Citations

AbstractBackgroundExtracellular vesicles (EVs) carry pathogenic molecules and play a role in the disease spread, including aggregated tau proteins. The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery is responsible for the biogenesis of small EVs (exosomes), thus targeting critical ESCRT molecules can disrupt EV synthesis. We hypothesize that microglia‐specific targeting of ESCRT‐I molecule Tsg101 suppresses microglia‐derived EV‐mediated propagation of tau pathology, leading to amelioration of the disease phenotype of the tauopathy mouse model.MethodsPS19 tau transgenic mouse line was crossed with Cx3cr1CreERT2/+:Tsg101fl/fl lines for tamoxifen‐inducible Cx3cr1‐specific deletion of Tsg101 (Tsg101 cKO) in PS19 mice to generate WT, Tsg101 cKO, PS19, and PS19:Tsg101cKO groups. The animals were treated with tamoxifen or corn oil at 2 months of age and subjected to comprehensive behavioral, neuropathological, biochemical, and molecular biological assessments at 6‐7 months of age. The microglia isolated from Cx3cr1CreERT2/+:Tsg101fl/fl pups were subjected to in vitro synaptosome uptake analysis with or without 4‐Hydroxytamoxifen treatment.ResultsPS19 mice develop cognitive impairment as determined by Y‐maze, forced alternation, novel object recognition and fear conditioning, which are reversed in PS19:Tsg101cKO mice. This is correlated with reduced Alz50+ tau accumulation, neurodegenerative microglial activation, neuroinflammation and complement pathway activation as determined by bulk RNA sequencing, ELISA and neuropathology. Primary cultured Cx3cr1CreERT2/+:Tsg101fl/fl microglia with 4‐Hydroxytamoxifen treatment show reduced phagocytosis of E. coli particles and synaptosome in C1q dependent manner. Tsg101 cKO microglia show reduced expression of C3aR1 and CD68, and secretion of total and Tau+ EVs in vivo.ConclusionMicroglia‐specific targeting of Tsg101 show beneficial effect for ameliorating the disease progression of tauopathy mouse model via suppression of EV secretion, microglial activation, tau accumulation and complement‐dependent synaptic pruning. Microglial Tsg101 is a potential therapeutic target of Alzheimer’s disease and related tauopathy.

AbstractBackgroundAutosomal dominant progranulin (GRN) mutations are a common genetic cause of frontotemporal lobar degeneration. Though clinical trials for GRN‐related therapies are underway, there is an unmet need for biomarkers that can predict symptom onset and track disease progression. We previously showed that presymptomatic GRN carriers exhibit thalamocortical hyperconnectivity that increases with age when they are presumably closer to symptom onset. However, whether hyperconnectivity arises concomitantly with markers of neurodegeneration remains unclear.MethodUtilizing T1 and task‐free functional magnetic resonance imaging (tf‐fMRI) from 49 presymptomatic and 26 symptomatic GRN mutation carriers, we determined the relationships between functional connectivity as measured by voxel‐wise whole brain degree and GRN‐relevant markers of disease progression, which included plasma neurofilament light chain (NfL) concentrations, CSF complement C1q and C3b protein levels, grey matter atrophy, and OCD symptom severity.ResultNfL concentrations were associated with frontotemporoparietal and thalamic hyperconnectivity in presymptomatic GRN carriers and extensive regions of atrophy in symptomatic carriers. Complement levels were associated with regions of hyperconnectivity, but not gray matter, in symptomatic carriers. Presymptomatic carriers with thalamic hyperconnectivity tended to have lower grey matter volume in bilateral insula and left lateral parietal cortex, which are among regions that deteriorate in GRN‐FTD. OCD symptom severity was associated with hypoconnectivity across all GRN carriers.ConclusionIn presymptomatic carriers, the co‐occurrence of hyperconnectivity, high NfL, and low gray matter suggests that tf‐fMRI hyperconnectivity may portend the onset of the neurodegenerative phase. These findings point toward hyperconnectivity as an indicator of approaching symptomatic onset.

