Search

Abstract Background Variants of the gene triggering receptor expressed on myeloid cells-2 (TREM2) increase the risk of Alzheimer’s disease (AD) and other neurodegenerative disorders. Signaling by TREM2, an innate immune receptor expressed by microglia, is thought to enhance phagocytosis of amyloid beta (Aβ) and other damaged proteins, promote microglial proliferation, migration, and survival, and regulate inflammatory signaling. Thus, TREM2 activation has potential to alter the progression of AD. AL002 is an investigational, engineered, humanized monoclonal immunoglobulin G1 (IgG1) antibody designed to target TREM2. In AD mouse models, an AL002 murine variant has been previously shown to induce microglial proliferation and reduce filamentous Aβ plaques and neurite dystrophy. Methods Preclinical studies assessed the safety, tolerability, pharmacokinetics, and pharmacodynamics of AL002 in cynomolgus monkeys. INVOKE-1 (NCT03635047) was a first-in-human phase 1, randomized, placebo-controlled, double-blind study assessing the safety, tolerability, PK, and PD of AL002 administered as single ascending doses (SAD) in healthy volunteers. Results In cynomolgus monkeys, weekly intravenous injections of AL002 for 4 weeks were well tolerated, dose-dependently decreased soluble TREM2 (sTREM2) in cerebrospinal fluid (CSF) and total TREM2 in hippocampus and frontal cortex, and increased biomarkers of TREM2 signaling in CSF and brain. In the phase 1 study of 64 healthy volunteers, a single intravenous infusion of AL002 demonstrated brain target engagement based on a dose-dependent reduction of sTREM2 in CSF and parallel increases in biomarkers of TREM2 signaling and microglia recruitment. Single-dose AL002 showed central nervous system penetrance and was well tolerated, with no treatment-related serious adverse events over 12 weeks. Conclusions These findings support the continued clinical development of AL002 for AD and other neurodegenerative diseases in which TREM2 activation may be beneficial. AL002 is currently being tested in a phase 2, randomized, double-blind, placebo-controlled study in early AD. Trial registration Clinicaltrials.gov, NCT03635047. Registered on August 15, 2018, https://www.clinicaltrials.gov/study/NCT03635047 .

Abstract Background Blood-derived mitochondrial DNA copy number (mtDNA-CN) is a proxy measurement of mitochondrial function in the peripheral and central systems. Abnormal mtDNA-CN not only indicates impaired mtDNA replication and transcription machinery but also dysregulated biological processes such as energy and lipid metabolism. However, the relationship between mtDNA-CN and Alzheimer disease (AD) is unclear. Methods We performed two-sample Mendelian randomization (MR) using publicly available summary statistics from GWAS for mtDNA-CN and AD to investigate the causal relationship between mtDNA-CN and AD. We estimated mtDNA-CN using whole-genome sequence data from blood and brain samples of 13,799 individuals from the Alzheimer’s Disease Sequencing Project. Linear and Cox proportional hazards models adjusting for age, sex, and study phase were used to assess the association of mtDNA-CN with AD. The association of AD biomarkers and serum metabolites with mtDNA-CN in blood was evaluated in Alzheimer’s Disease Neuroimaging Initiative using linear regression. We conducted a causal mediation analysis to test the natural indirect effects of mtDNA-CN change on AD risk through the significantly associated biomarkers and metabolites. Results MR analysis suggested a causal relationship between decreased blood-derived mtDNA-CN and increased risk of AD (OR = 0.68; P = 0.013). Survival analysis showed that decreased mtDNA-CN was significantly associated with higher risk of conversion from mild cognitive impairment to AD (HR = 0.80; P = 0.002). We also identified significant associations of mtDNA-CN with brain FDG-PET (β = 0.103; P = 0.022), amyloid-PET (β = 0.117; P = 0.034), CSF amyloid-β (Aβ) 42/40 (β=-0.124; P = 0.017), CSF t-Tau (β = 0.128; P = 0.015), p-Tau (β = 0.140; P = 0.008), and plasma NFL (β=-0.124; P = 0.004) in females. Several lipid species, amino acids, biogenic amines in serum were also significantly associated with mtDNA-CN. Causal mediation analyses showed that about a third of the effect of mtDNA-CN on AD risk was mediated by plasma NFL (P = 0.009), and this effect was more significant in females (P

Abstract Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder characterized by brain aggregation of β-amyloid (Aβ) peptides and phosphorylated tau (P-tau) proteins. Extracellular vesicles (EVs) can be isolated and studied for potential roles in disease. While several studies have tested plasma-derived EVs in AD, few have analyzed EVs from cerebrospinal fluid (CSF), which are potentially more closely related to brain changes. This study included 20 AD patients and 20 cognitively unimpaired (CU) participants. Using a novel EV isolation method based on acoustic trapping, we isolated and purified EVs from minimal CSF volumes. EVs were lysed and analyzed by immunoassays for P-tau217 and P-tau181. Isolation was confirmed through transmission electron microscopy and the presence of EV-specific markers (CD9, CD63, CD81, ATP1A3). Nanoparticle tracking analysis revealed a high variance in EV distribution. AD patients exhibited increased P-tau181 and decreased P-tau217 in EVs, leading to a higher EV P-tau181/P-tau217 ratio compared to CU. No significant differences in EV counts or sizes were observed between AD and CU groups. This study is the first to use acoustic trapping to isolate EVs from CSF and demonstrates differential P-tau content in AD-derived EVs, warranting further research to understand the relationship between these EV changes and brain pathology.

Abstract Disruptions in pericyte and endothelial cell communication can compromise the integrity of the blood-brain barrier (BBB), leading to neurovascular dysfunction and the development of neurological disorders. However, the evaluation of microvessel RNAs has been limited to tissue homogenates, with spatial visualization only available for protein targets. The aim of the present study is the development of an innovative microvessel isolation technique that is RNA-friendly for the purpose of coupling with in situ hybridization RNAscope analysis. RNA-friendly microvessel isolation combined with RNAscope analysis enables the visualization of cell-specific RNA within the spatial and histological context of the BBB. Using this approach, we have gained valuable insights into the structural and functional differences associated with the microvessels of 5xFAD mice, a mouse model of Alzheimer’s disease (AD). RNAscope analysis revealed a decrease in pericytes from microvessels isolated from 5xFAD mice in comparison to wild-type mice. Additionally, the microvessels of 5xFAD mice exhibited an increase in TYRO protein tyrosine kinase binding protein (TYROBP) mRNA expression. These findings significantly advance our understanding of neurovascular interactions and hold great promise for guiding the development of targeted therapeutic interventions. This innovative approach enables visualization of cell RNA while preserving the spatial and histological context of the BBB, shedding light on the mechanisms underlying neurovascular unit communication.

Abstract Alzheimer’s disease (AD) poses a major challenge due to its impact on the elderly population and the lack of effective early diagnosis and treatment options. In an effort to address this issue, a study focused on identifying potential biomarkers and therapeutic agents for AD was carried out. Using RNA-Seq data from AD patients and healthy individuals, 12 differentially expressed genes (DEGs) were identified, with 9 expressing upregulation (ISG15, HRNR, MTATP8P1, MTCO3P12, DTHD1, DCX, ST8SIA2, NNAT, and PCDH11Y) and 3 expressing downregulation (LTF, XIST, and TTR). Among them, TTR exhibited the lowest gene expression profile. Interestingly, functional analysis tied TTR to amyloid fiber formation and neutrophil degranulation through enrichment analysis. These findings suggested the potential of TTR as a diagnostic biomarker for AD. Additionally, druggability analysis revealed that the FDA-approved drug Levothyroxine might be effective against the Transthyretin protein encoded by the TTR gene. Molecular docking and dynamics simulation studies of Levothyroxine and Transthyretin suggested that this drug could be repurposed to treat AD. However, additional studies using in vitro and in vivo models are necessary before these findings can be applied in clinical applications.

