically-encoded noncanonical amino acid (ncAA). The ncAA was introduced at specific positions in AβPP using a genetic code expansion strategy and afterwards, the reactive side-chain of the ncAA was coupled to the dye using a bioorthogonal labeling chemistry. In proof-of-concept experiments, HEK293T cells were transfected with plasmids containing engineered AβPP harboring an amber mutation and an amber codon suppression system with an evolved tRNA synthetase/tRNA pair and grown in the presence of a lysine-derived ncAA. Processing of the AβPP variants was validated with ELISA and immunoblotting, and seven AβPP mutants that showed similar cleavage pattern as wild-type AβPP were identified. The AβPP mutant was fluorescently labeled with 6-methyl-tetrazine-BDP-FL and TMR-Star at the ncAA and SNAP-tag, respectively. Using this approach, AβPP was fluorescently labeled at two sites in living cells with minimal background to allow monitoring of Aβ and C-terminal cleavage products simultaneously. The method described provides a powerful tool to label Aβ with minimal perturbations of its processing, thus enabling studies of the trafficking of the cleavage products of AβPP.

Pages 549-562
Nathan M. D’Cunha, Andrew J. McKune, Stephen Isbel, Jane Kellett, Ekavi N. Georgousopoulou, Nenad Naumovski (Handling Associate Editor: Sandra Garrido)
Psychophysiological Responses in People Living with Dementia after an Art Gallery Intervention: An Exploratory Study
Abstract: The use of existing public spaces by people living with dementia, such as museums and art galleries, are becoming popular due to their ability to facilitate programs which promote social engagement and inclusion. However, few studies have investigated physiological outcomes of art gallery-based programs. Using a quasi-experimental design, the present study aimed to investigate the levels of salivary biomarkers of cortisol and interleukin-6, quality of life (QoL), depressive symptoms, cognition, and wellbeing, after attending the National Gallery of Australia (NGA) Art and Dementia program. Twenty-eight people living with dementia, each supported by a carer or family member, were recruited for a six-week program and were followed up at twelve weeks. In total, 25 participants (17 female; mean age 84.6±7.27 years) completed the study, and 22 provided viable saliva samples. The waking to evening salivary cortisol ratio was higher post-intervention (p=0.033), and returned to baseline levels at follow-up (p=1.00), indicating a more dynamic salivary cortisol rhythm in response to the six-week program. Interleukin-6 levels remained unchanged (p=0.664). No improvements in QoL (DEMQOL-Carer) were observed between baseline and post-intervention (p=0.076). However, self-reported depressive symptoms decreased post-intervention compared with baseline (p=0.015), and memory (immediate recall) (p=0.009) and verbal fluency (p=0.027) improved between the same timepoints. The NGA Art and Dementia program appears to have quantifiable benefits, including improved hypothalamic-pituitary-adrenal axis function, justifying a need for longer controlled trial inclusive of physiological outcomes.

Pages 563-574
Vasiliki Orgeta, Remco Tuijt, Phuong Leung, Elisabet Sole Verdaguer, Rebecca L. Gould, Rebecca Jones, Gill Livingston
Behavioral Activation for Promoting Well-Being in Mild Dementia: Feasibility and Outcomes of a Pilot Randomized Controlled Trial
Abstract: Engaging in meaningful and enjoyable activities is an important contributor to well-being and maintaining good quality of life. There is a paucity of randomized controlled trials of interventions supporting people with mild dementia to engage in meaningful and purposeful activity. The aim of this study was to assess whether Behavioral Activation (BA) is an acceptable psychological intervention for people with mild dementia and whether a large-scale trial is feasible. Participants were randomly assigned to BA (n = 42) or treatment as usual (TAU) (n = 21). BA aimed at increasing engagement in enjoyable and meaningful activity, and preventing low mood. Follow-up was at 3 and 6 months. Assessors were blind to treatment allocation (trial registration number: ISRCTN75503960). Retention rate was above 80% at both assessment time points. Treatment acceptability and credibility were high. Depressive symptoms remained unchanged in both groups. There was evidence of improvement associated with BA for every day function (-3.92, 95% Confidence Interval (CI) -6.87 to -0.97), and engagement in meaningful and enjoyable activity (5.08, 95% CI 0.99 to 9.16) post-treatment (3 months) in comparison to TAU. Both carer-rated patient health-related quality of life (0.16, 95% CI 0.04 to 0.28) and physical health (11.31, 95% CI 2.03 to 20.59) showed evidence of improvement at 3 months. Improvements in meaningful and enjoyable activity were maintained at 6 months. BA for people with mild dementia is feasible and acceptable and may be associated with clinically significant changes in function and quality of life. A full scale randomized controlled trial of clinical effectiveness is now needed.

