Kanekiyo, Takahisa

AbstractBackgroundThe immune system is substantially involved in the development and progression of age‐related cognitive decline and Alzheimer’s disease (AD).MethodAs genetic and environmental factors interactively impact these conditions, we investigated how risk factors such as APOE genotype, age, and sex influence immune activation markers and AD biomarkers in cerebrospinal fluid (CSF) in elderly individuals enrolled in the Mayo Clinic Study of Aging cohort. Among cognitively unimpaired individuals aged over 65 at the baseline visit (N=298), we measured 365 CSF immune activation markers using the proximity extension assay.ResultWe found that age, sex, and diabetes status are associated with altered CSF levels of immune activation markers independently of other factors. For CSF AD biomarkers, we observed significant positive correlations between age and total tau, phosphorylated tau‐181 (p‐tau181), neurofilament light (NfL), and YKL40. APOE4 was also associated with lower Aß42 and higher SNAP25 in CSF. We further examined whether baseline visit variables can predict cognitive decline, represented by the conversion from CDR=0 to CDR>0. We found that age, Aß42, NfL, and REG4 were independently correlated with CDR conversion risk. When the cohort was dichotomized by their median values, older participants with lower Aß42, higher NfL, and higher REG4 at baseline developed cognitive impairment during the follow up with a c‐index of 0.762 while age alone had a c‐index of 0.699.ConclusionTogether, our results suggest that assessing CSF immune activation markers and AD biomarkers can improve the prediction of cognitive impairment risk in the elderly.

AbstractBackgroundHomozygosity for the rare APOE3‐Christchurch (APOE3Ch) variant, encoding for apoE3‐R136S (apoE3‐Ch), was linked to resistance against an aggressive form of familial Alzheimer’s disease (AD). Carrying two copies of APOE3Ch was sufficient to delay autosomal AD onset by 30 years. This remarkable protective effect makes it a strong candidate for uncovering new therapies against AD. Thus, we aim to explore the protective mechanisms of APOE3Ch against AD onset, to inform therapy.MethodsWe used both amyloid mouse models and human induced‐pluripotent stem cells (iPSC) models to address this aim. We examined whether astrocytic expression of apoE3‐Ch through an AAV‐mediated approach can mitigate AD‐related pathology and toxicity in early (3.5 months old) and late (8 months old) amyloid pathology in 5xFAD mice. We also used isogenic iPSC lines carrying the APOE3Ch variant that were generated from APOE3 parental lines using CRISPR/Cas9 technology. Biochemical and biophysical properties of both recombinant and native apoE3‐Ch lipoprotein particles secreted by iPSC‐derived astrocytes have been investigated using different techniques including heparin affinity chromatography and size‐exclusion chromatography.ResultsWe investigated the impact of astrocytic apoE3‐Ch on amyloid pathology and toxicity in 3.5‐month‐old and 8‐month‐old 5xFAD mice. We confirmed successful AAV‐mediated overexpression of apoE3 and apoE3‐Ch in astrocytes. We found apoE3‐Ch specific changes in protein levels and solubility. Astrocytic expression of apoE3‐Ch reduced soluble Aβ oligomer levels and ameliorated ER stress response compared to apoE3 in the 8‐month‐old 5xFAD mice. Furthermore, our findings showed altered biochemical properties of HEK cell‐derived apoE3‐Ch protein, including decreased heparin binding, altered receptor binding, and enhanced lipid accepting capacity. We also characterized APOE3Ch iPSC‐derived cell models, including iPSC‐derived astrocytes, neurons, and cerebral organoids, and found differences between APOE3 and APOE3Ch at the functional level in these iPSC models.ConclusionsThe proposed work and the developed methods to study apoE3‐Ch promises to provide new insights into the possible roles of this rare, mutated protein in protecting against AD, offering new therapeutic avenues for AD treatment.

AbstractBackgroundAlzheimer’s disease (AD) is the most common cause of age‐related dementia, and the presence of amyloid‐β (Aβ) plaques and tau‐containing neurofibrillary tangles is associated with the neurodegeneration and cognitive impairment in this incurable disease. Growing evidence shows that epigenetic dysregulation through histone deacetylases (HDACs) plays a critical role in synaptic dysfunction and memory loss in AD, and HDACs have been highlighted as a novel class of anti‐Alzheimer targets. Moreover, restoring Wnt/β‐catenin signaling, which is greatly suppressed in AD brains, is a promising therapeutic strategy for AD. CI‐994 is an orally active class I HDAC inhibitor that has undergone several phase II/III clinical trials on cancer treatment. Importantly, CI‐994 can cross the blood‐brain barrier and is a cognitive enhancer.MethodWe evaluated the therapeutic potential of CI‐994 in human patient‐specific iPSC‐derived neurons and cerebral organoids with APOE ε4/ε4 genotype or carrying MAPT p.P301L mutation.ResultWe found that CI‐994 is not only a potent class I HDAC inhibitor but also a novel modulator of Wnt/β‐catenin signaling, and that activation of Wnt/β‐catenin signaling by CI‐994 is associated with elevated Wnt co‐receptor LRP6 protein level. Moreover, CI‐994 increased synaptic protein levels, enhanced spontaneous synaptic firing and network formation, and decreased tau phosphorylation in iPSC‐derived neurons. Finally, CI‐994 significantly increased histone acetylation, activated Wnt/β‐catenin signaling, and decreased Aβ production and tau phosphorylation in AD patient‐specific iPSC‐derived cerebral organoids.ConclusionOur findings indicate that CI‐994 is a novel potential therapeutic candidate for AD.