Abstract Background Tauopathies, including Alzheimer’s disease, are characterized by the pathological aggregation of tau protein, which is strongly linked to dysregulation of the autophagy-lysosomal degradation pathway. However, therapeutic strategies targeting this pathway remain limited. Methods We used both in vitro and in vivo models to investigate the role of Raptor in tau pathology. Knockdown of Raptor was performed to assess its impact on mTORC1 activation, autophagy, and tau accumulation. The relationship between USP9X and Raptor was also examined. Pharmacological inhibition of USP9X with WP1130 was employed to further confirm the involvement of the USP9X-Raptor-mTORC1 axis in tau degradation. Results Elevated Raptor levels in the hippocampus of P301S mice led to hyperactivation of mTORC1, impairing autophagy flux. Knockdown of Raptor effectively suppressed mTORC1 activation, promoted autophagy, and mitigated the accumulation of tau and its phosphorylated isoforms. This reduction in tau pathology was accompanied by decreased neuronal loss in the hippocampus, amelioration of synaptic damage, and improvement in cognitive function. The increased Raptor protein observed in the hippocampus of P301S mice was likely attributable to elevated USP9X content, which enhanced Raptor deubiquitination and protected it from proteasomal degradation. Pharmacological inhibition of USP9X with WP1130 in vitro effectively suppressed Raptor, promoted autophagy, and accelerated the degradation of tau and phosphorylated tau. Conclusions Our findings highlight Raptor and USP9X as promising molecular targets for therapeutic intervention in tauopathies. Targeting the USP9X-Raptor-mTORC1 axis may provide a novel strategy for promoting autophagy and mitigating tau pathology in Alzheimer’s disease and other tauopathies. Graphical Abstract

Abstract Background Alzheimer’s disease (AD) is a neurodegenerative disease that causes cognitive dysfunction in older adults. One of the AD pathological factors, β-Amyloid (Aβ), triggers inflammatory responses and phagocytosis of microglia. C-type lectin domain family 5 member A (CLEC5A) induces over-reactive inflammatory responses in several virus infections. Yet, the role of CLEC5A in AD progression remains unknown. This study aimed to elucidate the contribution of CLEC5A to Aβ-induced microglial activation and behavioral deficits. Methods The AD mouse model was crossed with Clec5a knockout mice for subsequent behavioral and pathological tests. The memory deficit was revealed by the Morris water maze, while the nociception abnormalities were examined by the von Frey filament and hotplate test. The Aβ deposition and microglia recruitment were identified by ELISA and immunohistochemistry. The inflammatory signals were identified by ELISA and western blotting. In the Clec5a knockdown microglial cell model and Clec5a knockout primary microglia, the microglial phagocytosis was revealed using the fluorescent-labeled Aβ. Results The AD mice with Clec5a knockout improved Aβ-induced memory deficit and abnormal nociception. These mice have reduced Aβ deposition and increased microglia coverage surrounding the amyloid plaque, suggesting the involvement of CLEC5A in AD progression and Aβ clearance. Moreover, the phagocytosis was also increased in the Aβ-stressed Clec5a knockdown microglial cell lines and Clec5a knockout primary microglia. Conclusion The Clec5a knockout ameliorates AD-like deficits by modulating microglial Aβ clearance. This study implies that targeting microglial Clec5a could offer a promising approach to mitigate AD progression.

Abstract Preclinical methods are needed for screening potential Alzheimer’s disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer’s and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ42-induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases.

Abstract Alzheimer’s disease (AD) is a progressive neurodegenerative condition and the primary form of dementia among elderly people. The amyloidogenic hypothesis is the main theory that explains this phenomenon and describes the extracellular accumulation of amyloid beta (Aβ) peptides. Model organisms such as Drosophila melanogaster have been utilized to improve the understanding of this disease and its treatment. This study evaluated the effects of peptide and metabolic fractions of Brazilian kefir on a strain of D. melanogaster that expresses human Aβ peptide 1–42 in the eye. The parameters assessed included ommatidial organization, vacuole area, retinal thickness, and Aβ peptide quantification. The present study revealed that the fractions, particularly the peptidic fraction, significantly reduced the vacuole area and increased the retina thickness in treated flies, indicating an improvement in neurodegeneration phenotype. The peptidic fraction was also found to alter Aβ aggregation dynamics, inhibiting Aβ fibril formation, as revealed by dynamic light scattering. This study demonstrated that kefir fractions, particularly the peptidic fraction

Abstract Background Mild cognitive impairment (MCI) and Alzheimer’s disease (AD) are significant neurodegenerative disorders with increasing prevalence worldwide. Lifestyle and dietary factors, including micronutrients, have been suggested as modifiable risk factors for disease development. This study aims to investigate the association between micronutrients and cognitive ability in these diseases. Methods A cross-sectional study involving 105 participants with MCI and AD was conducted. Dietary assessments were performed using a validated food frequency questionnaire (FFQ), and micronutrient intake was calculated based on nutrient content. Disease severity was evaluated using the Functional Assessment Staging Tool (FAST). Statistical analyses, including correlation coefficients and multiple regression models, were employed to examine the association between micronutrients and disease progression. Results The results revealed significant correlations between disease severity and several micronutrients, including omega-3 fatty acids (B = -0.2, P = 0.01), carotenoids (B = -0.19, P = 0.02), dietary antioxidant compounds, including vitamins A, C, D, E (B = -0.19, P = 0.02), selenium (B = -0.17, P = 0.03), alpha-carotene (B = -0.16, P = 0.04), beta-carotene (B = -0.17, P = 0.03), and lycopene (B = -0.16, P = 0.04). Multivariate regression analysis showed that higher intake of omega-3 fatty acids was associated with slower disease progression. Furthermore, the levels of these micronutrients declined in advanced stages of the disease. Conclusion Omega-3 fatty acids and carotenoids may affect the cognitive ability and disease progression. Further longitudinal studies are warranted to establish causality and explore the therapeutic implications of these findings for the prevention and management of MCI and AD.

Abstract Brain microvascular dysfunction is an important feature of Alzheimer’s disease (AD). To better understand the brain microvascular molecular signatures of AD, we processed and analyzed isolated human brain microvessels by data-independent acquisition liquid chromatography with tandem mass spectrometry (DIA LC–MS/MS) to generate a quantitative dataset at the peptide and protein level. Brain microvessels were isolated from parietal cortex grey matter using protocols that preserve viability for downstream functional studies. Our cohort included 23 subjects with clinical and neuropathologic concordance for Alzheimer’s disease, and 21 age-matched controls. In our analysis, we identified 168 proteins whose abundance was significantly increased, and no proteins that were significantly decreased in AD. The most highly increased proteins included amyloid beta, tau, midkine, SPARC related modular calcium binding 1 (SMOC1), and fatty acid binding protein 7 (FABP7). Additionally, Gene Ontology (GO) enrichment analysis identified the enrichment of increased proteins involved in cellular detoxification and antioxidative responses. A systematic evaluation of protein functions using the UniProt database identified groupings into common functional themes including the regulation of cellular proliferation, cellular differentiation and survival, inflammation, extracellular matrix, cell stress responses, metabolism, coagulation and heme breakdown, protein degradation, cytoskeleton, subcellular trafficking, cell motility, and cell signaling. This suggests that AD brain microvessels exist in a stressed state of increased energy demand, and mount a compensatory response to ongoing oxidative and cellular damage that is associated with AD. We also used public RNAseq databases to identify cell-type enriched genes that were detected at the protein level and found no changes in abundance of these proteins between control and AD groups, indicating that changes in cellular composition of the isolated microvessels were minimal between AD and no-AD groups. Using public data, we additionally found that under half of the proteins that were significantly increased in AD microvessels had concordant changes in brain microvascular mRNA, implying substantial discordance between gene and protein levels. Together, our results offer novel insights into the molecular underpinnings of brain microvascular dysfunction in AD.

Abstract The recently discovered interaction between presenilin 1 (PS1), a subunit of γ-secretase involved in amyloid-β (Aβ) peptide production, and GLT-1, the major brain glutamate transporter (EAAT2 in the human), may link two pathological aspects of Alzheimer’s disease: abnormal Aβ occurrence and neuronal network hyperactivity. In the current study, we employed a FRET-based fluorescence lifetime imaging microscopy (FLIM) to characterize the PS1/GLT-1 interaction in brain tissue from sporadic AD (sAD) patients. sAD brains showed significantly less PS1/GLT-1 interaction than those with frontotemporal lobar degeneration or non-demented controls. Familial AD (fAD) PS1 mutations, inducing a “closed” PS1 conformation similar to that in sAD brain, and gamma-secretase modulators (GSMs), inducing a “relaxed” conformation, respectively reduced and increased the interaction. Furthermore, PS1 influences GLT-1 cell surface expression and homomultimer formation, acting as a chaperone but not affecting GLT-1 stability. The diminished PS1/GLT-1 interaction suggests that these functions may not work properly in AD.

Neurodegenerative disorders, particularly Alzheimer’s and Parkinson’s diseases, continue to challenge modern medicine despite therapeutic advances. Orphan G-protein-coupled receptors (GPCRs) have emerged as promising targets in the central nervous system, offering new avenues for drug development. This review focuses on the structural biology of orphan GPCRs implicated in these disorders, providing a comprehensive analysis of their molecular architecture and functional mechanisms. We examine recent breakthroughs in structural determination techniques, such as cryo-electron microscopy and X-ray crystallography, which have elucidated the intricate conformations of these receptors. The review highlights how structural insights inform our understanding of orphan GPCR activation, ligand binding and signaling pathways. By integrating structural data with molecular pharmacology, we explore the potential of structure-guided approaches in developing targeted therapeutics toward orphan GPCRs. This structural-biology-centered perspective aims to deepen our comprehension of orphan GPCRs and guide future drug discovery efforts in neurodegenerative disorders.

Alzheimer’s disease (AD) is the leading cause of dementia worldwide. The disease is characterized by the abnormal accumulation of amyloid β (Aβ) protein creating neuritic plaques, hyperphosphorylated tau (p-tau) protein forming intracellular tangles, and neuronal degeneration. Pathological changes related to abnormal Aβ and p-tau accumulation may begin more than fifteen years before the clinical diagnosis of AD is made. The glymphatic system is the brain’s waste clearance pathway that prevents the accumulation of these abnormal proteins and macromolecules. Glymphatic clearance is negatively affected by physiological conditions such as sleep deprivation, and pathological conditions such as traumatic brain injury and hemorrhagic strokes. These physiological and pathological conditions are strong risk factors for AD. In conclusion, impaired glymphatic clearance is an important pathogenetic mechanism for AD.