Pages 575-585
Brent Aulston, Qing Liu, Michael Mante, Jazmin Florio, Robert A. Rissman, Shauna H. Yuan
Extracellular Vesicles Isolated from Familial Alzheimer’s Disease Neuronal Cultures Induce Aberrant Tau Phosphorylation in the Wild-Type Mouse Brain
Abstract: Extracellular vesicles (EVs) are a heterogeneous group of secreted particles consisting of microvesicles, which are released by budding of the cellular membrane, and exosomes, which are secreted through exocytosis from multivesicular bodies. EV cargo consists of a wide range of proteins and nucleic acids that can be transferred between cells. Importantly, EVs may be pathogenically involved in neurodegenerative diseases such as Alzheimer’s disease (AD). While EVs derived from AD neurons have been found to be neurotoxic in vitro, little is known about the pathological consequences of AD EVs in vivo. Furthermore, although all known familial AD (fAD) mutations involve either amyloid-β protein precursor (AβPP) or the machinery that processes AβPP, hyperphosphorylation of the microtubule associated protein tau appears to play a critical role in fAD-associated neurodegeneration, and previous reports suggest EVs may propagate tau pathology in the AD brain. Therefore, we hypothesized that fAD EVs may have a mechanistic involvement in the development of fAD-associated tau pathology. To test this, we isolated EVs from iPSC-derived neuronal cultures generated from an fAD patient harboring a A246E mutation to presenilin-1 and stereotactically injected these EVs into the hippocampi of wild-type C57BL/6 mice. Five weeks after injection, mice were euthanized and pathology evaluated. Mice injected with fAD EVs displayed increased tau phosphorylation at multiple sites relative to PBS and non-disease control EV injected groups. Moreover, fAD EV injected hippocampi contained significantly more tau inclusions in the CA1 hippocampal neuronal field than controls. In total, these findings identify EVs as a potential mediator of fAD-associated tau dysregulation and warrant future studies to investigate the therapeutic potential of EV-targeted treatments for fAD.

Pages 587-604
Keren Nitzan*, Sandrine Benhamron*, Michael Valitsky, Eyal E. Kesner, Michal Lichtenstein, Ayal Ben-Zvi, Ezra Ella, Yehudit Segalstein, Saada Ann, Haya Lorberboum-Galski, Hanna Rosenmann *These authors contributed equally to this work.
Mitochondrial Transfer Ameliorates Cognitive Deficits, Neuronal Loss, and Gliosis in Alzheimer’s Disease Mice
Abstract: Pathogenesis of neurodegenerative diseases involves dysfunction of mitochondria, one of the most important cell organelles in the brain, with its most prominent roles in producing energy and regulating cellular metabolism. Here we investigated the effect of transferring active intact mitochondria as a potential therapy for Alzheimer’s disease (AD), in order to correct as many mitochondrial functions as possible, rather than a mono-drug related therapy. For this purpose, AD-mice (amyloid-β intracerebroventricularly injected) were treated intravenously (IV) with fresh human isolated mitochondria. One to two weeks later, a significantly better cognitive performance was noticed in the mitochondria treated AD-mice relative to vehicle treated AD-mice, approaching the performance of non-AD mice. We also detected a significant decrease in neuronal loss and reduced gliosis in the hippocampus of treated mice relative to untreated AD-mice. An amelioration of the mitochondrial dysfunction in brain was noticed by the increase of citrate-synthase and cytochrome c oxidase activities relative to untreated AD-mice, reaching activity levels of non-AD-mice. Increased mitochondrial activity was also detected in the liver of mitochondria treated mice. No treatment-related toxicity was noted. Thus, IV mitochondrial transfer may possibly offer a novel therapeutic approach for AD.

Pages 605-612
Taiki Sugimoto, Rei Ono, Ai Kimura, Naoki Saji, Shumpei Niida, Kenji Toba, Takashi Sakurai
Cross-Sectional Association Between Cognitive Frailty and White Matter Hyperintensity Among Memory Clinic Patients
Abstract: Background: Cognitive frailty (CF) is defined as simultaneous presence of physical frailty (PF) and cognitive impairment among older adults without dementia. Although white matter hyperintensities (WMH) as expressions of cerebral small vessel disease are associated with physical and cognitive decline and could manifest as CF, this association remains yet to be clarified. Objects: To clarify the association between CF and WMH among memory clinic patients. Methods: The subjects of this cross-sectional study were 121 cognitively normal (CN) and 212 mildly cognitively impaired (MCI) patients who presented to the Memory Clinic at the National Center for Geriatrics and Gerontology of Japan. PF status was defined based on the definition proposed by Fried and colleagues. CF was defined as simultaneous presence of pre-PF or PF and MCI. WMH volumes were measured using an automatic segmentation application. Multiple liner regression analyses with adjustment for cardiovascular risk factors were performed. Results: Of all subjects, 77 (63.6%) and 22 (18.2%) CN patients and 132 (62.3%) and 65 (30.7%) MCI patients were categorized into pre-PF and PF, respectively. Multiple liner regression analysis showed that those with CF had higher WMH volumes than those without (β = 0.23). When categorized into six groups according to PF and cognitive status, the PF/CN (β = 0.15), pre-PF/MCI (β = 0.41), and PF/MCI (β = 0.34) groups had higher WMH volumes than the non-PF/CN group. Conclusions: This study showed increased WMH volumes in CF and PF, indicating that WMH could be one of the key underlying brain pathologies of CF.

