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Background: No treatments exist for apathy in people with frontotemporal dementia. Previously, in a randomised double-blind, placebo-controlled, dose-finding study, intranasal oxytocin administration in people with frontotemporal dementia improved apathy ratings on the Neuropsychiatric Inventory over 1 week and, in a randomised, double-blind, placebo-controlled, crossover study, a single dose of 72 IU oxytocin increased blood-oxygen-level-dependent signal in limbic brain regions. We aimed to determine whether longer treatment with oxytocin improves apathy in people with frontotemporal dementia., Methods: We conducted a multicentre, randomised, double-blind, placebo-controlled, adaptive, crossover, phase 2a/2b trial, enrolling participants from 11 expert frontotemporal dementia outpatient clinics across Canada and the USA. People aged 30-80 years with a diagnosis of probable frontotemporal dementia, a Neuropsychiatric Inventory apathy score of 2 or higher, a study partner who interacted with them for at least 3 h per day, and stable cognitive and behavioural medications for 30 days were eligible for inclusion. In stage 1, participants were randomly assigned (1:1:1:1:1:1) to one of three dose schedules (every day, every other day, and every third day) of 72 IU intranasal oxytocin or placebo and to the order they would received the intervention in the crossover; intranasal oxytocin or placebo were administered twice daily for 6 weeks, with a 6-week washout and then crossover to the other intervention. In stage 2, new participants were randomised (1:1) to the dose that had been determined as optimal in stage 1 or to placebo, with crossover as in stage 1. Randomisation used variable block sizes and was stratified by participant sex and Clinical Dementia Rating severity score. All kits of investigational product were identical and produced centrally, and all local teams, study staff, and participants were masked to treatment allocation and order. The primary outcome was difference in the change in Neuropsychiatric Inventory apathy scores for oxytocin versus placebo periods in the per-protocol population after 6 weeks of treatment. Safety was assessed at each visit via electrocardiogram, blood work, and collection of data on adverse events. This trial is registered at ClinicalTrials.gov (NCT03260920)., Findings: Between Jan 31, 2018, and Dec 11, 2020, 70 patients were screened for stage 1 and 60 (86%) were enrolled. 45 (75%) completed both treatment periods of stage 1. 72 IU oxytocin every third day was the optimal dose schedule from stage 1 based on its Bayesian posterior probability (Pr(Best)=0·478). Between June 28, 2021, and Jan 31, 2023, 42 patients were screened for stage 2, and 34 (81%) were enrolled. 28 (82%) completed both treatment periods in stage 2. 38 (40%) of 94 participants were female and 56 (60%) were male (mean age 65·9 years, SD 8·2) Treatment with oxytocin every third day resulted in an improved Neuropsychiatric Inventory apathy score, with an estimated -1·32 points (95% CI -2·43 to -0·21) relative to placebo (one sided p=0·010). Two adverse events were reported in at least 5% of participants: upper respiratory tract infection (five [6%] of 78 participants on placebo and three [5%] on every third day at all doses of oxytocin) and headache (two [3%] participants on placebo, one [7%] of 15 participants on oxytocin every day, and two [4%] of 55 participants on oxytocin every third day). No adverse events were attributed to oxytocin treatment., Interpretation: Intranasal oxytocin given every third day was well tolerated and was associated with a small reduction in apathy in patients with frontotemporal dementia. Future trials might investigate intermittent dosing of more potent formulations than in this study, to establish whether larger effects are possible., Funding: Canadian Institutes of Health Research and Weston Foundation., Competing Interests: Declaration of interests ECF received grant support for the FOXY trial from the Canadian Institutes for Health Research, the Weston Foundation, and the Physician Services Incorporated Foundation, and serves as a scientific advisor for and has received consulting fees from Psilera. AdLB has received consulting fees from AGTC, Alchemab, Alector, Alzprotect, Amylyx, Arkuda, Arrowhead, Arvinas, Aviado, Eli Lilly, GSK, Humana, Merck, Modalis, Muna, Oligomerix, Oscotec, Pfizer, Roche, Switch, Transposon, and UnlearnAI and stock or stock options from Alector, and Arvinas. SHP has received consulting fees from Zywie Bio and honorarium from Eli Lilly and has a leadership role in the Consortium of Canadian Centers for Clinical Cognitive Research. CO has received clinical trial funding, trial drug, and materials from Alector, Transposon Therapeutics, and Denali Therapeutics; consulting fees from Acadia Pharmaceuticals, Reata Pharmaceuticals, Otsuka Pharmaceutical, Eisai Pharmaceutical, Lykos Therapeutics, and Zevra Therapeutics; honoraria from the Philadelphia Psychiatric Society, the American Academy of Neurology Institute, and the Lewy body Dementia Association; and support for travel from Cure VCP and the Lewy Body Dementia Association. He has had leadership roles in the Association for Frontotemporal Degeneration Medical Advisory Council, the FTD Disorders Scientific Advisory Board, the Tau Consortium Scientific Advisory Board, the International Society for Frontotemporal Dementias Executive Committee, International Society to Advance Alzheimer's Research and Treatment Frontotemporal Dementia Professional Interest Area Executive Committee, and the International Society for Neurodegenerative Diseases. MCT has received consulting fees from Eisai, Eli Lilly, and Novo Nordisk and served as a scientific advisor to the Women's Brain Foundation, Brain Injury Canada, and Progressive Supranuclear Palsy Society of Canada; her institution has received support to serve as a clinical trial site for Janssen, Biogen, Avanex, Green Valley, UCB, Novo Nordisk, GSK, Bristol Myers Squibb, and Passage Bio and materials and funding for research from Roche. Berry Consultants (SB, MAD, AnLB, and AM) received consulting fees paid from the Canadian Institutes of Health Research and Weston grants through the Lawson Health Research Institute for the FOXY trial design and analysis. KD-R has received grant funding to her institution from US National Institutes of Health (NIH) grants U19 AG063911 and P30 AG066509. EH has received grant funding from the NIH/National Institute on Aging (NIA) R01 AG062268, U01 AG079850, and NIH/National Institute of Mental Health (NIMH) R01 MH120794. He participated on a DSMB for R21AG07895. AP has received grant funding from Alector, Athira, Biogen, Cassava, Eisai, Eli Lilly, Genentech/Roche, Vaccinex, NIA, NIMH, and Department of Defense; received consulting fees from Athira, Biogen, Eisai, IQVIA, Lundbeck, Otsuka, ONO Pharmaceuticals, WIRB-Copernicus Group received honoraria from WebMD; and participated on DSMB or advisory boards for Acadia, Bristol Myers Squibb, Cognitive Research Corporation, Functional Neuromodulation, Novartis, and Xenon. He has held leadership for fiduciary roles for boards for Alzheon, Athira, and Cognition Therapeutics. SD has received grant and research funding from the Alzheimer Drug Discovery Foundation, Canadian Institutes for Health Research (CIHR), Fond de recherche du Québec, Novo Nordisk, Biogen, Janssen, Alnylam, and Innodem Neurosciences; received consulting fees from Eisai, QurAlis, and Eli Lilly; received honoraria from Eisai; participated on advisory boards for IntelGenX and Aviado Bio; and held stock or stock options as co-founder of AFX Medical. RRG has received honoraria for lectures from Carnot Pharma and Torrent Pharma and has served on an advisory board for Silanes Pharma. JC has received grant funding from National Institute of General Medical Services (grant P20GM109025), NIA (R35AG71476 and R25AG083721–01), and National Institute of Neurological Disorders and Stroke (RO1NS139383); received royalties from the Neuropsychiatric Inventory; consulting fees from Acadia, Acumen, ALZpath, Annovis, Aprinoia, Artery, Biogen, Biohaven, BioXcel, Bristol Myers Squib, Eisai, Fosun, GAP Foundation, Green Valley, Janssen, Kinoxis, Lighthouse, Lilly, Lundbeck, LSP/eqt, Merck, MoCA Cognition, New Amsterdam, Novo Nordisk, Optoceutics, Otsuka, Oxford Brain Diagnostics, Prothena, ReMYND, Roche, Scottish Brain Sciences, Signant Health, Simcere, Sinaptica, and Vaxxinity; received honoraria from Eisai; participated on a DSMB for Johns Hopkins University; and held stock options for Annovis, Artery, Vaxxinity, Behrens, Alzheon, MedAvante-Prophase, and Acumen. MaM has received grants from the University of Washington, CIHR, The Weston Brain Foundation, Brain Canada, and the Women's Brain Health Initiative; has research contracts with Roche and Alector; has received royalties from the Henry Stewart Talks; has received consulting fees from Eli Lilly Canada, Alector, Biogen Canada, Wave Life Sciences, Eisai Canada, and Novo-Nordisk Canada; has received honoraria from the MINT Memory Clinics and the ECHO Dementia Series; and has held unpaid leadership roles in the Alzheimer Society of Canada and Parkinson Canada. G-YRH has had research contracts with Biogen, Cassava, Eli Lilly, and Eisai; received research grants from the NIH (Asian Cohort on Alzheimer's Disease) and CIHR; received consulting fees from Biogen, Eli Lilly, Eisai, Novo-Nordisk, and Roche; and held the unpaid position of President of the Consortium of Canadian Centers for Clinical Cognitive Research. HF has received grant support from Allyx Therapeutics, Vivoryon (Probiodrug), Biohaven Pharmaceuticals, and LuMind Foundation; has had service agreements with US San Diego for consulting activities with LuMind Foundation, Novo Nordisk, Axon Neuroscience, and Arrowhead Pharmaceuticals; has received support for travel-related expenses to conferences from Novo Nordisk, the Royal Society of Canada, Translating Research in Elder Care, the Association for Frontotemporal Dementia, and the Rainwater Charitable Foundation. He holds US patent number PCT/US2007/07008; has participated on a DSMB and Data Safety Monitoring Committee for Roche/Genetch Pharmaceuticals and Janssen Research and Development; and served on the Scientific Advisory Board for the Tau Consortium. He has received philanthropic support for Alzheimer's therapeutic research from the Epstein Family Alzheimer's Research Collaboration. All other authors declare no competing interests., (Copyright © 2025 Elsevier Ltd. 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Lecanemab and donanemab are monoclonal antibody therapies that remove amyloid-beta from the brain. They are the first therapies that alter a fundamental mechanism, amyloid-beta deposition, in Alzheimer disease (AD). To inform Canadian decisions on approval and use of these drugs, the Canadian Consortium on Neurodegeneration in Aging commissioned Work Groups to review evidence on the efficacy and safety of these new therapies, as well as their projected impacts on Canadian dementia systems of care. We included persons with lived experience with Alzheimer disease in the discussion about the benefits and harms. Our review of the trial publications found high quality evidence of statistically significant group differences, but also recognized that there are mixed views on the clinical relevance of the observed differences and the value of therapy for individual patients. The drugs are intended for persons with early AD, at a stage of mild cognitive impairment or mild dementia. If patients are treated, then confirmation of AD by positron emission tomography or cerebrospinal fluid analysis and monitoring for risk of amyloid-related imaging abnormalities was recommended, as done in the clinical trials, although it would strain Canadian resource capacity. More data are needed to determine the size of the potentially eligible treatment population in Canada., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Howard Cherkow reports financial support was provided by Canadian Institutes of Health Research. Howard Chertkow reports financial support was provided by Alberta Prion Research Institute. Howard Chertkow reports financial support was provided by Alberta Innovates. Howard Chertkow reports financial support was provided by Alzheimer Research UK. Alzheimer Society of Canada reports financial support was provided by Alzheimer Society of Canada. Alzheimer Society of Canada reports financial support was provided by Brain Canada Foundation. Howard Chertkow reports financial support was provided by Canadian Nurses Foundation. Howard Chertkow reports financial support was provided by Eli Lilly and Company. Howard Chertkow reports financial support was provided by Quebec Health Research Fund. Howard Chertkow reports financial support was provided by Hypertension Canada. Howard Chertkow reports financial support was provided by Michael Smith Foundation for Health Research. Howard Chertkow reports financial support was provided by New Brunswick Health Research Foundation. Howard Chertkow reports financial support was provided by Nova Scotia Health Research Foundation. Howard Chertkow reports financial support was provided by Ontario Brain Institute. Howard Chertkow reports financial support was provided by Pfizer Canada Inc. Howard Chertkow reports financial support was provided by Robin and Barry Picov Family Foundation. Howard Chertkow reports financial support was provided by Sanofi. Howard Chertkow reports financial support was provided by Saskatchewan Health Research Foundation. AmanPreet Badhwar reports a grant from the Alzheimer Society of Canada, paid to her institution. Laura Barlow reports no disclosures. Robert Bartha reports no disclosures. Sarah Best reports honoraria from Eisai pharmaceuticals, paid to her. Jennifer Bethell reports no disclosures. Jaspreet Bhangu reports a contract from Eisai for site PI, paid to his institution; consulting for Eisai, unpaid; and consulting for Eli Lilly, paid to him. Sandra Black reports contracts with Genentech, Optina, Roche, Eli Lilly, Eisai/Biogen Idec, Novo Nordisk, Lilly Avid, and ICON, paid to her institution; grants from the Ontario Brain Institute, CIHR, Leducq Foundation, Heart and Stroke Foundation of Canada, NIH, Alzheimers Drug Discovery Foundation, Brain Canada, Weston Brain Institute, Canadian Partnership for Stroke Recovery, Canadian Foundation for Innovation, Focused Ultrasound Foundation, Alzheimers Association US, Queens University, Compute Canada Resources for Research Groups, CANARIE, and Networks of Centres of Excellence of Canada, paid to her institution; consulting for Novo Nordisk, Eisai, Eli Lilly, Roche, and Cpd network, paid to her; consulting for DSR: Diagnosis, Solutions & Results Inc., Conference Board Canada, World Dementia Council, University of Rochester Contribution to the Mission and Scientific Leadership of the Small Vessel VCID Biomarker Validation Consortium, National Institute of Neurological Disorders and Stroke, and Ontario Dementia Care Alliance (ODCA), paid to her institution; honoraria from Biogen, Roche, and Eisai, paid to her; and honoraria from Roche, unpaid. Christian Bocti reports stock in Imeka. Michael Borrie reports consulting for Eisai, Eli Lilly, Biogen, and Hoffmann-La Roche, paid to him; and contracts with Eisai, Eli Lilly, Biogen, Hoffmann-La Roche, and Alector, paid to his institution. Susan Bronskill reports grants from the Alzheimer Society of Canada, Brain Canada Foundation, Evaluative Clinical Sciences Research Platform of Sunnybrook Research Institute, Canadian Consortium on Neurodegeneration in Aging (CCNA), Canadian Institutes of Health Research, and Ontario Brain Institute, paid to her institution; and a contract with the Public Health Agency of Canada, paid to her institution. Amer Burhan reports a contract with the Toronto Memory Program (Headland Research), paid to him; consulting for Eisai, Avanir, Otsuka-Lundbeck, Boehringer-Ingelheim, and Roche Pharma, paid to him; and grants from the National Institute of Aging, Brain Canada Foundation, Alzheimer Drug Discovery Foundation, National Research Council, Weston Foundation, and Canada Institute for Health Research, paid to his institution. Frederic Calon reports meeting expenses from Eli Lilly, paid to him. Richard Camicioli reports grants from the Canadian Institutes of Health Research, National Institute of Health, Weston Foundation, paid to him; and support for attending meetings from Parkinson Canada, Canadian Movement Disorders Group, and the Canadian Consortium on Neurodegeneration in Aging, paid to him; and advisory boards for Ambroxal trial and Parkinson Canada, unpaid. Barry Campbell reports no disclosures. Howard Chertkow reports contracts as site PI for clinical trials with Hoffmann-La Roche Limited, TauRx, Eli Lilly Corp., Anavex Life Sciences, Alector LLC, Biogen MA Inc., IntelGenX Corp., and Immunocal, paid to his institution; and consulting for Eisai, Biogen, and Lilly Inc. in Canada, paid to his institution. D. Louis Collins reports no disclosures. Mahsa Dadar reports no disclosures. Mari DeMarco reports consulting for Roche and Eisai, paid to her; honoraria from Roche, paid to her; and a grant from Roche, paid to her institution. Philippe Desmarais reports consulting for Eisai, paid to him. Simon Ducharme reports contracts for clinical trials with Biogen, Janssen, Novo Nordisk, Alnylam, Passage Bio, and Innodem Neurosciences, paid to his institution; consulting for QuRALIS, Eisai, and Eli Lilly, paid to him; honoraria from Eisai, paid to him; and participation in a Data Safety Monitoring Board or Advisory Board for Aviado Bio and IntelGenX, paid to him. Simon Duchesne reports stock or stock options for True Positive Medical Devices Inc., paid to him; honoraria from Novo Nordisk, paid to him; and travel expenses from Eisai, paid to him. Gillian Einstein reports no disclosures. Howard Feldman reports consulting for Eisai Inc., Biogen, LuMind, and Novo Nordisk, paid to his institution; grants from Allyx Therapeutics, Vivoryon Therapeutics, Biohaven Pharmaceuticals, and LuMind Foundation, paid to his institution; royalties from the University of British Columbia, paid to him; a patent held by the University of British Columbia, paid to him; consulting for Axon Neuroscience, Arrowhead Pharmaceuticals, and Biosplice Therapeutics, paid to his institution; support for attending meetings or travel from Novo Nordisk Inc., Royal Society of Canada, Translating Research in Elder Care (TREC), and the Association for Frontotemporal Dementia (AFTD), with payments made to his institution; participation in a Data Safety Monitoring Board or Advisory Board for the Tau Consortium and Janssen Research & Development LLC, paid to his institution; and philanthropic support from the Epstein Family Alzheimers Research Collaboration, paid to his institution. John Fisk reports no disclosures. Andrew Frank reports consulting for Eli Lilly Canada, Eisai Canada, and Novo Nordisk, paid to him. Aravind Ganesh reports grants from the Alzheimer Society of Canada and Alzheimer Society of Alberta and Northwest Territories, paid to his institution; and consulting for Biogen and Eisai, paid to him. Jodie Gawryluk reports no disclosures. Linda Grossman reports no disclosures. Arthur Harrison reports no disclosures. Alexandre Henri-Bhargava reports consulting for Eisai Canada and Eli Lilly, paid to him; contracts for clinical trials with Novo Nordisk, Cerevel, Anavex, IntelGenX, and Green Valley (Shanghai), paid to his institution; honoraria from the Canadian Coalition for Seniors Mental Health, paid to him; leadership in the Consortium of Canadian Centres for Clinical Cognitive Research and the Canadian Neurological Society, unpaid; and other financial or non-financial interests through the Neil and Susan Manning Cognitive Health Initiative, paid to his institution. Zahinoor Ismail reports consulting for CADTH, Eisai, Lilly, Lundbeck, Novo Nordisk, Otsuka, and Roche, paid to him. Inbal Itzhak reports no disclosures. Manish Joshi reports consulting for Eisai, Biogen, Eli Lilly, and Clario, paid to him. Edeltraut Kroger reports no disclosures. Sanjeev Kumar reports no disclosures. Robert Laforce reports consulting for Eisai and Eli Lilly, paid to him. Krista Lanctot reports grants from the Canadian Institutes of Health Research, Alzheimers Drug Discovery Foundation, Weston Foundation, Alzheimers Association (US), and Pooler Charitable Fund, paid to her institution; contracts with Cerevel and BioXcel, paid to her institution; consulting for Boehringer Ingelheim, Bright Minds, Bristol Myers Squibb, Cerevel, Eisai Co. Ltd, Exciva, Ironshore Pharmaceuticals, Kondor Pharma, H Lundbeck A/S, Novo Nordisk, and Praxis Therapeutics, paid to her; honoraria from Novo Nordisk, H Lundbeck, and Eisai, paid to her; support for attending meetings or travel from H Lundbeck and Eisai, paid to her institution; participation in a Data Safety Monitoring Board or advisory board for the PAS-MCI Study, unpaid; leadership in the American Association of Geriatric Psychiatry, unpaid; and receipt of equipment, materials, drugs, gifts, or services from PBG, paid to her institution. Meghan Lau reports no disclosures. Linda Lee reports consulting for Eisai, Lilly, Novo Nordisk, Lundbeck, and Roche, paid to her. Mario Masellis reports grants from the Canadian Institutes of Health Research, Weston Brain Institute, Ontario Brain Institute, Washington University, Women's Brain Health Initiative, Brain Canada, and EU Joint Program for Neurodegenerative Disease Research, paid to him; contracts for clinical trials with Roche and Alector, paid to his institution; consulting fees from Ionis, Alector, Biogen Canada, Wave Life Science, Eisai Canada, and Novo Nordisk Canada, paid to him; royalties from the Henry Stewart Talks, paid to him; honoraria from MINT Memory Clinics and ECHO Dementia Series, paid to him; and membership in Scientific Advisory Boards of the Alzheimer's Society Canada and Parkinson Canada, unpaid. Fadi Massoud reports honoraria from Astellas and Pfizer, paid to him; and consulting for Eisai and Novo Nordisk, paid to him. Sara Mitchell reports consulting for Eisai, paid to her; honoraria from Eisai and Eli Lilly, paid to her; support for attending meetings or travel from Eli Lilly, paid to her; and leadership in the Novo Nordisk Advisory Board, paid to her. Manuel Montero-Odasso reports no disclosures. Karen Myers Barnett reports no disclosures. Haakon Nygaard reports consulting for Eisai, paid to him; and participation in advisory boards for Biogen and Hoffmann-La Roche, paid to him. Stephen Pasternak reports a grant from Zywie Bio LLC and the Weston Foundation, paid to his institution; site investigator for contracts with Cassava and AriBio, paid to his institution; consulting for Eisai, unpaid; an advisory board role for Zywie Bio LLC, unpaid; stock in Zywie Bio LLC; a patent through Zywie Bio LLC, unpaid; and leadership in the C5R Board, unpaid. Jody Peters reports no disclosures. Natalie Phillips reports no disclosures. M. Natasha Rajah reports no disclosures. Julie Robillard reports no disclosures. Kenneth Rockwood reports royalties from The Clinical Frailty Scale and the Pictorial Fit-Frail Scale, paid to his institution; honoraria from the Burnaby Family Practice, Chinese Medical Association, University of Nebraska-Omaha, the Australia New Zealand Society of Geriatric Medicine, the Atria Institute, University of British Columbia, McMaster University, and Fraser Health Authority, paid to him; a US patent application submitted for Electronic Goal Attainment Scaling (patent number US20230402138A1); consulting for EIP Pharma Inc., the ADMET-2 advisory board (Johns Hopkins), and the Wake Forest University Medical School Centre advisory board, unpaid; advisory board roles with Ardea Outcomes, Danone, Hollister, INmune, Novartis, Takeda, and Nutricia, unpaid. Pedro Rosa-Neto reports no disclosures. Dallas Seitz reports a grant from the University Health Foundation - Alberta Roche Collaboration in Health, Alzheimers Association, and Alzheimer Society of Canada, paid to his institution; honoraria from the Canadian Coalition for Seniors Mental Health, Alberta Health Services, and Recovery Alberta, paid to him; and leadership on the Board of Directors for the Alzheimer Society of Alberta and Northwest Territories, unpaid. Eric Smith reports grants from the Canadian Institutes of Health Research, Weston Family Foundation, and Weston Brain Institute, paid to his institution; contracts for clinical trials from Biogen, paid to his institution; contracts with Sense Diagnostics and SFJ Pharmaceuticals, paid to his institution; royalties from UpToDate and UTI Limited Partnership, paid to him; consulting for Eisai, Eli Lilly, and Alnylam, unpaid; and participation in a Data Safety and Monitoring Board for NINDS, paid to him. Jean-Paul Soucy reports grants from the Weston Foundation and Canadian Institutes of Health Research, paid to his institution; a contract with Charles River, paid to his institution; and consulting for Biogen, paid to him. Shanna Trenaman reports being a patient advisor for a Delphi panel developing best practices for antipsychotic use in long-term care, unpaid. Cheryl Wellington reports grants from the Heart and Stroke Foundation of Canada, BrightFocus Foundation, and Cure Alzheimer Fund, paid to her institution. Aicha Zadem reports no disclosures. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Masson SAS.)