The aggregation of Amyloid-β (Aβ) peptides is associated with neurodegeneration in Alzheimer's disease (AD). We previously identified novel naphtalene derivatives, including the lead compound Amylovis-201, able to form thermodynamically stable complexes with Aβ species, peptides and fibrils. As the drug showed a chemical scaffold coherent for an effective interaction with the σ1 receptor chaperone and as σ1 agonists are currently developed as potent neuroprotectants in AD, we investigated the pharmacological action of Amylovis-201 on the σ1 receptor. We report that Amylovis-201 is a potent σ1 agonist by several in silico, in vitro and in vivo assays and that its anti-amnesic and neuroprotective effects involve a pharmacological action at σ1 receptors. Furthermore, we show for the first time that classical σ1 receptor agonist (PRE-084), and antagonist (NE-100) are able to interact and disaggregate Aβ25–35 fibrils. Interestingly, Amylovis-201 was the only compound inhibiting Aβ25–35 aggregates formation. Our results therefore highlight a dual action of Amylovis-201 as anti-aggregating agent and σ1 receptor agonist that could be highly effective in long-term treatment against neurodegeneration in AD.

Abstract Background Multiple psychosocial factors have been associated with dementia, while the individual or joint effects of various psychosocial states on dementia remain unrevealed due to the complex interplay between those factors. Here, the authors examined the associations of psychosocial factors and patterns with subsequent risk of dementia, and if the associations could be modified by genetic susceptibility to dementia. Methods UK Biobank dementia-free participants were followed from one year after recruitment (median date: 24 January, 2010) until 31 October, 2022. Psychosocial states were measured by 22 items related to five dimensions, including psychiatric history, recent stressful life events, current psychiatric symptoms, social contact, and individual socioeconomic state. We identified clusters of individuals with distinct psychosocial patterns using latent class analysis. Cox proportional hazards models were used to evaluate the association between psychosocial items, as well as psychosocial patterns, and risk of dementia. We further performed stratification analyses by apolipoprotein E (APOE) genotype, polygenic risk score (PRS) of dementia, and family history of dementia. Results Of 497,787 included participants, 54.54% were female. During a median follow-up of 12.70 years, we identified 9,858 (1.98%) patients with newly diagnosed dementia. We identified seven clusters with distinct psychosocial patterns. Compared to individuals with a pattern of ‘good state’, individuals with other unfavorable patterns, featured by varying degrees of poor psychological state (‘fair state’ and ‘mildly, moderately, and extremely poor psychological state’), low social contact or socioeconomic state (‘living alone’ and ‘short education years’), were all at an increased risk of dementia (hazard ratios [HR] between 1.29 and 2.63). The observed associations showed no significant differences across individuals with varying APOE genotypes, levels of PRS, and family histories of dementia. Conclusion Unfavorable psychosocial patterns are associated with an increased risk of dementia, independent of genetic susceptibility. The findings highlight the importance of surveillance and prevention of cognitive decline among individuals with suboptimal psychosocial state.

Abstract Background The potential of microglia as a target for Alzheimer’s disease (AD) treatment is promising, yet the clinical and pathological diversity within microglia, driven by genetic factors, poses a significant challenge. Subtyping AD is imperative to enable precise and effective treatment strategies. However, existing subtyping methods fail to comprehensively address the intricate complexities of AD pathogenesis, particularly concerning genetic risk factors. To address this gap, we have employed systems biology approaches for AD subtyping and identified potential therapeutic targets. Methods We constructed patient-specific microglial molecular regulatory network models by utilizing existing literature and single-cell RNA sequencing data. The combination of large-scale computer simulations and dynamic network analysis enabled us to subtype AD patients according to their distinct molecular regulatory mechanisms. For each identified subtype, we suggested optimal targets for effective AD treatment. Results To investigate heterogeneity in AD and identify potential therapeutic targets, we constructed a microglia molecular regulatory network model. The network model incorporated 20 known risk factors and crucial signaling pathways associated with microglial functionality, such as inflammation, anti-inflammation, phagocytosis, and autophagy. Probabilistic simulations with patient-specific genomic data and subsequent dynamics analysis revealed nine distinct AD subtypes characterized by core feedback mechanisms involving SPI1, CASS4, and MEF2C. Moreover, we identified PICALM, MEF2C, and LAT2 as common therapeutic targets among several subtypes. Furthermore, we clarified the reasons for the previous contradictory experimental results that suggested both the activation and inhibition of AKT or INPP5D could activate AD through dynamic analysis. This highlights the multifaceted nature of microglial network regulation. Conclusions These results offer a means to classify AD patients by their genetic risk factors, clarify inconsistent experimental findings, and advance the development of treatments tailored to individual genotypes for AD.

Abstract Background The research community has historically failed to enroll diverse groups of participants in dementia clinical trials. A unique aspect of dementia care research is the requirement of a study partner, who can attest to the care recipient’s clinical and functional capacity. The aim of this study is to assess racial and ethnic differences and the importance of various trial considerations among dementia caregivers, in their decision to participate in clinical research as study partners. Method We embedded a vignette about a hypothetical dementia clinical trial in a nationally representative survey of U.S. dementia caregivers, oversampling non-Hispanic Black and Hispanic caregivers. Dementia caregivers were asked about their willingness to participate in the trial with their care recipient and rated the importance of nine considerations in hypothetical decisions to participate. Caregiver demographic characteristics were analyzed as predictors of trial participation in a base demographic model. In a second reasons model caregiver demographic characteristics and the rated importance of the nine considerations were separately analyzed as predictors; both models used survey-weighted logistic regression. Result The sample consisted of 610 dementia caregivers, including 156 non-Hispanic Black and 122 Hispanic caregiver participants. In the base demographic model, hypothetical trial participation was negatively associated with older caregiver age (OR (odds ratio) = 0.72, p =

Abstract Background Dementia with Lewy Bodies (DLB) is a complex neurodegenerative disorder that often overlaps clinically with Alzheimer’s disease (AD), presenting challenges in accurate diagnosis and underscoring the need for novel biomarkers. Lipidomic emerges as a promising avenue for uncovering disease-specific metabolic alterations and potential biomarkers, particularly as the lipidomics landscape of DLB has not been previously explored. We aim to identify potential diagnostic biomarkers and elucidate the disease's pathophysiological mechanisms. Methods This study conducted a lipidomic analysis of plasma samples from patients with DLB, AD, and healthy controls (HCs) at Xuanwu Hospital. Untargeted plasma lipidomic profiling was conducted via liquid chromatography coupled with mass spectrometry. Machine learning methods were employed to discern lipidomic signatures specific to DLB and to differentiate it from AD. Results The study enrolled 159 participants, including 57 with AD, 48 with DLB, and 54 HCs. Significant differences in lipid profiles were observed between the DLB and HC groups, particularly in the classes of sphingolipids and phospholipids. A total of 55 differentially expressed lipid species were identified between DLB and HCs, and 17 between DLB and AD. Correlations were observed linking these lipidomic profiles to clinical parameters like Unified Parkinson’s Disease Rating Scale III (UPDRS III) and cognitive scores. Machine learning models demonstrated to be highly effective in distinguishing DLB from both HCs and AD, achieving substantial accuracy through the utilization of specific lipidomic signatures. These include PC(15:0_18:2), PC(15:0_20:5), and SPH(d16:0) for differentiation between DLB and HCs; and a panel includes 13 lipid molecules: four PCs, two PEs, three SPHs, two Cers, and two Hex1Cers for distinguishing DLB from AD. Conclusions This study presents a novel and comprehensive lipidomic profile of DLB, distinguishing it from AD and HCs. Predominantly, sphingolipids (e.g., ceramides and SPHs) and phospholipids (e.g., PE and PC) were the most dysregulated lipids in relation to DLB patients. The lipidomics panels identified through machine learning may serve as effective plasma biomarkers for diagnosing DLB and differentiating it from AD dementia.