AbstractBackgroundProgressive supranuclear palsy (PSP) is a devastating primary tauopathy with rapid progression to death. Although several therapies currently in the development pipeline show promising safety profiles and robust target engagement, few demonstrated significant efficacy in patients, underscoring the need to interrogate additional targets with novel therapeutic modalities to expand the potential therapeutic arsenal. To diversify the therapeutic avenues for PSP and related tauopathies (e.g. Alzheimer’s disease), we systematically integrated multi‐omics data from human brains of PSP and control donors with cross‐species validation to nominate high‐confidence therapeutic targets. We plan to translate our findings into safe and effective treatments for PSP using antisense oligonucleotides (ASO).MethodWe analyzed brain gene expression profiles in PSP and control individuals at bulk tissue (N = 408) and single‐cell levels (snRNAseq, N = 36). Cell‐type‐specific expression perturbations were systematically prioritized using a cross‐species validation paradigm, including rTG4510 tau mice and tau‐overexpressing Drosophila models. We conducted in vitro screening of ASO candidates against our prioritized target genes. ASO efficacy and toxicity were measured. RNAseq experiments will be performed to interrogate off‐target effects. The safety and efficacy of the lead ASOs will be assessed in PSP‐patient‐derived iPSC models.ResultWe previously reported discovery of novel genes with significant differential expression in PSP brains, characterized their brain cell‐specificity and validated them using snRNAseq data. We prioritized 21 genes using gene expression data from a mouse tauopathy model. Validation of these 21 high‐priority genes using a Drosophila tau model nominated astrocytic STOM, KANK2, and DDR2 as potential therapeutic targets for PSP. Knocking down their expression in Drosophila significantly rescues tau‐mediated neurodegenerative pathology. In vitro screening identified ASO leads that reduced the target expressions at mRNA and protein levels with low cellular toxicity.ConclusionWe developed a systems biology pipeline and nominated STOM, KANK2, and DDR2 as candidate gene targets for PSP. We identified ASOs that modulate these targets’ expression without cellular toxicity, suggesting they may be suitable as potential therapeutic candidates. Importantly, the shared pathophysiology and molecular aberrations between PSP and other tauopathies, such as Alzheimer’s disease, suggests that such therapeutic candidates may be repurposed for multiple neurodegenerative diseases, further accelerating, and streamlining the therapeutic pipeline.

AbstractBackgroundAlzheimer’s disease (AD) risk and progression are significantly influenced by ApoE genotypes, with ApoE4 increasing and ApoE2 decreasing the susceptibility compared to ApoE3. Understanding metabolic pathways affected by ApoE genotypes will help decipher disease development and identify new therapeutic targets.MethodThis study investigates the impact of ApoE genotypes on aging brain metabolic trajectories using human ApoE‐targeted replacement mice. Applying Biocrates P180 targeted metabolomics platform, we analyzed the metabolic impact of ApoE2/2, ApoE3/3, and ApoE4/4 on fatty acid β‐oxidation, amino acids, and phospholipids, which are known to be altered in AD. Furthermore, we compared our rodent model results with human dorsolateral prefrontal cortex data from the Religious Orders Study/Memory and Aging Project (ROS‐MAP).ResultWe found aging mice carrying ApoE2/2 had altered branch‐chain amino acid metabolism and increased C5 acylcarnitine and its ratio to precursor isoleucine, pointing towards increased β‐oxidation and branched‐chain amino acid (BCAA) utilization. Furthermore, ROS‐MAP data revealed the ApoE2 genotype affects similar areas of metabolism in humans. Additionally, our data provide comprehensive insight into age‐related metabolic changes of the current mice model, independent of ApoE genotype, among phospholipids, sphingomyelins, amino acids, and biogenic amines. Many of the observed differences are also known aging markers in humans, with a connection to cognition and Alzheimer’s disease.ConclusionTogether, these results suggest a potential involvement of ApoE2/2 genotype in energy metabolism and characterize the current mice model for further study of ApoE in AD, brain aging, and brain BCAA utilization for energy.