Pages 613-622
Patrizia Turriziani, Daniela Smirni, Giuseppa Renata Mangano, Giuseppe Zappalà, Andreina Giustiniani, Lisa Cipolotti, Massimiliano Oliveri
Low-Frequency Repetitive Transcranial Magnetic Stimulation of the Right Dorsolateral Prefrontal Cortex Enhances Recognition Memory in Alzheimer’s Disease
Abstract: Background: The lack of effective pharmacological or behavioral interventions for memory impairments associated with Alzheimer's disease (AD) emphasizes the need for the investigation of approaches based on neuromodulation. Objective: This study examined the effects of inhibitory repetitive transcranial magnetic stimulation (rTMS) of prefrontal cortex on recognition memory in AD patients. Methods: In a first experiment, 24 mild AD patients received sham and real 1 Hz rTMS over the left and right dorsolateral prefrontal cortex (DLPFC), in different sessions, between encoding and retrieval phases of a non-verbal recognition memory task. In a second experiment, another group of 14 AD patients underwent sham controlled repeated sessions of 1 Hz rTMS of the right DLPFC across a two week treatment. Non-verbal recognition memory task was performed at baseline, at the end of the two weeks period and at a follow up of 1 month. Results: Right real rTMS significantly improved memory performance compared to right sham rTMS (p=0.001). Left real rTMS left the memory performance unchanged as compared with left sham rTMS (p=0.46). The two sham conditions did not differ between each other (p=0.24). In the second experiment, AD patients treated with real rTMS showed an improvement of memory performance at the end of the two weeks treatment (p = 0.0009), that persisted at 1-month follow-up (p = 0.002). Conclusion: These findings provide evidence that inhibitory rTMS over the right DLPFC can improve recognition memory function in AD patients. They also suggest the importance of a new approach of non-invasive brain stimulation as a promising treatment in AD.

Pages 623-631
Guoyu Zhou, Xinjing Zhao, Zhiyin Lou, Shengnian Zhou, Peiyan Shan, Ning Zheng, Xiaolin Yu, Lin Ma
Impaired Cerebral Autoregulation in Alzheimer’s Disease: A Transcranial Doppler Study
Abstract: Background: Vasculature changes have been observed in Alzheimer’s disease (AD). AD-related vascular pathology might impair cerebral autoregulation (CA). Objective: This study was designed to evaluate CA of AD patients by using transcranial doppler (TCD). Methods: A total of 61 participants were included in the study, including 31 AD patients and 30 controls. The trend curves of cerebral blood flow velocities (CBFV), pulsatility index, and resistance index were obtained using TCD during supine-to-standing posture changes. CA was measured by the changes of CBFV curves during supine-to-standing test. Results: There were two spikes named X spike and W spike that appeared in the CBFV curve when the subjects stood abruptly. The slope of the X spike descending branch, the slope of the W spike ascending branch, and the angle between X and W spikes (α angle), showed significant differences between the experimental and control groups (2.34±0.99 versus 3.15±1.61 cm/s2, p=0.021; 2.31±0.81 versus 3.38±1.18 cm/s2, p<0.001; and 52.71±20.26 versus 41.4±12.87 degrees, p=0.012, respectively). ROC analysis showed that AUCα angle is 0.664 (p=0.028) and that AUCSAB and AUCadjusted SAB are 0.775 and 0.738, respectively (both p<0.001). Conclusions: Our study demonstrated that supine-to-standing TCD test is a valuable tool for the evaluation of CA in AD patients. Impaired CA in AD patients manifested as decreased efficiency of changes in the CBFV curve. Neurovascular units were involved in the pathogenesis of AD.