Abstract Background Tau-PET is a diagnostic tool with high sensitivity and specificity for discriminating Alzheimer’s disease (AD) dementia from other neurodegenerative disorders in well-controlled research environments. The role of tau-PET in real-world clinical practice, however, remains to be established. The aim of the TAP-TAU study is therefore to investigate the impact of tau-PET in clinical practice. Methods TAP-TAU is a prospective, longitudinal multi-center study in 300 patients (≥ 50 years old) with mild cognitive impairment or mild dementia across five Dutch memory clinics. Patients are eligible if diagnostic certainty is

Abstract Background Analysis of selected research cohorts has highlighted an association between plasma neurofilament light (NfL) protein and cross-sectional cognitive impairment as well as longitudinal cognitive decline. However, the findings have yielded inconsistent results regarding its possible application in clinical practice. Despite its potential prognostic significance, the role of plasma NfL in daily clinical practice with unselected patients suffering from cognitive impairment remains largely unexplored. Methods This retrospective, cross-sectional and longitudinal monocentric study enrolled 320 patients with Alzheimer’s disease ([AD], n = 158), dementia with Lewy body ([DLB], n = 30), frontotemporal dementia ([FTD], n = 32), non-neurodegenerative diseases ([NND], n = 59) or subjective cognitive decline ([SCD], n = 41). Plasma NfL levels were measured at baseline on the Simoa platform. AD, DLB, and FTD patients were also analyzed altogether as a ‘degenerative conditions’ subgroup, whereas SCD and NND were grouped as a ‘non-degenerative conditions’ subgroup. We assessed the relationship between plasma NfL levels and cross-sectional cognitive performance, including global cognition and six specific cognitive domains. A subset of 239 patients had follow-up mini-mental state examinations (MMSE) up to 60 months. Models were adjusted on age, education level, glomerular filtration rate and body mass index. Results In 320 patients, baseline plasma NfL levels were negatively associated with global cognition (β=-1.28 (-1.81 ; -0.75) P

Abstract The Bio-Hermes Study was a cross-sectional observational study designed to develop a database of blood-based and digital biomarkers to improve detection of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). We examined the ability of Cognivue Clarity to (a) detect MCI and AD in clinical diagnostics groups, (b) determine the presence of amyloid, and (c) distinguish between biomarker-confirmed groups. Bio-Hermes enrolled 887 participants who completed both Cognivue Clarity and amyloid PET scans (388 Cognitively Normal, 282 MCI, 217 Probable AD). Cognivue Clarity differentiated between Cognitively Normal, MCI, and probable AD in clinical cohorts, amyloid positive from amyloid negative individuals, and True Controls from MCI due to AD and AD in biomarker-confirmed cohorts (all p

Abstract Convolutional neural networks (CNNs) for extracting structural information from structural magnetic resonance imaging (sMRI), combined with functional magnetic resonance imaging (fMRI) and neuropsychological features, has emerged as a pivotal tool for early diagnosis of Alzheimer’s disease (AD). However, the fixed-size convolutional kernels in CNNs have limitations in capturing global features, reducing the effectiveness of AD diagnosis. We introduced a group self-calibrated coordinate attention network (GSCANet) designed for the precise diagnosis of AD using multimodal data, including encompassing Haralick texture features, functional connectivity, and neuropsychological scores. GSCANet utilizes a parallel group self-calibrated module to enhance original spatial features, expanding the field of view and embedding spatial data into channel information through a coordinate attention module, which ensures long-term contextual interaction. In a four-classification comparison (AD vs. early MCI (EMCI) vs. late MCI (LMCI) vs. normal control (NC)), GSCANet demonstrated an accuracy of 78.70%. For the three-classification comparison (AD vs. MCI vs. NC), it achieved an accuracy of 83.33%. Moreover, our method exhibited impressive accuracies in the AD vs. NC (92.81%) and EMCI vs. LMCI (84.67%) classifications. GSCANet improves classification performance at different stages of AD by employing group self-calibrated to expand features receptive field and integrating coordinated attention to facilitate significant interactions among channels and spaces. Providing insights into AD mechanisms and showcasing scalability for various disease predictions.

Abstract Although Alzheimer’s disease (AD) and intracranial aneurysm (IA) were two different types of diseases that occurred in the brain, ruptured IA (RIA) survivors may experience varying degrees of cognitive dysfunction. Neither AD nor IA is easily recognizable by an early onset so that the incidence of adverse clinical outcomes would be on the rise. Therefore, we focused on the exploration of the shared genes and molecular mechanisms between AD and IA, which would be significant for the efficiency of co-screening and co-diagnosis. Two GEO datasets were selected for the weighted gene co-expression network analysis (WGCNA) and differentially expressed gene screening, obtaining 78 overlapped genes. Next, 9 hub genes were identified by the protein-protein interaction network, including PIK3CA, GAB1, IGF1R, PLCB1, PGR, PDGFRB, PLCE1, FGFR3, and SYNJ1. The interactions among the hub genes, miRNA, and TFs were also explored. Meanwhile, we performed GO and KEGG pathway enrichment analyses for the results of WGCNA and hub genes, which showed that the Ras signaling and Rap1 signaling were the main shared pathogenesis. In conclusion, the present bioinformatics analysis revealed that AD and IA had the shared genes and molecular mechanisms, and these outcomes were associated with inflammation and calcium homeostasis, which could provide research clues for further studies.

Abstract Observational studies have shown some association between cannabis use disorder (CUD) and neurological disorders, but their causal relationship is unclear. In this study, we tested the potential causal relationship between CUD and three common neurological disorders using two-sample Mendelian randomization (TSMR) and multivariate MR (MVMR) methods. Thirty-two genetic loci were extracted as exposure factors from the largest genome-wide association study (GWAS) summary statistics for CUD to date. TSMR results showed that genetic prediction of CUD with all stroke, ischemic stroke, large-artery atherosclerotic stroke, migraine with aura, and Alzheimer’s disease (AD) had a positive causal relationship (P 0.05). Subgroup analyses highlighted a causal relationship between genetically predicted CUD and large-artery atherosclerotic stroke (OR = 1.169; 95%CI 1.030–1.328; P = 0.016) and migraine with aura (OR = 1.142; 95% 1.021–1.278; P = 0.020). Our further functional mapping and annotation enrichment analyses using FUMA suggest that the brain-gut axis may serve as another layer of explanation for the existence of an association between CUD and neurological disorders.

Abstract Background Synaptic degeneration, axonal injury, and white matter disintegration are among the pathological events in Alzheimer’s disease (AD), for which growth-associated protein 43 (GAP-43) and diffusion tensor imaging (DTI) could be an indicator. In this study, the cerebrospinal fluid (CSF) GAP-43 clinical trajectories and their association with progression and AD hallmarks with white matter microstructural changes were evaluated. Methods A total number of 133 participants were enrolled in GAP-43 and DTI values were compared between groups, both cross-sectionally and longitudinally with two and four-year follow-ups. Subsequently, the correlation between GAP-43 levels in the CSF and DTI values was investigated using Spearman’s correlation. Results The CSF level of GAP-43 is negatively correlated with the mean diffusivity measures in Fornix (Cres)/Stria terminals in early and late MCI (rs=-0.478 p = 0.021 and rs=-0.425 p = 0.038). Additionally, the CSF level of GAP-43 is negatively correlated with fractional anisotropy in the cingulum in late MCI (rs=-0.437 p = 0.033). Moreover, the axial diffusivity in superior corona radiate (rs=-0.562 p = 0.005 and rs=-0.484 p = 0.036) and radial diffusivity in superior fronto-occipital fasciculus was negatively correlated with GAP-43 level in the early and mid-MCI participants (rs=-0.520 p = 0.011 and rs=-0.498 p = 0.030). Conclusions Presynaptic marker GAP-43 in combination with DTI can be used as a novel biomarker to identify microstructural synaptic degeneration in the early MCI. In addition, it can be used as a biomarker for tracking the progression of AD and monitoring treatment efficacy.

Abstract Periodontitis exacerbates Alzheimer’s disease (AD) through multiple pathways. Both periodontitis and AD are intricately correlated to intestinal homeostasis, yet there is still a lack of direct evidence regarding whether periodontitis can regulate the progression of AD by modulating intestinal homeostasis. The current study induced experimental periodontitis in AD mice by bilaterally ligating the maxillary second molars with silk and administering Pg-LPS injections in APPswe/PS1ΔE9 (APP/PS1) mice. Behavioral tests and histological analyses of brain tissue were conducted after 8 weeks. Gut microbiota was analyzed and colon tissue were also evaluated. Then, fecal microbiota from mice with periodontitis was transplanted into antibiotic-treated mice to confirm the effects of periodontitis on AD and the potential mechanism was explored. The results indicated periodontitis exacerbated cognitive impairment and anxious behaviour in APP/PS1 mice, with increased Aβ deposition, microglial overactivation and neuroinflammation in brain. Moreover, the intestinal homeostasis of AD mice was altered by periodontitis, including affecting gut microbiota composition, causing colon inflammation and destroyed intestinal epithelial barrier. Furthermore, AD mice that underwent fecal transplantation from mice with periodontitis exhibited worsened AD progression and disrupted intestinal homeostasis. It also impaired intestinal barrier function, elevated peripheral inflammation, damaged blood-brain barrier (BBB) and caused neuroinflammation and synapses impairment. Taken together, the current study demonstrated that periodontitis could disrupt intestinal homeostasis to exacerbate AD progression potential via causing gut microbial dysbiosis, intestinal inflammation and intestinal barrier impairment to induce peripheral inflammation and damage BBB, ultimately leading to neuroinflammation and synapse impairment. It underscores the importance of maintaining both periodontal health and intestinal homeostasis to reduce the risk of AD.