Introduction: Accurate testing for Alzheimer's disease (AD) represents a crucial step for therapeutic advancement. Currently, tests are expensive and require invasive sampling or radiation exposure., Methods: We developed a nanoscale flow cytometry (nFC)-based assay of extracellular vesicles (EVs) to screen biomarkers in plasma from mild cognitive impairment (MCI), AD, or controls., Results: Circulating amyloid beta (Aβ), tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, p-tauS235, ubiquitin, and lysosomal-associated membrane protein 1-positive EVs distinguished AD samples. p-tau181, p-tau217, p-tauS235, and ubiquitin-positive EVs distinguished MCI samples. The most sensitive marker for AD distinction was p-tau231, with an area under the receiver operating characteristic curve (AUC) of 0.96 (sensitivity 0.95/specificity 1.0) improving to an AUC of 0.989 when combined with p-tauS235., Discussion: This nFC-based assay accurately distinguishes MCI and AD plasma without EV isolation, offering a rapid approach requiring minute sample volumes. Incorporating nFC-based measurements in larger populations and comparison to "gold standard" biomarkers is an exciting next step for developing AD diagnostic tools., Highlights: Extracellular vesicles represent promising biomarkers of Alzheimer's disease (AD) that can be measured in the peripheral circulation. This study demonstrates the utility of nanoscale flow cytometry for the measurement of circulating extracellular vesicles (EVs) in AD blood samples. Multiple markers including amyloid beta, tau, phosphorylated tau (p-tau)181, p-tau231, p-tau217, and p-tauS235 accurately distinguished AD samples from healthy controls. Future studies should expand blood and cerebrospinal fluid-based EV biomarker development using nanoflow cytometry approaches., (© 2024 The Author(s). Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Objectives: To investigate whether a history of traumatic brain injury (TBI) is associated with greater long-term grey-matter loss in patients with mild cognitive impairment (MCI)., Methods: 85 patients with MCI were identified, including 26 with a previous history of traumatic brain injury (MCI[TBI-]) and 59 without (MCI[TBI+]). Cortical thickness was evaluated by segmenting T1-weighted MRI scans acquired longitudinally over a 2-year period. Bayesian multilevel modelling was used to evaluate group differences in baseline cortical thickness and longitudinal change, as well as group differences in neuropsychological measures of executive function., Results: At baseline, the MCI[TBI+] group had less grey matter within right entorhinal, left medial orbitofrontal and inferior temporal cortex areas bilaterally. Longitudinally, the MCI[TBI+] group also exhibited greater longitudinal declines in left rostral middle frontal, the left caudal middle frontal and left lateral orbitofrontal areas sover the span of 2 years (median = 1-2%, 90%HDI [-0.01%: -0.001%], probability of direction (PD) = 90-99%). The MCI[TBI+] group also displayed greater longitudinal declines in Trail-Making-Test (TMT)-derived ratio (median: 0.737%, 90%HDI: [0.229%: 1.31%], PD = 98.8%) and differences scores (median: 20.6%, 90%HDI: [-5.17%: 43.2%], PD = 91.7%)., Conclusions: Our findings support the notion that patients with MCI and a history of TBI are at risk of accelerated neurodegeneration, displaying greatest evidence for cortical atrophy within the left middle frontal and lateral orbitofrontal frontal cortex. Importantly, these results suggest that long-term TBI-mediated atrophy is more pronounced in areas vulnerable to TBI-related mechanical injury, highlighting their potential relevance for diagnostic forms of intervention in TBI., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