AbstractBackgroundAlzheimer’s disease (AD) is complex and multifactorial. Precision medicine approaches are needed to capture the basis of heterogeneity in AD pathogenesis, clinical presentation and neuropathology. Large‐scale molecular, deep phenotypic and exposomal data necessary to enable precision medicine research requires team‐based, interdisciplinary programs. Accelerating Medicines Partnership in Alzheimer’s Disease (AMP‐AD) was launched in 2014 with the goals to characterize disease heterogeneity and discover precision‐medicine therapeutic targets and biomarkers for AD and related disorders (ADRD).MethodAMP‐AD consortium, comprising eight teams addresses the barriers that historically limited therapeutic target and biomarker discovery in ADRD by characterizing the molecular disease complexity in deeply phenotyped human biospecimens and experimental disease models. Over the past decade, AMP‐AD teams generated multi‐omics data in ∼2,000 human brain samples from largely non‐Hispanic white donors and from >600 African American and Latin American donors. AMP‐AD datasets, including bulk and single cell molecular profiles from brain and peripheral tissues are analyzed using innovative analytic approaches generated and applied by these teams. Findings are validated with a broad array of cross‐species experimental platforms including human iPSC, fly and rodent models. These outcomes are integrated with deep phenotypic and exposomal measures for precision medicine discoveries and broadly shared.ResultAMP‐AD generates high‐dimensional molecular data and applies novel systems biology approaches bringing new insights into ADRD. AMP‐AD teams discovered molecular subtypes of AD and characterized the cell‐type specificity of these molecular changes. Discovery of disease signatures in brain and peripheral tissues enabled nominations of precision medicine therapeutic targets and biomarkers. Over 600 targets, annotated with human and cross‐species data, are shared with the research community on the Agora Platform of AD Knowledge portal that also houses all research data and outputs for rapid and responsible dissemination in a centralized manner.ConclusionFirst decade of AMP‐AD led to unprecedented data and knowledge for rigorous and reproducible ADRD research, accelerated the delivery of many new disease insights, and expanded the target and biomarker landscape. Future directions include discovery of subtypes for disease specificity and progression, contextualized by multi‐ethnic, molecular, exposomal data and validation platforms well‐integrated into therapeutic development pipelines to deliver new cures to ADRD.

AbstractBackgroundNeuropathologic inclusions formed by hyperphosphorylated protein tau in the brain are a hallmark of Alzheimer’s disease and other human neurodegenerative disorders commonly referred to as tauopathies. Tau lesions differ in their disease‐specific morphological presentations, affected cell type, subcellular compartments and tau isoforms present in the inclusions. In addition, tau filaments isolated from different tauopathies have distinct fibrillar structures that potentially underlie the morphological diversity of tau lesions. The factors that govern the formation of disease‐specific tau lesions and distinct patterns of neurodegeneration and clinical presentations are unknown. To address this gap of knowledge, we performed profiling of proteomes associated with phospho‐tau aggregates in 4 major tauopathies.MethodWe utilized the in‐house developed Probe‐dependent Proximity Profiling (ProPPr) method combined with data‐independent acquisition mass spectrometry to profile phospho‐tau associate proteomes in formalin‐fixed paraffin‐embedded (FFPE) frontal cortex tissue sections from the Mayo Clinic Florida Brain Bank. Selected candidates were validated by fluorescent co‐immunostaining of patient brains with corresponding antibodies.ResultIn situ proximity proteomic profiling revealed over 1300 protein associated with phospho‐tau pathology, including 229 proteins that were identified in all tauopathies. The most significantly enriched GO terms in this protein set common to all the tauopathies were related to chaperone activity, guanyl nucleotide binding, cadherin binding, cytoskeleton, and ion transporter activity, highlighting biological processes and pathways affected by tau aggregation regardless of the type of tau lesion. In addition, we identified 32 proteins with significantly different association with phospho‐tau between tauopathies. Immunostaining of post‐mortem patient frontal cortices confirmed some of the findings from proteomics and revealed differences in abundance and localization patterns of several tau‐associated proteins between tauopathies, uncovering a potential of using these proteins as biomarkers for antemortem diagnosis.ConclusionTau lesions in different tauopathies have differences in their associated proteomes that can be assessed by the ProPPr assay.