Abstract Background It is now realized that peripheral inflammation and abnormal immune responses, especially T cells, contribute to the development of Alzheimer’s disease (AD). Gasdermin D (GSDMD) -mediated pyroptosis has been associated with several neuroinflammatory diseases, but whether GSDMD is involved in the peripheral inflammation and T cell immunity during AD remains unclear. Methods We dynamically investigated GSDMD activation in the peripheral and central nervous system of 5×FAD mouse model and dissected the role of myeloid GSDMD using genetic knockout mice, especially its influence on peripheral T cell responses and AD inflammation. RNA sequencing and in vitro coculture were used to elucidate the underlying immune mechanisms involved. Targeted inhibitor experiments and clinical correlation analysis were used to further verify the function of GSDMD in AD. Results In the present study, caspase activated GSDMD in the spleen of 5×FAD mice earlier than in the brain during disease progression. Loss of myeloid cell GSDMD was shown to impair early-stage effector T cell activation in the periphery and prevent T cell infiltration into the brain, with an overall reduction in neuroinflammation. Furthermore, myeloid cell GSDMD induced T cell PD-1 expression through the IL-1β/NF-κB pathway, restricting regulatory T cells. The administration of a GSDMD inhibitor combined with an anti-PD-1 antibody was found to mitigate the development of AD-associated inflammation. In some AD patients, plasma sPD-1 is positively correlated with IL-Iβ and clinical features. Conclusions Our study systematically identified a role for GSDMD in the AD-related peripheral inflammation and early-stage T cell immunity. These findings also suggest the therapeutic potential of targeting GSDMD for the early intervention in AD.

Abstract The NLR family pyrin domain containing 3 (NLRP3) inflammasome in microglia is intimately linked to the pathogenesis of Alzheimer’s disease (AD). Although NLRP3 inflammasome activity is regulated by cellular metabolism, the underlying mechanism remains elusive. Here, we found that under the pathological conditions of AD, the activation of NLRP3 inflammasome in microglia is accompanied by increased glutamine metabolism. Suppression of glutaminase, the rate limiting enzyme in glutamine metabolism, attenuated the NLRP3 inflammasome activation both in the microglia of AD mice and cultured inflammatory microglia. Mechanistically, inhibiting glutaminase blocked the anaplerotic flux of glutamine to the tricarboxylic acid cycle and amino acid synthesis, down-regulated mTORC1 signaling by phosphorylating AMPK, which stimulated mitophagy and limited the accumulation of intracellular reactive oxygen species, ultimately prevented the activation of NLRP3 inflammasomes in activated microglia during AD. Taken together, our findings suggest that glutamine metabolism regulates the activation of NLRP3 inflammasome through mitophagy in microglia, thus providing a potential therapeutic target for AD treatment.

Abstract Alzheimer’s disease (AD) is a common neurodegenerative disease characterized by progressive cognitive and physical impairment. Neuroinflammation is related to AD, and the misfolding and aggregation of amyloid protein in the brain creates an inflammatory microenvironment. Microglia are the predominant contributors to neuroinflammation, and abnormal activation of microglia induces the release of a large amount of inflammatory factors, promotes neuronal apoptosis, and leads to cognitive impairment. In this study, we used microglial membranes containing caffeic acid-coupled carbon quantum dots to prepare a novel biomimetic nanocapsule (CDs-CA-MGs) for the treatment of AD. The application of CDs-CA-MGs via nasal administration can bypass the blood‒brain barrier (BBB) and directly target the site of inflammation. After treatment with CDs-CA-MGs, AD mice showed reduced inflammation in the brain, decreased neuronal apoptosis, and significantly improved learning and memory abilities. In addition, CDs-CA-MGs affect inflammation-related JAK-STAT and Toll-like receptor signaling pathways in AD mice. CDs-CA-MGs significantly downregulated interleukins (IL-1β and IL-6) and tumor necrosis factor (TNF-α). This finding suggested that CDs-CA-MGs may improve cognitive impairment by modulating inflammatory responses. In conclusion, the use of CDs-CA-MGs provides a possible therapeutic strategy for the treatment of AD. Graphical abstract

Abstract Background The activation of endoplasmic reticulum (ER) stress is an early pathological hallmark of Alzheimer’s disease (AD) brain, but how ER stress contributes to the onset and development of AD remains poorly characterized. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a non-canonical neurotrophic factor and an ER stress inducible protein. Previous studies reported that MANF is increased in the brains of both pre-symptomatic and symptomatic AD patients, but the consequence of the early rise in MANF protein is unknown. Methods We examined the expression of MANF in the brain of AD mouse models at different pathological stages. Through behavioral, electrophysiological, and neuropathological analyses, we assessed the level of synaptic dysfunctions in the MANF transgenic mouse model which overexpresses MANF in the brain and in wild type (WT) mice with MANF overexpression in the hippocampus. Using proteomic and transcriptomic screening, we identified and validated the molecular mechanism underlying the effects of MANF on synaptic function. Results We found that increased expression of MANF correlates with synapse loss in the hippocampus of AD mice. The ectopic expression of MANF in mice via transgenic or viral approaches causes synapse loss and defects in learning and memory. We also identified that MANF interacts with ELAV like RNA-binding protein 2 (ELAVL2) and affects its binding to RNA transcripts that are involved in synaptic functions. Increasing or decreasing MANF expression in the hippocampus of AD mice exacerbates or ameliorates the behavioral deficits and synaptic pathology, respectively. Conclusions Our study established MANF as a mechanistic link between ER stress and synapse loss in AD and hinted at MANF as a therapeutic target in AD treatment.

Zixuan Zhang,1 Jing Bai,1 Siqi Zhang,1 Renzhen Wang,1 Shaobing Zhu,1 Tianyue Li,1 Miao Zhang2 1Graduate Schools, Heilongjiang University of Traditional Chinese Medicine, Harbin, Heilongjiang, People’s Republic of China; 2Acupuncture and Moxibustion 10 Departments, The Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin, Heilongjiang, People’s Republic of ChinaCorrespondence: Miao Zhang, Acupuncture and Moxibustion 10 Departments, The Second Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin, Heilongjiang, 150001, People’s Republic of China, Tel +86-13845088833, Fax +86-13114600132, Email hljzmzjzy@163.comAbstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disease caused by multiple causes. The main pathological features of AD are β-amyloid (Aβ) deposition, hyperphosphorylation of Tau protein, and progressive neuronal loss. Pyroptosis is one of the main forms of neuronal death, which is mainly caused by the activation of Gasdermin protein by upstream signals and the release of its N-terminal domain on the cell membrane. Studies have shown that there is a close relationship between Alzheimer’s disease (AD) and pyroptosis. Therefore, this paper summarizes the relationship between pyroptosis and its molecular mechanism and AD, as well as the related research of traditional Chinese medicine in the treatment of AD by regulating pyroptosis, in order to provide a new direction for the study of AD pathogenesis based on pyroptosis pathway.Keywords: Alzheimer’s disease, cell pyroptosis, inflammasome, traditional Chinese medicine

Alzheimer’s disease (AD), a common neurodegenerative disease, has been linked to periodontitis, especially Porphyromonas gingivalis (P. gingivalis) infection. This review summarizes the potential mechanisms and pathways through which P. gingivalis and its virulence factors are involved in AD pathogenesis, aiming to provide the scientific basis for the development of novel prevention and treatment strategies for AD. P. gingivalis can promote AD by exacerbating neuroinflammation, facilitating amyloid beta and Tau deposition, and disrupting the blood-brain barrier. Gingipains, secreted by P. gingivalis, serve as core effector molecules by increasing the blood-brain barrier permeability. The association between P. gingivalis and its effectors and AD pathology has been validated by metagenomic analysis and sample detection, indicating that P. gingivalis may be an environmental susceptibility factor or modifiable risk factor for AD. However, the precise mechanisms by which P. gingivalis influences AD, and its interactions with other potential AD-related factors, remain unclear. Moreover, further research needs to be conducted on the therapeutic potential of P. gingvalis intervention in improving AD.

Abstract Background SHR-1707 is a novel humanized anti-Aβ IgG1 monoclonal antibody that binds to Aβ fibrils and monomers to block the formation of Aβ plaques or to promote the microglial phagocytosis of Aβ. Preclinical studies showed that SHR-1707 reduced brain Aβ deposition in 5xFAD transgenic mice. Herein, we conducted two phase 1 studies to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of a single intravenous dose of SHR-1707 in healthy adult subjects. Methods Two randomized, double-blind, single-ascending-dose, phase 1 studies were conducted in China (Study CHN) and Australia (Study AUS). Study CHN consisted of 2 parts. In Part 1, eligible healthy young adults (18–45 years) were sequentially randomized 8:2 to receive SHR-1707 (five cohorts: 2, 6, 20, 40, and 60 mg/kg) or placebo in each cohort; in Part 2, elderly subjects (55–80 years) were randomized 8:4 to receive SHR-1707 (20 mg/kg) or placebo. A similar design was used in Study AUS, but with only healthy young adults enrolled across three dosing cohorts (2, 20, and 60 mg/kg). Results Sixty-two (part 1/2, n = 50/12; age range, 18–42/55–63 years) and 30 subjects (age range, 18–42 years) received SHR-1707 or placebo in Study CHN and Study AUS, respectively. In Study CHN, all treatment-related adverse events (TRAEs) were mild, with the most common being transient laboratory abnormalities. In Study AUS, TRAEs were mostly mild (1 moderate event each with SHR-1707/placebo); the most common TRAEs with SHR-1707 were dysgeusia and fatigue (8.3% each). In both studies, the exposure of SHR-1707 increased in a slightly greater than dose-proportional manner over the dose range of 2–60 mg/kg in young adults; there was a dose-dependent increase in plasma Aβ42 concentration following SHR-1707 administration compared with the placebo group. The safety and PK and PD profiles of SHR-1707 in the elderly subjects were consistent with the younger counterpart at the same dose level. No ethnic difference in safety, PK and PD of SHR-1707 was observed. Conclusions A single intravenous dose of SHR-1707 at 2–60 mg/kg was safe and well tolerated in healthy young adult and elderly subjects. The PK and PD profiles are supportive for further clinical development. Trial registration NCT04973189 (retrospectively registered on Jul.21, 2021) and NCT04745104 (registered on Feb.6, 2021) on clinicaltrials.gov.