Alzheimer's disease (AD) is a progressive neurological condition characterized by impaired cognitive function and behavioral alterations. While AD research historically centered around mis-folded proteins, advances in mass spectrometry techniques have triggered increased exploration of the AD lipidome with lipid dysregulation emerging as a critical player in AD pathogenesis. Gangliosides are a class of glycosphingolipids enriched within the central nervous system. Previous work has suggested a shift in a-series gangliosides from complex (GM1) to simple (GM2 and GM3) species may be related to the development of neurodegenerative disease. In addition, complex gangliosides with 20 carbon sphingosine chains have been shown to increase in the aging brain. In this study, we utilized matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to interrogate the in situ relationship of a-series gangliosides with either 18 or 20 carbon sphingosine chains (d18:1 or d20:1, respectively) in the post-mortem human AD brain. Here, we expanded upon previous literature and demonstrated a significant decrease in the GM1 d20:1 to GM1 d18:1 ratio in regions of the dentate gyrus and entorhinal cortex in AD relative to control brain tissue. Then, we demonstrated that the MALDI-MSI profile of GM3 co-localizes with histologically confirmed amyloid beta (Aβ) plaques and found a significant increase in both GM1 and GM3 in proximity to Aβ plaques. Collectively, this study demonstrates a perturbation of the ganglioside profile in AD, and validates a pipeline for MALDI-MSI and classic histological staining in the same tissue sections. This demonstrates feasibility for integrating untargeted mass spectrometry imaging approaches into a digital pathology framework., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The overproduction of the toxic peptide amyloid-beta (Aβ) generated from the cleavage of amyloid precursor protein (APP) is proposed to be a critical event in the development of Alzheimer's disease. Evidence suggests that the cleavage of APP occurs after its internalization from the cell surface. Previously, we identified a novel pathway for APP internalization, which trafficks cell surface APP directly to lysosomes by macropinocytosis, leading to its processing into Aβ. We also demonstrated that ADP-ribosylation factor 6 (Arf6) is required for the macropinocytosis of APP. Here, we characterized the roles of Arf6's downstream effectors Rac1, Cdc42 and RhoA. Both pharmacological inhibition and siRNA knockdown of these proteins reduced the amount of APP colocalized with LAMP1-labeled lysosomes without affecting APP transport to early endosomes. Decreases in the production of both Aβ40 and Aβ42 were also observed by ELISA in response to inhibitor treatment. These findings together demonstrate that Rac1, Cdc42 and RhoA are components of the mechanism regulating the macropinocytosis of APP and targeting these components can reduce the production of Aβ., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephen Pasternak reports financial support was provided by The Canadian Institute for Health Research (10.13039/501100000024CIHR) Canada. Stephen Pasternak reports financial support was provided by Beaconbright Foundation. London, Canada. Stephen Pasternak reports a relationship with Zywie Bio LLC, Princeton NJ, USA that includes: shareholding, consulting or advisory and grant funding., (© 2024 The Authors. Published by Elsevier Ltd.)