AbstractBackgroundThe efficacy of Calorie Restriction (CR) in enhancing cognition, promoting healthy aging, and extending lifespan is well‐established. Yet, it remains unclear whether the apolipoprotein E (APOE) genotype, a known modifier for aging and age‐related disorders, influences the beneficial effects of CR in countering aging.MethodsTo investigate this question, we utilized humanized APOE mouse models, which express APOE2, APOE3, or APOE4 alleles systematically (refer to as E2, E3, and E4 mice). These mice were subjected to either ad libitum (AL) feeding or 30% CR feeding, starting from 12 months and continuing up to 20 months of age (N = 11‐20/ genotype /group, mixed sex). We monitored energy expenditure using the Comprehensive Laboratory Animal Monitoring System (CLAMS). Additionally, we evaluated anxiety levels and memory capabilities through various behavioral tests. Finally, we collected brain, blood, and feces samples for transcriptomic, lipidomic, and microbiomic analyses.ResultsCLAMS measurements showed that CR significantly decreased the metabolic rate in E3 and E4 mice, but not E2 mice, during the non‐feeding period of the CR group, compared to the AL group. Furthermore, CR reduced anxiety and enhanced associative memory in E3 and E4 mice relative to the AL group. Brain transcriptomics revealed that CR upregulated the cholesterol synthesis pathway, potentially improving myelination‐related pathways in E3 and E4 mice. This finding was further validated by the increased staining intensity of myelin basic protein (MBP) observed in E3 and E4 mice from an independent cohort. In contrast, these effects were not pronounced in E2 mice. Additionally, while CR’s impact on the brain lipid profile was minor across all APOE genotypes, it significantly altered blood lipid profile of E2 mice, with minimal changes observed in E3 and E4 mice. Finally, in microbiomics, CR led to notable changes in the abundance of Bifidobacterium species in E3 and E4 mice and Lactobacillus species in E2 mice.ConclusionsOur study highlights the role of APOE genotypes in modulating the diverse effects of CR on cognition, brain transcriptomics, lipid metabolism, and the microbiome, providing a crucial foundation for considering APOE genotypes in developing prevention strategies for aging and age‐related disorders.

AbstractBackgroundWhile disease‐modifying treatments that reduce Aβ have been recently approved by the FDA, the identification of novel therapeutic targets and strategies that target underlying mechanisms to delay the AD development are still needed. Abnormal brain energy homeostasis and mitochondria dysfunction are observed early in AD. Therefore, the development of treatments to restore these defects could be beneficial. We identified small molecule (code name CP2) as a mild and specific mitochondrial complex I (MCI) inhibitor. Application of CP2 improved brain energy homeostasis, restored synaptic and cognitive function in APP/PS1 and 3xTgAD mice. However, mechanistic relationship between MCI inhibition, glucose uptake/utilization and mitochondrial function remains to be determined.MethodCellular energy metabolism was assessed in the neuroblastoma SH‐SY5Y cells that express either mutant human APP protein (APPswe) or empty vector (control) treated with vehicle or CP2. A Seahorse Extracellular Flux Analyzer was used to measure glycolysis, oxygen consumption rate, and fatty acids β‐oxidation (FAO). Flow cytometry allowed determining the translocation of glucose transporters to the cell surface. Changes in protein expression in response to treatment were assessed using Western Blot analysis. Changes in mitochondrial morphology were monitored using electron microscopy. The non‐radioactive Glucose Uptake‐Glo™ assay was utilized for measuring glucose uptake in cells. Metabolic flux analysis was done using 13C D‐Glucose stable isotope‐labeling.ResultsAPPswe cells have a significant decrease in glycolysis and spare respiratory capacity, an indicator of the mitochondrial ability to produce energy under stress conditions. These observations were consistent with decreased glucose uptake, which was compensated by an increased FAO to provide axillary fuel for ATP production. Mechanistically, at the concentrations relevant to in vivo treatment in APP/PS1 and 3xTgAD mice, acute CP2 treatment increased glucose uptake and utilization through the translocation of glucose transporters to the plasma membrane while prolong CP2 treatment activated metabolic sensors and mitochondrial morphofunctional pathways (e.g., fission, fusion, biogenesis, and turnover) consistent with the improved cellular bioenergetics in the AMPK‐dependent pathway.ConclusionData suggested that mild MCI inhibition activates multiple neuroprotective mechanisms improving cellular energy homeostasis and mitochondrial function in vivo and in vitro, representing promising strategy that target early AD mechanisms.