Abstract Background The accumulation of amyloid-β (Aβ) peptide in the brain is a hallmark of Alzheimer’s disease (AD), occurring years before symptom onset. Current methods for quantifying in vivo amyloid load involve invasive or costly procedures, limiting accessibility. Early detection of amyloid positivity in non-demented individuals is crucial for aiding early AD diagnosis and for initiating anti-amyloid immunotherapies at early stages. This study aimed to develop and validate predictive models to identify brain amyloid positivity in non-demented patients, using routinely collected clinical data. Methods Predictive models for amyloid positivity were developed using data from 853 non-demented participants in the MEMENTO cohort. Amyloid levels were measured potentially repeatedly during study course through Positron Emision Tomography or CerebroSpinal Fluid analysis. The probability of amyloid positivity was modelled using mixed-effects logistic regression. Predictors included demographic information, cognitive assessments, visual brain MRI evaluations of hippocampal atrophy and lobar microbleeds, AD-related blood biomarkers (Aβ42/40 and P-tau181), and ApoE4 status. Models were subjected to internal cross-validation and external validation using data from the Amsterdam Dementia Cohort. Performance also was evaluated in a subsample that met the main criteria of the Appropriate Use Recommendations (AUR) for lecanemab. Results The most effective model incorporated demographic data, cognitive assessments, ApoE status, and AD-related blood biomarkers, achieving AUCs of 0.82 [95%CI 0.81-0.82] in MEMENTO sample and 0.90 [95%CI 0.86-0.94] in the external validation sample. This model significantly outperformed a reference model based solely on demographic and cognitive data, with an AUC difference in MEMENTO of 0.10 [95%CI 0.10-0.11]. A similar model without ApoE genotype achieved comparable discriminatory performance. MRI markers did not improve model performance. Performances in AUR of lecanemab subsample were comparable. Conclusion A predictive model integrating demographic, cognitive, and blood biomarker data offers a promising method to help identify amyloid status in non-demented patients. ApoE genotype and brain MRI data were not necessary for strong discriminatory ability, suggesting that ApoE genotyping may be deferred when assessing the risk-benefit ratio of immunotherapies in amyloid-positive patients who desire treatment. The integration of this model into clinical practice could reduce the need for lumbar puncture or PET examinations to confirm amyloid status.

Abstract Background Alzheimer’s Disease (AD) is the most common form of dementia. Its early stage, amnestic Mild Cognitive Impairment (aMCI), is characterized by disrupted information flow in the brain. Previous studies have yielded inconsistent results when using electrophysiological techniques to investigate functional connectivity changes in AD, and a contributing factor may be the study of brain activity divided into frequencies. Methods Our study aimed to address this issue by employing a cross-frequency approach to compare the functional networks of 172 healthy subjects and 105 aMCI patients. Using magnetoencephalography, we constructed source-based multilayer graphs considering both intra- and inter-frequency functional connectivity. We then assessed changes in network organization through three centrality measures, and combined them into a unified centrality score to provide a comprehensive assessment of centrality disruption in aMCI. Results The results revealed a noteworthy shift in centrality distribution in aMCI patients, both in terms of spatial distribution and frequency. Posterior brain regions decrease synchrony between their high-frequency oscillations and other regions’ activity across all frequencies, while anterior regions increase synchrony between their low-frequency oscillations and other regions’ activity across all frequencies. Thus, posterior regions reduce their relative importance in favor of anterior regions. Conclusions Our findings provide valuable insights into the intricate changes that occur in functional brain networks during the early stages of AD, demonstrating that considering the interplays between different frequency bands enhances our understanding of AD network dynamics and setting a precedent for the study of functional networks using a multilayer approach.

Abstract Background Early detection of Alzheimer’s disease (AD) is essential for timely management and consideration of therapeutic options; therefore, detecting the risk of conversion from mild cognitive impairment (MCI) to AD is crucial during neurodegenerative progression. Existing neuroimaging studies have mostly focused on group differences between individuals with MCI (or AD) and cognitively normal (CN), discarding the temporal information of conversion time. Here, we aimed to develop a prognostic model for AD conversion using functional connectivity (FC) and Cox regression suitable for conversion event modeling. Methods We developed a prognostic model using a large-scale Alzheimer’s Disease Neuroimaging Initiative dataset, and it was validated using external data obtained from the Open Access Series of Imaging Studies. We considered individuals who were initially CN or had MCI but progressed to AD and those with MCI with no progression to AD during the five-year follow-up period. As the exact conversion time to AD is unknown, we inferred this information using imputation approaches. We generated cortex-wide principal FC gradients using manifold learning techniques and computed subcortical-weighted manifold degrees from baseline functional magnetic resonance imaging data. A penalized Cox regression model with an elastic net penalty was adopted to define a risk score predicting the risk of conversion to AD, using FC gradients and clinical factors as regressors. Results Our prognostic model predicted the conversion risk and confirmed the role of imaging-derived manifolds in the conversion risk. The brain regions that largely contributed to predicting AD conversion were the heteromodal association and visual cortices, as well as the caudate and hippocampus. Our risk score based on Cox regression was consistent with the expected disease trajectories and correlated with positron emission tomography tracer uptake and symptom severity, reinforcing its clinical usefulness. Our findings were validated using an independent dataset. The cross-sectional application of our model showed a higher risk for AD than that for MCI, which correlated with symptom severity scores in the validation dataset. Conclusion We proposed a prognostic model predicting the risk of conversion to AD. The associated risk score may provide insights for early intervention in individuals at risk of AD conversion.

Abstract Background In Alzheimer’s disease (AD), the associations between tau pathology and brain atrophy and cognitive decline are well established, but imperfect. We investigate whether cerebrospinal fluid (CSF) biomarkers of biological processes (vascular, synaptic, and axonal integrity, neuroinflammation, neurotrophic factors) explain the disconnection between tau pathology and brain atrophy (brain resilience), and tau pathology and cognitive decline (cognitive resilience). Methods We included 428 amyloid positive participants (134 cognitively unimpaired (CU), 128 with mild cognitive impairment (MCI), 166 with AD dementia) from the BioFINDER-2 study. At baseline, participants underwent tau positron emission tomography (tau-PET), magnetic resonance imaging (MRI), cognitive testing, and lumbar puncture. Longitudinal data were available for MRI (mean (standard deviation) follow-up 26.4 (10.7) months) and cognition (25.2 (11.4) months). We analysed 18 pre-selected CSF proteins, reflecting vascular, synaptic, and axonal integrity, neuroinflammation, and neurotrophic factors. Stratifying by cognitive status, we performed linear mixed-effects models with cortical thickness (brain resilience) and global cognition (cognitive resilience) as dependent variables to assess whether the CSF biomarkers interacted with tau-PET levels in its effect on cortical atrophy and cognitive decline. Results Regarding brain resilience, interaction effects were observed in AD dementia, with vascular integrity biomarkers (VEGF-A (βinteraction = -0.009, pFDR = 0.047) and VEGF-B (βinteraction = -0.010, pFDR = 0.037)) negatively moderating the association between tau-PET signal and atrophy. In MCI, higher NfL levels were associated with more longitudinal cortical atrophy (β = -0.109, pFDR = 0.033) and lower baseline cortical thickness (β = -0.708, pFDR = 0.033) controlling for tau-PET signal. Cognitive resilience analyses in CU revealed interactions with tau-PET signal for inflammatory (GFAP, IL-15; βinteraction -0.073–-0.069, pFDR 0.001–0.045), vascular (VEGF-A, VEGF-D, PGF; βinteraction -0.099–-0.063, pFDR

Abstract Objective To explore the role of cuproptosis in Alzheimer’s disease (AD). Methods An AD-related microarray dataset was downloaded from the Gene Expression Omnibus (GEO) database (GSE140830). Weighted gene co-expression network analysis was used to identify AD-related modular genes. The Venn analysis was performed to obtain module genes associated with apoptosis and cuproptosis. Besides, we conducted an enrichment analysis of overlapped genes and constructed the protein–protein interaction (PPI) network, followed by screening hub genes and those significantly associated with AD were used to construct models of apoptosis and cuproptosis, respectively. Further, receiver operating characteristic (ROC) curve analysis, decision curve analysis (DCA), and subgroup analysis were used to compare the AD prediction performance of two models. Finally, the accuracy and reliability of AD prediction models were verified by GSE26927. Results We obtained 42 module genes related to apoptosis and 9 module genes related to cuproptosis. The enrichment analysis results revealed MAPK signaling pathway as the common signaling pathway of apoptosis- and cuproptosis-related genes. Next, the hub genes associated with apoptosis (TRADD, FADD, BIRC2, and CASP2) and cuproptosis (MAP2K1, SLC31A1, and PDHB) in AD were identified, which were used to construct apoptosis and cuproptosis models to distinguish AD patients from the control group (P