Guilt is a negative emotion elicited by realizing one has caused actual or perceived harm to another person. One of guilt's primary functions is to signal that one is aware of the harm that was caused and regrets it, an indication that the harm will not be repeated. Verbal expressions of guilt are often deemed insufficient by observers when not accompanied by nonverbal signals such as facial expression, gesture, posture, or gaze. Some research has investigated isolated nonverbal expressions in guilt, however none to date has explored multiple nonverbal channels simultaneously. This study explored facial expression, gesture, posture, and gaze during the real-time experience of guilt when response demands are minimal. Healthy adults completed a novel task involving watching videos designed to elicit guilt, as well as comparison emotions. During the video task, participants were continuously recorded to capture nonverbal behaviour, which was then analyzed via automated facial expression software. We found that while feeling guilt, individuals engaged less in several nonverbal behaviours than they did while experiencing the comparison emotions. This may reflect the highly social aspect of guilt, suggesting that an audience is required to prompt a guilt display, or may suggest that guilt does not have clear nonverbal correlates., (© 2024. The Author(s).)

Introduction: We investigated whether novel plasma biomarkers are associated with cognition, cognitive decline, and functional independence in activities of daily living across and within neurodegenerative diseases., Methods: Glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau (p-tau)181 and amyloid beta (Aβ) 42/40 were measured using ultra-sensitive Simoa immunoassays in 44 healthy controls and 480 participants diagnosed with Alzheimer's disease/mild cognitive impairment (AD/MCI), Parkinson's disease (PD), frontotemporal dementia (FTD) spectrum disorders, or cerebrovascular disease (CVD)., Results: GFAP, NfL, and/or p-tau181 were elevated among all diseases compared to controls, and were broadly associated with worse baseline cognitive performance, greater cognitive decline, and/or lower functional independence. While GFAP, NfL, and p-tau181 were highly predictive across diseases, p-tau181 was more specific to the AD/MCI cohort. Sparse associations were found in the FTD and CVD cohorts and for Aβ 42/40 ., Discussion: GFAP, NfL, and p-tau181 are valuable predictors of cognition and function across common neurodegenerative diseases, and may be useful in specialized clinics and clinical trials., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Objectives: Neuropsychiatric symptoms (NPS) increase risk of developing dementia and are linked to various neurodegenerative conditions, including mild cognitive impairment (MCI due to Alzheimer's disease [AD]), cerebrovascular disease (CVD), and Parkinson's disease (PD). We explored the structural neural correlates of NPS cross-sectionally and longitudinally across various neurodegenerative diagnoses., Methods: The study included individuals with MCI due to AD, (n = 74), CVD (n = 143), and PD (n = 137) at baseline, and at 2-years follow-up (MCI due to AD, n = 37, CVD n = 103, and PD n = 84). We assessed the severity of NPS using the Neuropsychiatric Inventory Questionnaire. For brain structure we included cortical thickness and subcortical volume of predefined regions of interest associated with corticolimbic and frontal-executive circuits., Results: Cross-sectional analysis revealed significant negative correlations between appetite with both circuits in the MCI and CVD groups, while apathy was associated with these circuits in both the MCI and PD groups. Longitudinally, changes in apathy scores in the MCI group were negatively linked to the changes of the frontal-executive circuit. In the CVD group, changes in agitation and nighttime behavior were negatively associated with the corticolimbic and frontal-executive circuits, respectively. In the PD group, changes in disinhibition and apathy were positively associated with the corticolimbic and frontal-executive circuits, respectively., Conclusions: The observed correlations suggest that underlying pathological changes in the brain may contribute to alterations in neural activity associated with MBI. Notably, the difference between cross-sectional and longitudinal results indicates the necessity of conducting longitudinal studies for reproducible findings and drawing robust inferences., (© 2024 John Wiley & Sons Ltd.)