AbstractBackgroundDespite recent FDA approvement of disease‐modifying treatments that reduce Aβ, the identification of novel therapeutic strategies that could delay the Alzheimer’s disease (AD) development are needed. We identified and developed novel small molecule compounds that mildly inhibit mitochondrial complex I (MCI). Chronic treatment with a tool compound CP2 in 4 mouse models of familial AD was efficacious protecting against synaptic dysfunction and memory impairment, improving brain energetics and cognitive performance, reducing levels of human pTau and Ab. The objective of this study was to validate efficacy of this approach in a human brain‐like environment to support its translation into clinical trials. Human induced pluripotent stem cells (hiPSC) retain the host’s endogenous AD‐associated genetic alterations and recapitulate multiple hallmarks of late‐onset Alzheimer’s disease (LOAD).MethodUsing pure human induced neurons (iNs) from ApoE4/4‐carrying LOAD patients, we evaluated mitochondrial bioenergetics and morphology using a Seahorse XFe96 flux analyzer and electron microscopy. Axonal trafficking was assessed using MitoTracker Red and live confocal imaging. Immunohistochemistry, quantitative‐PCR, and western blot were carried out to assess changes in the expression of genes and proteins, and cell viability assays were used to perform H2O2 challenge.ResultsCultures of hiPSC‐derived neurons were established using a lentiviral delivery for tetracycline‐inducible expression of NGN2‐GFP fusion gene driven by tetracycline‐responsive promoter. The validation of NGN2‐iNs confirmed the development of mature molecular, cellular, and synaptic properties. LOAD iNs have a significant reduction in the motile mitochondria in axons, increased accumulation of pTau, and altered bioenergetics compared to the control neurons. At CP2 concentrations relevant to in vivo treatment in APP/PS1 and 3xTgAD mice, significant reduction of pTau was associated with improved cellular bioenergetics, restored mitochondrial motility, and increased resistance to H2O2 induced oxidative stress.ConclusionLOAD neurons carrying ApoE4/4 alleles recapitulate LOAD phenotype allowing validation of a novel mitochondria‐targeted therapeutics. These data support previous observations in AD mice demonstrating that mild inhibition of MCI induce mild energetic stress with subsequent activation of multifaceted adaptive stress response and neuroprotective mechanisms improving cellular energetics and mitochondrial morphofunction in LOAD human neurons. Further efforts will be focused on the determination of mechanisms of neuroprotection.

AbstractBackgroundAlzheimer’s disease (AD) is heterogeneous in both its clinical and neuropathologic course. Age at onset and distribution of corticolimbic tangles can vary widely among individuals. Genetic risk factors APOE ε4 and MAPT H1 increase AD risk. However, the association with the spatial distribution of pathology remains a key gap in knowledge. We investigated the effects of age at onset, APOE ε4, MAPT H1, and family history of cognitive symptoms on corticolimbic tangle density variability to better distinguish how each factor contributes.MethodThe FLorida Autopsied Multi‐Ethnic (FLAME) cohort was queried for neuropathologically confirmed AD cases (n = 1233). Tangle density (count per 0.125mm2) was quantified using thioflavin‐S staining in nucleus basalis of Meynert (nbM), hippocampal CA1 and subiculum, association cortices, and primary cortices. Cortical thickness was measured using digital pathology on hematoxylin‐eosin‐stained sections. We employed univariate and multivariate negative binomial regression models to evaluate the association of each factor on tangle density across the corticolimbic axis. Multivariate analysis was adjusted for significant variables from univariate analyses.ResultDonors with young‐onset (<65) AD (YOAD; n = 314) had more tangles than those with late‐onset AD (LOAD; n = 919) across most corticolimbic regions (Fig. 1). Except for CA1, younger age at onset was associated with a higher expected tangle density with an increase ranging from 8% in subiculum up to 99% in motor cortex (Table 1). Male sex was associated with lower expected tangle density throughout all corticolimbic regions. Being male associated the strongest in visual cortex with a 41% tangle density decrease. The APOE ε4 allele associated with an increase of tangle density in the nbM, CA1, subiculum, and visual cortex, whereas the MAPT H1 haplotype associated with a decrease in association and motor cortices. A positive family history for cognitive problems was associated with a 16% decrease of tangle density in visual cortex. Tangle density correlated closely with (sub)cortical thickness in the CA1+subiculum, superior temporal, inferior parietal, and middle frontal cortex (Spearman Rho = ‐0.39 ‐ ‐0.58, p<0.001).ConclusionThese data contribute to the concept that brain regions may experience aging and genetics differentially, underlining the need to distinguish these aspects when investigating AD disease mechanisms.