Abstract We aimed to assess the association of tramadol use with the risk of dementia. This population-based retrospective cohort study using the Korean National Health Insurance Service database included a total of 1,865,827 older adult patients aged 60 years or older with common musculoskeletal pain between January 1, 2003, and December 31, 2007. Individuals who were newly dispensed tramadol (N = 41,963) were identified and propensity score–matched with those who were not (N = 41,963). Over a maximum of 14-year follow-up, the incidence rates (events per 1000 person-years) of all-cause dementia were 6.1 for nonusers, 6.2 for those with cumulative tramadol use of 1–14 days, 7.7 for those with 15–90 days of use, and 8.0 for those with > 90 days of use. Longer cumulative duration of tramadol use was associated with an increased risk of all-cause dementia compared with nonuse (1 to 14 days: aHR 1.06, 95% CI 0.96–1.17; 15 to 90 days: aHR 1.14, 95% CI 1.10–1.35; and more than 90 days: aHR 1.18, 95% CI 1.00–1.39; test for trend: P

Abstract Sensory stimulations at 40 Hz gamma (but not any other frequency), have shown promise in reversing Alzheimer’s disease (AD)-related pathologies. What distinguishes 40 Hz? We hypothesized that stimuli at 40 Hz might summate more efficiently (temporal summation) or propagate more efficiently between cortical layers (vertically), or along cortical laminas (horizontally), compared to inputs at 20 or 83 Hz. To investigate these hypotheses, we used brain slices from AD mouse model animals (5xFAD). Extracellular (synaptic) stimuli were delivered in cortical layer 4 (L4). Leveraging a fluorescent voltage indicator (VSFP) expressed in cortical pyramidal neurons, we simultaneously monitored evoked cortical depolarizations at multiple sites, at 1 kHz sampling frequency. Experimental groups (AD-Female, CTRL-Female, AD-Male, and CTRL-Male) were tested at three stimulation frequencies (20, 40, and 83 Hz). Despite our initial hypothesis, two parameters—temporal summation of voltage waveforms and the strength of propagation through the cortical neuropil—did not reveal any distinct advantage of 40 Hz stimulation. Significant physiological differences between AD and Control mice were found at all stimulation frequencies tested, while the 40 Hz stimulation frequency was not remarkable.

Abstract Alzheimer’s Disease (AD) is a neurodegenerative disorder, and various molecules associated with PANoptosis are involved in neuroinflammation and neurodegenerative diseases. This work aims to identify key genes, and characterize PANoptosis-related molecular subtypes in AD. Moreover, we establish a scoring system for distinguishing PANoptosis molecular subtypes and constructing diagnostic models for AD differentiation. A total of 5 hippocampal datasets were obtained from the Gene Expression Omnibus (GEO) database. In total, 1324 protein-encoding genes associated with PANoptosis (1313 apoptosis genes, 11 necroptosis genes, and 31 pyroptosis genes) were extracted from the GeneCards database. The Limma package was used to identify differentially expressed genes. Weighted Gene Co-Expression Network Analysis (WGCNA) was conducted to identify gene modules significantly associated with AD. The ConsensusClusterPlus algorithm was used to identify AD subtypes. Gene Set Variation Analysis (GSVA) was used to assess functional and pathway differences among the subtypes. The Boruta, Least Absolute Shrinkage and Selection Operator (LASSO), Random Forest (RF), and Support Vector Machine Recursive Feature Elimination (SVM-RFE) algorithms were used to select the three PANoptosis-related Key AD genes (PKADg). A scoring model was constructed based on the Boruta algorithm. PANoptosis diagnostic models were developed using the RF, SVM-RFE, and Logistic Regression (LR) algorithms. The ROC curves were used to assess the model performance. A total of 48 important genes were identified by intersecting 725 differentially expressed genes and 2127 highly correlated module genes from WGCNA with 1324 protein-encoding genes related to PANoptosis. Machine learning algorithms identified 3 key AD genes related to PANoptosis, including ANGPT1, STEAP3, and TNFRSF11B. These genes had strong discriminatory capacities among samples, with Receiver Operating Characteristic Curve (ROC) analysis indicating Area Under the Curve (AUC) values of 0.839, 0.8, and 0.868, respectively. Using the 48 important genes, the ConsensusClusterPlus algorithm identified 2 PANoptosis subtypes among AD patients, i.e., apoptosis subtype and mild subtype. Apoptosis subtype patients displayed evident cellular apoptosis and severe functionality damage in the hippocampal tissue. Meanwhile, mild subtype patients showed milder functionality damage. These two subtypes had significant differences in apoptosis and necroptosis; however, there was no apparent variation in pyroptosis functionality. The scoring model achieved an AUC of 100% for sample differentiation. The RF PANoptosis diagnostic model demonstrated an AUC of 100% in the training set and 85.85% in the validation set for distinguishing AD. This study identified two PANoptosis-related hippocampal molecular subtypes of AD, identified key genes, and established machine learning models for subtype differentiation and discrimination of AD. We found that in the context of AD, PANoptosis may influence disease progression through the modulation of apoptosis and necrotic apoptosis.

Abstract Cyanobacteria, especially Arthrospira, are valuable resources of nutrients and natural pigments with many beneficial health-related properties. This study optimized the extraction conditions of Arthrospira to achieve high phenolic contents and antioxidant activities. Under optimized extraction conditions, the bioactive compounds (phenolics and pigment components), antioxidant activities, and inhibitions of the key enzymes relevant to some non-communicable diseases were compared between Arthrospira platensis and Arthrospira maxima. Optimized extraction conditions were determined as 2 h shaking time, 50 °C extraction temperature, and 1% (w/v) solid-to-liquid ratio, giving effective phenolic and phycocyanin contents using aqueous extraction, while 80% (v/v) aqueous ethanolic extraction provided high total chlorophyll content. Most antioxidant activities were higher using 80% (v/v) aqueous ethanolic extracts. Both Arthrospira species inhibited the key enzymes involved in controlling non-communicable diseases including hyperlipidemia (lipase), diabetes (α-amylase, α-glucosidase, and dipeptidyl peptidase-IV), Alzheimer’s disease (acetylcholinesterase, butyrylcholinesterase and β-secretase), and hypertension (angiotensin-converting enzyme). High inhibitory activities were detected against β-secretase (BACE-1), the enzyme responsible for β-amyloid plaque formation in the brain that acts as a significant hallmark of Alzheimer’s disease. Arthrospira extract and donepezil (Alzheimer’s disease drug) synergistically inhibited BACE-1, suggesting the potential of Arthrospira extracts as effective BACE-1 inhibitors. Interestingly, A. maxima exhibited higher bioactive compound contents, antioxidant activities, and key enzyme inhibitions than A. platensis, indicating high potential for future food and medicinal applications.

Abstract Alzheimer disease (AD) is characterized by progressive loss of memory. Synaptic loss is now the best correlate of cognitive dysfunction in patients with Alzheimer’s disease. Thus, restoration or limitation of synapse loss is a promising strategy for pharmacotherapy of AD. N–N substituted piperazines are widely used chemical compounds for drug interventions to treat different illnesses including CNS diseases such as drug abuse, mental and anxiety disorders. Piperazine derivatives are small molecules that are usually well tolerated and cross blood brain barrier (BBB). Thus, disubstituted piperazines are good tools for searching and developing novel disease-modifying drugs. Previously, we have determined the piperazine derivative, 51164, as an activator of TRPC6 in dendritic spines. We have demonstrated synaptoprotective properties of 51164 in AD mouse models. However, 51164 was not able to cross BBB. Within the current study, we identified a novel piperazine derivative, cmp2, that is structurally similar to 51164 but is able to cross BBB. Cmp2 binds central part of monomeric TRPC6 in similar way as hypeforin does. Cmp2 selectively activates TRPC6 but not structurally related TRPC3 and TRPC7. Novel piperazine derivative exhibits synaptoprotective properties in culture and slices and penetrates the BBB. In vivo study indicated cmp2 (10 mg/kg I.P.) reversed deficits in synaptic plasticity in the 5xFAD mice. Thus, we suggest that cmp2 is a novel lead compound for drug development. The mechanism of cmp2 action is based on selective TRPC6 stimulation and it is expected to treat synaptic deficiency in hippocampal neurons.

Abstract Background Three common isoforms of the apolipoprotein E (APOE) gene - APOE2, APOE3, and APOE4 - hold varying significance in Alzheimer’s Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer’s Disease (AD), and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely, APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However, it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB. Methods To explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta, we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey’s post hoc testing as appropriate. Results Isogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance, tight junction integrity and efflux transporter gene expression. However, recombinant APOE4 protein significantly impeded the “brain-to-blood” amyloid beta 1–40 (Aβ40) transport capabilities of BMEC-like cells, suggesting a role in diminished amyloid clearance. Conversely, APOE2 increased amyloid beta 1–42 (Aβ42) transport in the model. Furthermore, we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways, mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes, yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition, while APOE2 pericyte-like cells displayed the least amyloid deposition, an observation in line with vascular pathologies in AD patients. Conclusions While APOE genotype did not directly impact general BMEC or pericyte properties, APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely, APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB, motivating further development of such in vitro models in AD modeling and drug development.