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments ( n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Pick's disease (PiD) is a rare form of frontal temporal lobar degeneration. The pathognomonic feature is atrophy of the frontotemporal lobes and intraneuronal deposits of 3R-τ inclusions, the Pick body. Corticobasal syndrome (CBS) is an atypical parkinsonian syndrome with a heterogeneous spectrum of underlying pathologies. We report a case of clinically diagnosed CBS with a post-mortem diagnosis of PiD and conduct a clinicopathological review of the literature on this unusual presentation.

Guilt is a negative emotion, elicited by realizing one has caused actual or perceived harm to another person. Anecdotally, guilt often is described as a visceral and physical experience. However, while the way that the body responds to and contributes to emotions is well known in basic emotions, little is known about the characteristics of guilt as generated by the autonomic nervous system. This study investigated the physiologic signature associated with guilt in adults with no history of psychological or autonomic disorder. Healthy adults completed a novel task, including an initial questionnaire about their habits and attitudes, followed by videos designed to elicit guilt, as well as the comparison emotions of amusement, disgust, sadness, pride, and neutral. During the video task, participants' swallowing rate, electrodermal activity, heart rate, respiration rate, and gastric activity rate were continuously recorded. Guilt was associated with alterations in gastric rhythms, electrodermal activity, and swallowing rate relative to some or all the comparison emotions. These findings suggest that there is a mixed pattern of sympathetic and parasympathetic activation during the experience of guilt. These results highlight potential therapeutic targets for modulation of guilt in neurologic and psychiatric disorders with deficient or elevated levels of guilt, such as frontotemporal dementia, posttraumatic stress disorder, and Obsessive-compulsive disorder., (© 2023. The Author(s).)

Oculomotor tasks generate a potential wealth of behavioural biomarkers for neurodegenerative diseases. Overlap between oculomotor and disease-impaired circuitry reveals the location and severity of disease processes via saccade parameters measured from eye movement tasks such as prosaccade and antisaccade. Existing studies typically examine few saccade parameters in single diseases, using multiple separate neuropsychological test scores to relate oculomotor behaviour to cognition; however, this approach produces inconsistent, ungeneralizable results and fails to consider the cognitive heterogeneity of these diseases. Comprehensive cognitive assessment and direct inter-disease comparison are crucial to accurately reveal potential saccade biomarkers. We remediate these issues by characterizing 12 behavioural parameters, selected to robustly describe saccade behaviour, derived from an interleaved prosaccade and antisaccade task in a large cross-sectional data set comprising five disease cohorts (Alzheimer's disease/mild cognitive impairment, amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease, and cerebrovascular disease; n = 391, age 40-87) and healthy controls ( n = 149, age 42-87). These participants additionally completed an extensive neuropsychological test battery. We further subdivided each cohort by diagnostic subgroup (for Alzheimer's disease/mild cognitive impairment and frontotemporal dementia) or degree of cognitive impairment based on neuropsychological testing (all other cohorts). We sought to understand links between oculomotor parameters, their relationships to robust cognitive measures, and their alterations in disease. We performed a factor analysis evaluating interrelationships among the 12 oculomotor parameters and examined correlations of the four resultant factors to five neuropsychology-based cognitive domain scores. We then compared behaviour between the abovementioned disease subgroups and controls at the individual parameter level. We theorized that each underlying factor measured the integrity of a distinct task-relevant brain process. Notably, Factor 3 (voluntary saccade generation) and Factor 1 (task disengagements) significantly correlated with attention/working memory and executive function scores. Factor 3 also correlated with memory and visuospatial function scores. Factor 2 (pre-emptive global inhibition) correlated only with attention/working memory scores, and Factor 4 (saccade metrics) correlated with no cognitive domain scores. Impairment on several mostly antisaccade-related individual parameters scaled with cognitive impairment across disease cohorts, while few subgroups differed from controls on prosaccade parameters. The interleaved prosaccade and antisaccade task detects cognitive impairment, and subsets of parameters likely index disparate underlying processes related to different cognitive domains. This suggests that the task represents a sensitive paradigm that can simultaneously evaluate a variety of clinically relevant cognitive constructs in neurodegenerative and cerebrovascular diseases and could be developed into a screening tool applicable to multiple diagnoses., Competing Interests: S.R.A. has consulted for Indoc Research Canada. C.E.F. receives commercial grant support from Vielight Inc. and Hoffman La Roche. S.K. has received research support from Brain and Behavior Foundation, National Institute on Ageing, BrightFocus Foundation, Brain Canada, Canadian Institute of Health Research, Canadian Consortium on Neurodegeneration in Aging, Centre for Ageing and Brain Health Innovation, Centre for Addiction and Mental Health; an Academic Scholars Award from the Department of Psychiatry, University of Toronto; and has received equipment support from Soterix Medical. S.H.P. has received research funding from Zywie Bio LLC. B.G.P. receives research support from the Peter & Shelagh Godsoe Endowed Chair in Late-Life Mental Health, CAMH Foundation, and Discovery Fund, National Institute of Aging, Brain Canada, the Canadian Institutes of Health Research, the Alzheimer’s Drug Discovery Foundation, the Ontario Brain Institute, the Centre for Aging and Brain Health Innovation, the Bright Focus Foundation, the Alzheimer’s Society of Canada, the W. Garfield Weston Foundation, the Weston Brain Institute, the Canadian Consortium on Neurodegeneration in Aging and Genome Canada; receives honoraria from the American Geriatrics Society for book authorship; and is listed on United States Provisional Patent Nos. 6/490,680 and 17/396030 and Canadian Provisional Patent No. 3054093 for a cell-based assay and kits for assessing serum anticholinergic activity. T.K.R. has received research support from Brain Canada, Brain and Behavior Research Foundation, BrightFocus Foundation, Canada Foundation for Innovation, Canada Research Chair, Canadian Institutes of Health Research, Centre for Aging and Brain Health Innovation, National Institutes of Health, Ontario Ministry of Health and Long-Term Care, Ontario Ministry of Research and Innovation, and the Weston Brain Institute; received for an investigator-initiated study in-kind equipment support from Newronika; in-kind research online accounts from Scientific Brain Training Pro; participated in 2021 in one advisory board meeting for Biogen Canada Inc; and is listed on United States Provisional Patent No. 17/396030 that describes cell-based assays and kits for assessing serum cholinergic receptor activity. D.P.S. receives research funding from CIHR, Alzheimer’s Association, University of Calgary, and Hotchkiss Brain Institute. M.C.T. receives grant support from NIH and CIHR and is a clinical trial investigator for Biogen, Janssen, Anavex, Green Valley and Roche. All other authors report no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

The brain's response to acute injury is characterized by increased permeability of the blood-brain barrier (BBB) and pro-inflammatory microglia signaling, both of which have been linked to poor cognitive outcomes and neurological disease. The damaged BBB has increased leakiness, allowing serum proteins like fibrinogen into the brain, which interacts with local cells in a deleterious manner. At the same time, in response to injury, microglia demonstrate increased NLRP3 inflammasome activity and heightened release of pro-inflammatory cytokines. The relationship between increased fibrinogen uptake and microglial inflammasome signaling in the injured brain has not been well described. In this work, we investigate fibrinogen mediated NLRP3 inflammasome priming of BV-2 cells and primary adult microglia and propose a role for extracellular vesicles (EVs) as propagators of this interaction. Following exposure to fibrinogen microglia significantly upregulate transcription of IL-1β, IL-6, NLRP3 and other pro-inflammatory cytokines which was sustained by repeated fibrinogen exposure. Inhibition of fibrinogen mediated NLRP3 signaling was achieved at the transcriptional and assembly level using cannabidiol (CBD) and the NLRP3 inhibitor MCC950, respectively. EVs released following NLRP3 priming carry IL-1β, IL-18 mRNA and fibrinogen, propagate inflammatory signaling and can be detected in the circulation following BBB disruption in a preclinical stroke model. In conclusion, the interplay between fibrinogen extravasation, microglial NLRP3 signaling, and EV release can perpetuate chronic pro-inflammatory signaling and represents a novel method of inflammatory propagation., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic microglia activation post-stroke is associated with worse neurological and cognitive outcomes. However, measurement of microglia activation in vivo is currently limited. Plasma derived extracellular vesicles (EVs) are cell-specific indicators that may allow for non-invasive measurement of microglia phenotype. The aim of this study was to identify activation-state specific microglia EVs (MEVs) in vitro followed by validation in an experimental stroke model. Following pro-inflammatory activation, MEVs contain the microglia protein TMEM119 alongside increased expression of the Toll-like receptor 4 co-receptor CD14. Immunoprecipitation followed by fluorescent nanoparticle tracking analysis (ONI Nanoimager) was used to confirm the isolation of TMEM119 + /CD14 + EVs from rat plasma. Electron microscopy confirmed that TMEM119 and CD14 localize to the MEV membrane. To model ischemia, plasma was collected from 3-month wildtype Fischer344 rats prior to, 7 and 28 days after endothelin-1 or saline injection into the dorsal right striatum. Fluorescently labelled MEVs were directly measured in the plasma using nanoflow cytometry (Apogee A60 Microplus). We report a significant increase in circulating TMEM119 + /CD14 + EVs 28-days post-stroke in comparison to baseline levels and saline-injected rats, which correlated weakly with stroke volume. TMEM119 + /MHC-II + EVs were also increased post-stroke in comparison to baseline and saline-injected animals. This study is the first to describe an EV biomarker of activated microglia detected directly in plasma following stroke and represents a future tool for the measurement of microglia activity in vivo., (© 2023. The Author(s).)