AbstractBackgroundThe Uniform Data Set (UDS) neuropsychological battery, administered across Alzheimer’s Disease Centers (ADC), includes memory tests but lacks a list‐learning paradigm. ADCs often supplement the UDS with their own preferred list‐learning task. Given the importance of list‐learning for characterizing memory, we aimed to develop a harmonized memory score that incorporates UDS memory tests while allowing centers to contribute differing list‐learning tasks.MethodWe applied item‐banking confirmatory factor analysis to develop a composite memory score in 5,287 participants (mean age 67.1; SD = 12.2) recruited through 18 ADCs and four consortia (DiverseVCID, MarkVCID, ALLFTD, LEADS) who completed UDS memory tasks (used as linking‐items) and one of five list‐learning tasks. All analyses used linear regression. We tested whether memory scores were affected by which list‐learning task was administered. To assess construct validity, we tested associations of memory scores with demographics, disease severity (CDR Box Score), an independent memory task (TabCAT Favorites, n = 675), and hippocampal volume (n = 811). We compared performances between cognitively unimpaired (n = 279), AD‐biomarker+ MCI (n = 26), and AD‐biomarker+ dementia (n = 98). In a subsample with amyloid‐ and tau‐PET (n = 49), we compared memory scores from participants with positive vs negative scans determined using established quantitative cutoffs.ResultModel fit indices were excellent (e.g., CFI = 0.998) and factor loadings were strong (0.43‐0.93). Differences in list‐learning task had a negligible effect on scores (average Cohen’s d = 0.11). Higher memory scores were significantly (p’s<.001) correlated with younger age (β = ‐0.18), lower CDR Box Scores (β = ‐0.63), female sex (β = 0.12), higher education (β = 0.19), larger hippocampal volume (β = 0.42), and an independent memory task (β = 0.71, p<0.001). The memory composite declined in a stepwise fashion by diagnosis (cognitively unimpaired>MCI>AD dementia, p<0.001). On average, amyloid‐PET positivity was associated with lower composite scores, but was not statistically significant (β = ‐0.34; p = 0.25; d = 0.40). Tau‐PET positivity was associated with worse performance, demonstrating a large effect size (β = ‐0.75; p<0.002; d = 0.91).ConclusionThe harmonized memory score developed in a large national sample was stable regardless of contributing list‐learning task and its validity for cross‐cohort ADRD research is supported by expected associations with demographics, clinical measures, and Alzheimer’s biomarkers. A processing script will be made available to enhance cross‐cohort ADRD research.

AbstractBackgroundThe newly proposed criteria by the AA working group incorporates both biological and clinical stages to characterize the progression of AD. In this study, we aim to evaluate the agreement between these two complementary systems.MethodsUsing 188 participants from McGill TRIAD and 139 from the HEAD cohorts, we categorized participants into biological (0‐4) and clinical (0‐4) stages using amyloid PET, tau PET(MK‐6240), and clinical measures as described by the working group. Participants were then stratified into three categories: congruent (clinical = biological), higher in the biological stage (clinical < biological), and higher in the clinical stage (clinical > biological). In the TRIAD cohort, we further compared these groups for age, sex, years of education, vascular burden (WMH), microglial activation (PBR scan), Astrocyte reactivity (GFAP), amyloid load, tau load, and NPI‐Q scores using Welch two‐sided t‐test with FDR multiple comparison correction.ResultsIn the TRIAD cohort, 34% of participants were congruent, 24.5% had a higher clinical stage, and 41.5% had a higher biological stage. Meanwhile in the HEAD cohort 68.2% were congruent, 20.7% had a higher clinical stage, and 17% had a higher biological stage. TRIAD participants with a higher clinical stage had lower education (P = 0.02), more neuropsychiatric symptoms (P = 0.03), and a higher vascular burden (P = 0.03) (Table 1). As expected, people with higher biological stage had more amyloid(P<0.001), tau(P<0.001), and astrocyte reactivity(P<0.001) (Table 1, Figure 2).ConclusionsOur findings highlight important discordance between clinical and biological stages, which could be partially explained by cognitive reserve. This was supported by the protective effects of educational attainment in participants with a higher biological stage. Vascular burden played a major role in the cognitive impairment of individuals with higher clinical stages. Future studies should replicate these findings in larger more representative population‐based cohorts.