Abstract Introduction Heparin binding proteins (HBPs) with roles in extracellular matrix assembly are strongly correlated to β-amyloid (Aβ) and tau pathology in Alzheimer’s disease (AD) brain and cerebrospinal fluid (CSF). However, it remains challenging to detect these proteins in plasma using standard mass spectrometry-based proteomic approaches. Methods We employed heparin-affinity chromatography, followed by off-line fractionation and tandem mass tag mass spectrometry (TMT-MS), to enrich HBPs from plasma obtained from AD (n = 62) and control (n = 47) samples. These profiles were then correlated to Aβ, tau and phosphorylated tau (pTau) CSF biomarkers and plasma pTau181 from the same individuals, as well as a consensus brain proteome network to assess the overlap with AD brain pathophysiology. Results Heparin enrichment from plasma was highly reproducible, enriched well-known HBPs like APOE and thrombin, and depleted high-abundant proteins such as albumin. A total of 2865 proteins, spanning 10 orders of magnitude in abundance, were measured across 109 samples. Compared to the consensus AD brain protein co-expression network, we observed that specific plasma proteins exhibited consistent direction of change in both brain and plasma, whereas others displayed divergent changes, highlighting the complex interplay between the two compartments. Elevated proteins in AD plasma, when compared to controls, included members of the matrisome module in brain that accumulate with Aβ deposits, such as SMOC1, SMOC2, SPON1, MDK, OLFML3, FRZB, GPNMB, and the APOE4 proteoform. Additionally, heparin-enriched proteins in plasma demonstrated significant correlations with conventional AD CSF biomarkers, including Aβ, total tau, pTau, and plasma pTau181. A panel of five plasma proteins classified AD from control individuals with an area under the curve (AUC) of 0.85. When combined with plasma pTau181, the panel significantly improved the classification performance of pTau181 alone, increasing the AUC from 0.93 to 0.98. This suggests that the heparin-enriched plasma proteome captures additional variance in cognitive dementia beyond what is explained by pTau181. Conclusion These findings support the utility of a heparin-affinity approach coupled with TMT-MS for enriching amyloid-associated proteins, as well as a wide spectrum of plasma biomarkers that reflect pathological changes in the AD brain.

Abstract Background Current AT(N) stratification for Alzheimer’s disease (AD) accounts for complex combinations of amyloid (A), tau proteinopathy (T) and neurodegeneration (N) signatures. Understanding the transition between these different stages is a major challenge, especially in view of the recent development of disease modifying therapy. Methods This is an observational study, CSF levels of Tau, pTau181, pTau217, Aβ38/40/42, sAPPα/β, BACE1 and neurogranin were measured in the BALTAZAR cohort of cognitively impaired patients and in the Alzheimer's Disease Neuroimaging Initiative (ADNI). Biomarkers levels were related to the AT(N) framework. (A) and (T) were defined in BALTAZAR with CSF Aβ42/40 ratio and pTau217 respectively, and in ADNI with amyloid and tau PET. (N) was defined using total CSF tau in both cohorts. Results As expected, CSF Aβ42 decreased progressively with the AD continuum going from the A-T-N- to the A + T + N + profile. On the other hand, Tau and pTau181 increased progressively with the disease. The final transition from A + T + N- to A + T + N + led to a sharp increase in Aβ38, Aβ42 and sAPP levels. Synaptic CSF biomarkers BACE1 and neurogranin, were lowest in the initial A + T-N- stage and increased with T + and N + . CSF pTau181 and total tau were closely related in both cohorts. Conclusions The early transition to an A + phenotype (A + T-N-) primarily impacts synaptic function. The appearance of T + and then N + is associated with a significant and progressive increase in pathological Alzheimer's disease biomarkers. Our main finding is that CSF pTau181 is an indicator of N + rather than T + , and that N + is associated with elevated levels of BACE1 protein and beta-amyloid peptides. This increase may potentially fuel the amyloid cascade in a positive feedback loop. Overall, our data provide further insights into understanding the interconnected pathological processes of amyloid, tau, and neurodegeneration underlying Alzheimer's disease.

Shenghao Zhang,1,* Zhejianyi Gao,2,* Lina Feng,1,3 Mingquan Li1 1Department of Neurology, The Third Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, Jilin Province, 130021, People’s Republic of China; 2Department of Orthopaedics, Fushun Hospital of Chinese Medicine, Fushun, Liaoning Province, 113008, People’s Republic of China; 3Shandong Key Laboratory of TCM Multi-Targets Intervention and Disease Control, The Second Affiliated Hospital of Shandong First Medical University, Taian, Shandong Province, 271000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lina Feng; Mingquan Li, Department of Neurology, the Third Affiliated Hospital of Changchun University of Chinese Medicine, Changchun, Jilin, 130117, People’s Republic of China, Tel +86-15543120222 ; +86-15948000577, Email Fenglina0001@163.com; Limingquan0001@126.comAbstract: Alzheimer’s disease (AD) is a fatal neurodegenerative disease characterized by its insidious onset and progressive development, making it the most common form of dementia. Despite its prevalence, the exact causes and mechanisms responsible for AD remain unclear. Recent studies have highlighted that inflammation in the central nervous system (CNS) plays a crucial role in both the initiation and progression of AD. Neuroinflammation, an immune response within the CNS triggered by glial cells in response to various stimuli, such as nerve injury, infection, toxins, or autoimmune reactions, has emerged as a significant factor alongside amyloid deposition and neurofibrillary tangles (NFTs) commonly associated with AD. This article aims to provide an overview of the most recent research regarding the involvement of neuroinflammation in AD, with a particular focus on elucidating the specific mechanisms involving microglia and astrocytes. By exploring these intricate processes, a new theoretical framework can be established to further probe the impact of neuroinflammation on the development and progression of AD. Through a deeper understanding of these underlying mechanisms, potential targets for therapeutic interventions and novel treatment strategies can be identified in the ongoing battle against AD.Keywords: alzheimer’s disease, neuroinflammation, microglia, astrocyte, review

Abstract Background Recent advances in blood-based biomarker discovery are paving the way for simpler, more accessible diagnostic tools that can detect early signs of Alzheimer’s disease (AD). Recent successes in the development of amyloid-targeting immunotherapy approaches mark an important advancement in providing new options for the treatment of AD. We have developed a set of high-affinity monoclonal antibodies (mAbs) to tau protein that have the potential as tools for diagnosis and treatment of AD. Methods Sheep were immunised with either full-length tau (1-441) or truncated paired helical filament (PHF)-core tau (297–391). A stringent bio-panning and epitope selection strategy, with a particular focus directed to epitopes within the disease-relevant PHF-core tau, was used to identify single-chain antibodies (scAbs). These scAbs were ranked by affinity for each epitope class, with leads converted to high-affinity mAbs. These antibodies and their potential utility were assessed by their performance in tau immunoassays, as well as their ability to prevent tau aggregation and propagation. Further characterisation of these antibodies was performed by immunohistochemical staining of brain sections and immuno-gold electronmicroscopy of isolated PHFs. Results Our work resulted in a set of high-affinity antibodies reacting with multiple epitopes spanning the entire tau protein molecule. The tau antibodies directed against the core tau unit of the PHF inhibited pathological aggregation and seeding using several biochemical and cell assay systems. Through staining of brain sections and PHFs, the panel of antibodies revealed which tau epitopes were available, truncated, or occluded. In addition, highly sensitive immunoassays were developed with the ability to distinguish between and quantify various tau fragments. Conclusion This article introduces an alternative immunodiagnostic approach based on the concept of a “tauosome” – the diverse set of tau fragments present within biological fluids. The development of an antibody panel that can distinguish a range of different tau fragments provides the basis for a novel approach to potential diagnosis and monitoring of disease progression. Our results further support the notion that tau immunotherapy targeting the PHF-core needs to combine appropriate selection of both the target epitope and antibody affinity to optimise therapeutic potential.

Abstract Background Glucagon-like peptide-1 receptor agonists are a viable option for the prevention of Alzheimer’s disease (AD) but the mechanisms of this potential disease modifying action are unclear. We investigated the effects of once-weekly exenatide (EQW) on AD associated proteomic clusters. Methods The Exenatide Study of Cardiovascular Event Lowering study compared the cardiovascular effects of EQW 2 mg with placebo in 13,752 people with type 2 diabetes mellitus. 4,979 proteins were measured (Somascan V0.4) on baseline and 1-year plasma samples of 3,973 participants. C-reactive protein (CRP), ficolin-2 (FCN2), plasminogen activator inhibitor 1 (PAI-1), soluble vascular cell adhesion protein 1 (sVCAM1) and 4 protein clusters were tested in multivariable mixed models. Results EQW affected FCN2 (Cohen’s d -0.019), PAI-1 (Cohen’s d -0.033), sVCAM-1 (Cohen’s d 0.035) and a cytokine-cytokine cluster (Cohen’s d 0.037) significantly compared with placebo. These effects were sustained in individuals over the age of 65 but not in those under 65. Conclusions EQW treatment was associated with significant change in inflammatory proteins associated with AD. Trial Registration EXSCEL is registered on ClinicalTrials.gov: NCT01144338 on 10th of June 2010.