Introduction: Understanding synergies between neurodegenerative and cerebrovascular pathologies that modify dementia presentation represents an important knowledge gap., Methods: This multi-site, longitudinal, observational cohort study recruited participants across prevalent neurodegenerative diseases and cerebrovascular disease and assessed participants comprehensively across modalities. We describe univariate and multivariate baseline features of the cohort and summarize recruitment, data collection, and curation processes., Results: We enrolled 520 participants across five neurodegenerative and cerebrovascular diseases. Median age was 69 years, median Montreal Cognitive Assessment score was 25, median independence in activities of daily living was 100% for basic and 93% for instrumental activities. Spousal study partners predominated; participants were often male, White, and more educated. Milder disease stages predominated, yet cohorts reflect clinical presentation., Discussion: Data will be shared with the global scientific community. Within-disease and disease-agnostic approaches are expected to identify markers of severity, progression, and therapy targets. Sampling characteristics also provide guidance for future study design., (© 2022 the Alzheimer's Association.)

Background: Although genetic factors are known to contribute to neurodegenerative disease susceptibility, there remains a large amount of heritability unaccounted for across the diagnoses. Copy number variants (CNVs) contribute to these phenotypes, but their presence and influence on disease state remains relatively understudied., Methods: Here, we applied a depth of coverage approach to detect CNVs in 80 genes previously associated with neurodegenerative disease within participants of the Ontario Neurodegenerative Disease Research Initiative (n = 519)., Results: In total, we identified and validated four CNVs in the cohort, including: (1) a heterozygous deletion of exon 5 in OPTN in an Alzheimer's disease participant; (2) a duplication of exons 1-5 in PARK7 in an amyotrophic lateral sclerosis participant; (3) a duplication of >3 Mb, which encompassed ABCC6, in a cerebrovascular disease (CVD) participant; and (4) a duplication of exons 7-11 in SAMHD1 in a mild cognitive impairment participant. We also identified 43 additional CNVs that may be candidates for future replication studies., Conclusion: The identification of the CNVs suggests a portion of the apparent missing heritability of the phenotypes may be due to these structural variants, and their assessment is imperative for a thorough understanding of the genetic spectrum of neurodegeneration., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Individuals with frontotemporal dementia (FTD) often present with poor decision-making, which can affect both their financial and social situations. Delineation of the specific cognitive impairments giving rise to impaired decision-making in individuals with FTD may inform treatment strategies, as different neurotransmitter systems have been associated with distinct patterns of altered decision-making., Objective: To use a reversal-learning paradigm to identify the specific cognitive components of reversal learning that are most impaired in individuals with FTD and those with Alzheimer disease (AD) in order to inform future approaches to treatment for symptoms related to poor decision-making and behavioral inflexibility., Method: We gave 30 individuals with either the behavioral variant of FTD or AD and 18 healthy controls a stimulus-discrimination reversal-learning task to complete. We then compared performance in each phase between the groups., Results: The FTD group demonstrated impairments in initial stimulus-association learning, though to a lesser degree than the AD group. The FTD group also performed poorly in classic reversal learning, with the greatest impairments being observed in individuals with frontal-predominant atrophy during trials requiring inhibition of a previously advantageous response., Conclusion: Taken together, these results and the reversal-learning paradigm used in this study may inform the development and screening of behavioral, neurostimulatory, or pharmacologic interventions aiming to address behavioral symptoms related to stimulus-reinforcement learning and response inhibition impairments in individuals with FTD., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Periventricular white matter hyperintensities (pvWMH) are neuroimaging abnormalities surrounding the lateral ventricles that are apparent on magnetic resonance imaging (MRI). They are associated with age, neurodegenerative disease, and cerebrovascular risk factors. While pvWMH ultimately represent a loss of white matter structural integrity, the pathological causes are heterogeneous in nature, and currently, cannot be distinguished using neuroimaging alone. pvWMH could occur because of a combination of small vessel disease (SVD), ependymal loss, blood-brain barrier dysfunction, and microgliosis. In this study we aimed to characterize microvascular stenosis, fibrinogen extravasation, and microgliosis within pvWMH with and without imaging evidence of periventricular infarction. Using postmortem neuroimaging of human brains (n = 20), we identified pvWMH with and without periventricular infarcts (PVI). We performed histological analysis of microvessel stenosis, perivascular spaces, microgliosis, and immunohistochemistry against fibrinogen as a measure of serum protein extravasation. Herein, we report distinctions between pvWMH with and without periventricular infarcts based on associations with microvessel stenosis, enlarged perivascular spaces, and fibrinogen IHC. Microvessel stenosis was significantly associated with PVI and with cellular deposition of fibrinogen in the white matter. The presence of fibrinogen was associated with PVI and increased number of microglia. These findings suggest that neuroimaging-based detection of infarction within pvWMH may help distinguish more severe lesions, associated with underlying microvascular disease and BBB dysfunction, from milder pvWMH that are a highly frequent finding on MRI., (© 2021 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology.)

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with ∼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.

Genetic factors contribute to neurodegenerative diseases, with high heritability estimates across diagnoses; however, a large portion of the genetic influence remains poorly understood. Many previous studies have attempted to fill the gaps by performing linkage analyses and association studies in individual disease cohorts, but have failed to consider the clinical and pathological overlap observed across neurodegenerative diseases and the potential for genetic overlap between the phenotypes. Here, we leveraged rare variant association analyses (RVAAs) to elucidate the genetic overlap among multiple neurodegenerative diagnoses, including Alzheimer's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), mild cognitive impairment, and Parkinson's disease (PD), as well as cerebrovascular disease, using the data generated with a custom-designed neurodegenerative disease gene panel in the Ontario Neurodegenerative Disease Research Initiative (ONDRI). As expected, only ~3% of ONDRI participants harboured a monogenic variant likely driving their disease presentation. Yet, when genes were binned based on previous disease associations, we observed an enrichment of putative loss of function variants in PD genes across all ONDRI cohorts. Further, individual gene-based RVAA identified significant enrichment of rare, nonsynonymous variants in PARK2 in the FTD cohort, and in NOTCH3 in the PD cohort. The results indicate that there may be greater heterogeneity in the genetic factors contributing to neurodegeneration than previously appreciated. Although the mechanisms by which these genes contribute to disease presentation must be further explored, we hypothesize they may be a result of rare variants of moderate phenotypic effect contributing to overlapping pathology and clinical features observed across neurodegenerative diagnoses., (© 2021. The Author(s).)