Pages 633-640
Xi-Xi Liu*, Bin Jiao*, Xin-Xin Liao, Li-Na Guo, Zhen-Hua Yuan, Xin Wang, Xue-Wen Xiao, Xin-Yue Zhang, Bei-Sha Tang, Lu Shen *These authors contributed equally to this work.
Analysis of Salivary Microbiome in Patients with Alzheimer’s Disease
Abstract: Recent studies found that poor oral hygiene was associated with increased risk of dementia, and the number of oral bacteria significantly increased in the brain tissues of patients with Alzheimer’s disease (AD), suggesting that the oral microbiota may play an important role in the pathogenesis of AD. However, the actual composition of oral bacteria communities in patients with AD and whether these oral bacteria are associated with disease severity remain largely unknown. Also, the APOE ε4 polymorphism is a strong risk factor for sporadic AD, and it would be pertinent to see if the bacterial flora was different in those patients who were APOE ε4 positive. A total of 78 subjects were recruited in this study, including 39 patients with AD and 39 healthy controls. Saliva was collected from each subject.16S ribosomal RNA (16S rRNA) sequencing was conducted to analyze the salivary microbiota, and Sanger sequencing was performed to analyze the APOE genotype. There was a significantly lower richness and diversity of saliva microbiota detected in AD patients than healthy controls. The relative abundance of Moraxella, Leptotrichia, and Sphaerochaeta in the saliva of AD patients greatly increased, whereas that of Rothia was significantly reduced. Compared with APOE ε4 (-) patients, the level of Abiotrophia and Desulfomicrobium was comparatively abundant, while Actinobacillus and Actinomyces decreased significantly in patients carrying the APOE ε4. No bacteria were found to be associated with the severity of AD. This is the first study to analyze the salivary microorganisms in patients with AD, and we discovered that the composition of salivary microbiome was altered in AD, providing further support for the role of the oral microbiome in AD development.

Pages 641-652
Koji Yoshida, Yukiko Hata, Shojiro Ichimata, Naoki Nishida
Tau and Amyloid-β Pathology in Japanese Forensic Autopsy Series Under 40 Years of Age: Prevalence and Association with APOE Genotype and Suicide Risk
Abstract: Background: Aggregation of abnormal phosphorylated tau in brain stem areas may be a possible early pathological manifestation of Alzheimer’s disease (AD). Objective: This study aimed to explore the prevalence of cases with AD-related pathology in subjects <40 years of age and to explore the association of such pathology, neuropsychiatric symptoms, and APOE genotype. Method: We conducted brain immunohistochemistry for 189 cases <40 years of age (mean±standard deviation age 25.3±13.1 years). Tau positive cases were then assessed for the distribution of tau pathology in the locus ceruleus (LC), raphe nucleus (RN), and entorhinal cortex (ErC), and the distinction between neuronal threads and cellular inclusions. Apolipoprotein E (APOE) genotype was also examined. Results: Tau pathology was detected in 135 cases (71.4%; 13–39 years; only LC, 23 cases; only RN, 4 cases; only ErC, 35 cases; LC+RN, 3 cases; LC+ErC, 57 cases; all three regions, 10 cases). The prevalence of thread pathology was higher than that of cellular inclusions. Significantly higher prevalence of the APOE ε2 allele were found in 10-39 years of age natural death cases (p<0.05). Amyloid-β deposition was found in only 7 cases, along with a significantly high frequency of the ε4 allele (p<0.05). While a past history of psychiatric disease was a significant risk factor for suicide, AD-related pathology was not associated with suicide. Conclusions: Both the brain stem and entorhinal cortex was the initial site of tau pathology in many younger subjects. AD-related pathology may not be a significant accelerating factor for suicide in younger subjects.

Pages 653-662
Harriet J. Forbes, Angel Y.S. Wong, Caroline Morton, Krishnan Bhaskaran, Liam Smeeth, Marcus Richards, Sigrun A.J. Schmidt, Sinéad M. Langan, Charlotte Warren-Gash
Partner Bereavement and Detection of Dementia: A UK-Based Cohort Study Using Routine Health Data
Abstract: Background: In the UK, an estimated one third of people with dementia have not received a diagnosis. Good evidence suggests that dementia risk is increased among widowed individuals; however, it is not clear if they are being diagnosed in routine primary care. Objective: This study aimed to investigate if bereavement influenced the probability of having received a dementia diagnosis. Methods: A population-based cohort study using UK electronic health records, between 1997 and 2017, among 247,586 opposite-sex partners. Those experiencing partner bereavement were matched (age, sex, and date of bereavement) to a non-bereaved person living in a partnership. Multivariate cox regression was performed. Results: Partner bereavement was associated with an increased risk of receiving a diagnosis of dementia in the first three months (hazard ratio (HR) 1.43, 95%CI 1.20-1.71) and first six months (HR 1.24, 95%CI 1.09-1.41), while there was a small reduced risk of getting a dementia diagnosis over all follow-up (HR 0.94, 95%CI 0.89-0.98). Conclusions: Partner bereavement appears to lead to a short-term increased risk of the surviving partner receiving a diagnosis of dementia, suggesting that bereavement unmasks existing undiagnosed dementia. Over the longer term, however, bereaved individuals are less likely to have a diagnosis of dementia in their health records than non-bereaved individuals.