Background: Perry syndrome is a rare genetic parkinsonian disorder with TAR DNA binding protein 43 (TDP-43) pathology clinically presenting with parkinsonism, neuropsychiatric features, weight loss, and central hypoventilation. As respiratory complications are often the cause of death, studies likely show the early stage of the neurodegenerative process. Because of the rarity of this condition, few studies exist, and each case provides insight into pathological findings in this neurodegenerative condition., Objective: To study the clinical and pathological correlations of an autopsy case of Perry syndrome., Methods: The patient was a woman in her 50s with Perry syndrome and a DCTN1 gene mutation. Between October 2016 and July 2019, she underwent postmortem and pathological examination at University Hospital in London, Ontario, Canada. Data were obtained through clinical pathological examination., Results: Microscopy showed significant neuronal loss with pigmentary incontinence and gliosis in the substantia nigra. There was no atrophy elsewhere, including the frontal and cingulate cortex. Intraneuronal cytoplasmic TDP-43 inclusions and neurites were noticed in a moderate number in the substantia nigra and midbrain and were sparsely noticed in the basal ganglia, thalamus, thoracic motor neuron, posterior nucleus of the hypothalamus, and rostral ventral medulla. β-Amyloid, Lewy body, and tau pathologies were absent. Rare axonal swelling was identified at the rostral ventrolateral medulla., Conclusions and Relevance: This study confirms that Perry syndrome is characterized by TDP-43 pathology with absent Lewy bodies or tau pathology. These findings support the hypothesis of dysfunctional neurons in the medulla and hypothalamus, which may respectively correlate to the clinical symptoms of hypoventilation and weight loss in Perry syndrome., Competing Interests: There were no funding sources or conflicts of interest for this study., (© 2021 International Parkinson and Movement Disorder Society.)

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and β cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Periventricular white matter hyperintensities (pvWMHs) are a neurological feature detected with magnetic resonance imaging that are clinically associated with an increased risk of stroke and dementia. pvWMHs represent white matter lesions characterized by regions of myelin and axon rarefaction and as such likely involve changes in lipid composition; however, these alterations remain unknown. Lipids are critical in determining cell function and survival. Perturbations in lipid expression have previously been associated with neurological disorders. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an emerging technique for untargeted, high-throughput investigation of lipid expression and spatial distribution in situ ; however, the use of MALDI IMS has been previously been limited by the need for non-embedded, non-fixed, fresh-frozen samples. In the current study, we demonstrate the novel use of MALDI IMS to distinguish regional lipid abnormalities that correlate with magnetic resonance imaging (MRI) defined pvWMHs within ammonium formate washed, formalin-fixed human archival samples. MALDI IMS scans were conducted in positive or negative ion detection mode on tissues sublimated with 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene matrices, respectively. Using a broad, untargeted approach to lipid analysis, we consistently detected 116 lipid ion species in 21 tissue blocks from 11 different post-mortem formalin-fixed human brains. Comparing the monoisotopic mass peaks of these lipid ions elucidated significant differences in lipid expression between pvWMHs and NAWM for 31 lipid ion species. Expanding our understanding of alterations in lipid composition will provide greater knowledge of molecular mechanisms underpinning ischemic white matter lesions and provides the potential for novel therapeutic interventions targeting lipid composition abnormalities.

Molecular chaperones and co-chaperones, which are part of the protein quality control machinery, have been shown to regulate distinct aspects of Alzheimer's Disease (AD) pathology in multiple ways. Notably, the co-chaperone STI1, which presents increased levels in AD, can protect mammalian neurons from amyloid-β toxicity in vitro and reduced STI1 levels worsen Aβ toxicity in C. elegans. However, whether increased STI1 levels can protect neurons in vivo remains unknown. We determined that overexpression of STI1 and/or Hsp90 protected C. elegans expressing Aβ (3-42) against Aβ-mediated paralysis. Mammalian neurons were also protected by elevated levels of endogenous STI1 in vitro, and this effect was mainly due to extracellular STI1. Surprisingly, in the 5xFAD mouse model of AD, by overexpressing STI1, we find increased amyloid burden, which amplifies neurotoxicity and worsens spatial memory deficits in these mutants. Increased levels of STI1 disturbed the expression of Aβ-regulating enzymes (BACE1 and MMP-2), suggesting potential mechanisms by which amyloid burden is increased in mice. Notably, we observed that STI1 accumulates in dense-core AD plaques in both 5xFAD mice and human brain tissue. Our findings suggest that elevated levels of STI1 contribute to Aβ accumulation, and that STI1 is deposited in AD plaques in mice and humans. We conclude that despite the protective effects of STI1 in C. elegans and in mammalian cultured neurons, in vivo, the predominant effect of elevated STI1 is deleterious in AD.

Background/objective: Structural brain magnetic resonance imaging (MRI) is not mandatory in Alzheimer's disease (AD) research or clinical guidelines. We aimed to explore the use of structural brain MRI in AD/mild cognitive impairment (MCI) trials over the past 10 years and determine the frequency with which inclusion of standardized structural MRI acquisitions detects comorbid vascular and non-vascular pathologies., Methods: We systematically searched ClinicalTrials.gov for AD clinical trials to determine their neuroimaging criteria and then used data from an AD/MCI cohort who underwent standardized MRI protocols, to determine type and incidence of clinically relevant comorbid pathologies., Results: Of 210 AD clinical trials, 105 (50%) included structural brain imaging in their eligibility criteria. Only 58 (27.6%) required MRI. 16,479 of 53,755 (30.7%) AD participants were in trials requiring MRI. In the observational AD/MCI cohort, 141 patients met clinical criteria; 22 (15.6%) had relevant MRI findings, of which 15 (10.6%) were exclusionary for the study., Discussion: In AD clinical trials over the last 10 years, over two-thirds of participants could have been enrolled without brain MRI and half without even a brain CT. In a study sample, relevant comorbid pathology was found in 15% of participants, despite careful screening. Standardized structural MRI should be incorporated into NIA-AA diagnostic guidelines (when available) and research frameworks routinely to reduce diagnostic heterogeneity.

Identification of biological changes underlying the early symptoms of Alzheimer's disease (AD) will help to identify and stage individuals prior to symptom onset. The limbic system, which supports episodic memory and is impaired early in AD, is a primary target. In this study, brain metabolism and microstructure evaluated by high field (7 Tesla) proton magnetic resonance spectroscopy (1H-MRS) and diffusion tensor imaging (DTI) were evaluated in the limbic system of eight individuals with mild cognitive impairment (MCI), nine with AD, and sixteen normal elderly controls (NEC). Left hippocampal glutamate and posterior cingulate N-acetyl aspartate concentrations were reduced in MCI and AD compared to NEC. Differences in DTI metrics indicated volume and white matter loss along the cingulum in AD compared to NEC. Metabolic and microstructural changes were associated with episodic memory performance assessed using Craft Story 21 Recall and Benson Complex Figure Copy. The current study suggests that metabolite concentrations measured using 1H-MRS may provide insight into the underlying metabolic and microstructural processes of episodic memory impairment.

White matter hyperintensities (WMH) occur in normal aging and across diagnostic categories of neurodegeneration. Ultra-high field imaging (UHF) MRI machines offer the potential to improve our understanding of WMH. Post-mortem imaging using UHF magnetic resonance imaging (MRI) is a useful way of assessing WMH, however, the responsiveness of UHF-MRI to pathological changes within the white matter has not been characterized. In this study we report post-mortem MRI sequences of white matter hyperintensities in normal aging, Alzheimer's disease, and cerebrovascular disease. Seven Tesla post-mortem MRI reliably detected periventricular WMH using both FLAIR and T2 sequences and reflects underlying pathology of myelin and axon density despite prolonged fixation time. Co-registration of histological images to MRI allowed for direct voxel- wise comparison of imaging findings and pathological changes. Myelin content and cerebrovascular pathology were the most significant predictors of MRI white matter intensity as revealed by linear mixed models. Future work investigating the utility of UHF- MRI in studying cell-specific changes within WMH is required to better understand radio-pathologic correlations., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Creutzfeldt-Jakob disease (CJD) is a fatal neurological illness for which accurate diagnosis is paramount. Real-time quaking-induced conversion (RT-QuIC) is a prion-specific assay with high sensitivity and specificity for CJD. The Canadian endpoint quaking-induced conversion (EP-QuIC) test is similar, but unlike RT-QuIC there is little data regarding its diagnostic utility in clinical practice. In this exploratory predictive value analysis of EP-QuIC in CJD, the negative predictive value (NPV) and positive predictive value (PPV) was 100% and 83%, respectively, with one false-positive result identified. Re-testing this sample with an optimized EP-QuIC protocol eliminated this false-positive result, leading to a PPV of 100%.

Background: Currently there are no disease-modifying treatments for Parkinson's disease dementia (PDD), a condition linked to aggregation of the protein α-synuclein in subcortical and cortical brain areas. One of the leading genetic risk factors for Parkinson's disease is being a carrier in the gene for β-Glucocerebrosidase (GCase; gene name GBA1). Studies in cell culture and animal models have shown that raising the levels of GCase can decrease levels of α-synuclein. Ambroxol is a pharmacological chaperone for GCase and is able to raise the levels of GCase and could therefore be a disease-modifying treatment for PDD. The aims of this trial are to determine if Ambroxol is safe and well-tolerated by individuals with PDD and if Ambroxol affects cognitive, biochemical, and neuroimaging measures., Methods: This is a phase II, single-centre, double-blind, randomized placebo-controlled trial involving 75 individuals with mild to moderate PDD. Participants will be randomized into Ambroxol high-dose (1050 mg/day), low-dose (525 mg/day), or placebo treatment arms. Assessments will be undertaken at baseline, 6-months, and 12-months follow up times. Primary outcome measures will be the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-Cog) and the ADCS Clinician's Global Impression of Change (CGIC). Secondary measures will include the Parkinson's disease Cognitive Rating Scale, Clinical Dementia Rating, Trail Making Test, Stroop Test, Unified Parkinson's disease Rating Scale, Purdue Pegboard, Timed Up and Go, and gait kinematics. Markers of neurodegeneration will include MRI and CSF measures. Pharmacokinetics and pharmacodynamics of Ambroxol will be examined through plasma levels during dose titration phase and evaluation of GCase activity in lymphocytes., Discussion: If found effective and safe, Ambroxol will be one of the first disease-modifying treatments for PDD., Trial Registration: ClinicalTrials.gov NCT02914366, 26 Sep 2016/retrospectively registered.

Apoptosis is a feature of stroke and Alzheimer's disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [ 68 Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and 68 Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [ 68 Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells. In vitro, Ga-TC3-OGDOTA labeled apoptotic neurons following exposure to camptothecin, oxygen-glucose deprivation, and β -amyloid oligomers. In vivo, PET showed accumulation of [ 68 Ga]Ga-TC3-OGDOTA in the brain of mouse models of stroke or AD. Optical clearing revealed colocalization of [ 68 Ga]Ga-TC3-OGDOTA and cleaved caspase-3 in brain cells. In stroke, [ 68 Ga]Ga-TC3-OGDOTA accumulated in neurons in the penumbra area, whereas in AD mice [ 68 Ga]Ga-TC3-OGDOTA was found in single cells in the forebrain and diffusely around amyloid plaques. In summary, this bifunctional tracer is selectively associated with apoptotic cells in vitro and in vivo in brain disease models and represents a novel tool for apoptosis detection that can be used in neurodegenerative diseases.

Spontaneous intracranial hypotension (SIH) is a progressive clinical syndrome characterized by orthostatic headaches, nausea, emesis, and occasionally focal neurological deficits. Rarely, SIH is associated with neurocognitive changes. An epidural blood patch (EBP) is commonly used to treat SIH when conservative measures are inadequate, although some patients require multiple EBP procedures or do not respond at all. Recently, the use of a large-volume (LV) EBP has been described to treat occult leak sites in treatment-refractory SIH. This article describes the management of a patient with profound neurocognitive decline associated with SIH, who was refractory to conservative management and multiple interventions. The authors describe the successful use of an ultra-LV-EBP of 120 ml across multiple levels, the largest volume reported in the literature, and describe the technical aspects of the procedure. This procedure has resulted in dramatic and sustained symptom resolution.

Background: Early detection of Alzheimer's disease (AD) pathology is a serious challenge for both diagnosis and clinical trials. The aspartyl protease, Cathepsin D (CatD), is overexpressed in AD and could be a biomarker of disease. We have previously designed a unique contrast agent (CA) for dual-optical and magnetic resonance imaging of the activity of the CatD class of enzymes., Objective: To compare the uptake and retention of a novel, more sensitive, and clinically-translatable 68Ga PET tracer targeting CatD activity in 5XFAD mice and non-Tg littermates., Methods: The targeted CA consisted of an HIV-1 Tat cell penetrating peptide (CPP) conjugated to a specialized cleavage sequence targeting aspartyl cathepsins and a DOTA conjugate chelating 68Ga. PET images were acquired using a Siemens Inveon preclinical microPET in female Tg AD mice and non-Tg age matched female littermates (n = 5-8) following intravenous CA administration at 2, 6, and 9 months of age. Additionally, 18F fluorodeoxyglucose (FDG) PET imaging was performed at 10 months to measure glucose uptake., Results: The Tg mice showed significantly higher relative uptake rate of the targeting CA in the forebrain relative to hindbrain at all ages compared to controls, consistent with histology. In contrast, no differences were seen in CA uptake in other organs. Additionally, the Tg mice did not show any differences in relative uptake of FDG at 10 months of age in the forebrain relative to the hindbrain compared to age matched non-Tg controls., Conclusions: Elevated aspartryl cathepsin activity was detected in vivo in the 5XFAD mouse model of AD using a novel targeted PET contrast agent.

Alzheimer's disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived β-amyloid peptides (Aβ) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aβ peptides released from neural cells. The Aβ-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA 1 ). BafA 1 also attenuated the Aβ-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aβ on CHT activity. Importantly, neutralizing Aβ using an anti-Aβ antibody directed at the N-terminal amino acids 1-16 of Aβ, but not by an antibody directed at the mid-region amino acids 22-35 of Aβ, attenuates the effect of Aβ on CHT activity and trafficking. This indicates that a specific N-terminal Aβ epitope, or specific conformation of soluble Aβ, may impair CHT activity. Therefore, Aβ immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.

The Montreal Cognitive Assessment (MoCA), a brief screening test developed to detect patients with mild cognitive impairment, is used in clinical settings across North America [Nasreddine et al.: J Am Geriatr Soc 2005;53:695-699]. The MoCA has been demonstrated to be sensitive to cognitive deficits in frontotemporal dementias (FTD) and related disorders [Coleman et al.: Alzheimer Dis Assoc Disord 2016;30:258-263]. Given attentional impairments in patients with FTD, whether and to what extent the abbreviated items on the MoCA may predict performance on corresponding assessments is not known. Testing and demographic data were extracted from a clinical database using a sample of 91 patients with FTD and related disorders. The relationship between MoCA items and corresponding neuropsychological tasks was assessed through McNemar tests and Spearman correlations. While some MoCA items such as letter fluency, orientation, and clock drawing were strongly correlated with the corresponding standard cognitive test, the MoCA trails were insensitive to impairment compared to the full Trail Making B Test (p = 0.01). In contrast, MoCA naming and delayed recall sub-items detected cognitive impairment more frequently than available comparison tests. The MoCA is a sensitive screening measure to detect impairment in patients with FTD and related disorders, but cognitive deficits specific to FTD result in differential performance on MoCA items compared to longer standard cognitive tests., (© 2017 S. Karger AG, Basel.)

The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer's disease., Competing Interests: The authors have declared that no competing interests exist.

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objective: The Montreal Cognitive Assessment (MoCA) is a general cognitive screening tool that has shown sensitivity in detecting mild levels of cognitive impairment in various clinical populations. Although mood dysfunction is common in referrals to memory clinics, the influence of mood on the MoCA has to date been largely unexplored., Method: In this study, we examined the impact of mood dysfunction on the MoCA using a memory clinic sample of individuals with depressive symptoms who did not meet criteria for a neurodegenerative disease., Results: Half of the group with depressive symptoms scored below the MoCA-suggested cutoff for cognitive impairment. As a group, they scored below healthy controls, but above individuals with Alzheimer's disease and frontotemporal dementia. A MoCA subtask analysis revealed a pattern of executive/attentional dysfunction in those with depressive symptoms., Conclusions: This observed negative impact of depressive symptomatology on the MoCA has interpretative implications for its utility as a cognitive screening tool in a memory clinic setting.

The Montreal Cognitive Assessment (MoCA) is a cognitive screening tool used by practitioners worldwide. The efficacy of the MoCA for screening frontotemporal dementia (FTD) and related disorders is unknown. The objectives were: (1) to determine whether the MoCA detects cognitive impairment (CI) in FTD subjects; (2) to determine whether Alzheimer disease (AD) and FTD subtypes and related disorders can be parsed using the MoCA; and (3) describe longitudinal MoCA performance by subtype. We extracted demographic and testing data from a database of patients referred to a cognitive neurology clinic who met criteria for probable AD or FTD (N=192). Logistic regression was used to determine whether dementia subtypes were associated with overall scores, subscores, or combinations of subscores on the MoCA. Initial MoCA results demonstrated CI in the majority of FTD subjects (87%). FTD subjects (N=94) performed better than AD subjects (N=98) on the MoCA (mean scores: 18.1 vs. 16.3; P=0.02). Subscores parsed many, but not all subtypes. FTD subjects had a larger decline on the MoCA within 13 to 36 months than AD subjects (P=0.02). The results indicate that the MoCA is a useful tool to identify and track progression of CI in FTD. Further, the data informs future research on scoring models for the MoCA to enhance cognitive screening and detection of FTD patients.

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer's disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied. While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells. We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.