AbstractBackgroundTauopathies are a group of neurodegenerative disorders which are characterized by the accumulation of abnormal tau protein in the brain. However, the mechanistic understanding of pathogenic tau formation and spread within the brain remains elusive. Astrocytes are major immune reactive cells in the brain and have been implicated in exacerbating tau pathology by releasing extracellular vesicles (AEVs) containing pro‐inflammatory cytokines and chemokines upon activation. Our prior investigation revealed a significant association between AEVs and tau pathology development, as well as cognitive function, by analyzing brain‐derived EV proteins from AD patients. In this study, we explore the potential roles of AEVs in tau pathogenesis using a human induced pluripotent stem cell (iPSC) model.MethodWe obtained two male P301L tau mutant iPSC lines from a Polish family with frontotemporal dementia. By including two male control lines, these iPSCs were differentiated into astrocytes (iAs) and characterized by immunocytochemistry and subjected for bulk RNAseq. Bioinformatics analysis was conducted to compare the transcriptome profile between wild‐type (WT) and P301L iAs. EVs from WT and P301L iAs were isolated by ultracentrifugation combined with size exclusion chromatography. Characterization of WT and P301L iAEVs involved nanoparticle tracking analysis, nano‐flow cytometry and super‐resolution microscopy.ResultWe successfully differentiated WT and P301L mutant iPSC lines into astrocytes with >99% purity. P301L iAs displayed distinctive astrocyte reactivity compared to WT cells, with elevated levels of pan‐reactive astrocyte genes (e.g., GFAP, CD44) and decreased expression of neuroprotective A2 astrocyte‐specific genes (e.g., TM4SF1, PTGS2). Additionally, gene enrichment set analysis of RNAseq data revealed dysregulation in the endo‐lysosomal pathway and extracellular matrix in P301L iAs compared to WT iAs. The count of intraluminal vesicles marked by CD9+ were reduced in P301L iAs compared to WT cells. Moreover, we observed a significant increase in the internalization of Tau by P301L iAs compared to WT iAs following incubation with preformed Tau fibrils, resulting in an augmented release of tau‐containing EVs from P301L iAs.ConclusionOur findings suggest a potential alteration in EV biogenesis in P301L iAs, potentially contributing to astrocyte‐mediated tau pathology. Future investigations will focus on understanding how AEVs contribute to tau propagation and accumulation.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disease and the most prevalent form of late‐life dementia. The ε2 allele of the APOE gene encoding apolipoprotein E (APOE2) is associated with lower susceptibility to AD among the three genotypes (ε2, ε3, ε4), while APOE4 is the strongest genetic risk factor for late‐onset AD. APOE plays a critical role in maintaining synaptic plasticity and neuronal function by controlling lipid homeostasis, with APOE2 having a superior function. Gene therapy that increases APOE2 levels in the brain has, therefore, emerged as a potential therapeutic strategy to treat AD.MethodWe conjugated PEGylated liposomes with transferrin and Penetratin, a cell‐penetrating peptide, sufficiently deliver chitosan‐APOE2 cDNA plasmid complex into amyloid model AppNL‐G‐F knockin mice at 12‐month‐old. Biochemical studies and brain transcriptome analysis were employed to investigate how brain‐targeting liposome‐based APOE2 gene delivery influences amyloid‐β (Aβ)‐related pathologies in AppNL‐G‐F knockin mice.ResultOne month after APOE2 gene therapy, there was a trend of reduced insoluble Aβ levels in the mouse cortices. Furthermore, in the AppNL‐G‐F knockin mice that received the APOE2 gene therapy, brain transcriptome analysis through RNA‐sequencing identified the upregulation of genes/pathways related to neuronal development. This was supported by increases of Dlg4 and Syp mRNAs coding synaptic proteins in the experimental group. On the other hand, we found that APOE2 gene delivery increased soluble Aβ levels, including oligomers, as well as exacerbated neurite dystrophy and decreased synaptophysin.ConclusionOur results suggest that brain‐targeting liposome‐based APOE2 gene therapy is potentially beneficial for synaptic formation at the transcriptional level. Forced APOE2 expressions, however, may exacerbate Aβ toxicity by increasing the dissociation of Aβ oligomers from aggregates in the presence of considerable amyloid burden.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm measures from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases was measured by reduced representation bisulfite sequencing (RRBS) from 471 TCX samples, 200 of which also had CER RRBS. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured previously by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged into CpGm clusters (rCpGm). rCpGms were tested for association with each AD endophenotype and expression levels of nearby genes through multi‐variable linear regression. Replication of significant rCpGms was performed in two independent datasets with AD‐related endophenotypes (Shireby 2022, De Jager 2014).ResultsOur innovative binning method demonstrated biologically relevant CpGm patterns. We found epigenome‐wide significant associations primarily with tau‐related endophenotypes including 93 that had significant and concordant associations in the replication datasets. These rCpGms also significantly associated with expression of nearby genes showing enrichment in oligodendrocyte marker genes including those related to myelination. In vitro validation of tau effects on oligodendrocyte gene expression through DNAm is ongoing.ConclusionsAlthough all endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. In particular, we found evidence of DNAm variability associating with brain oligodendrocyte gene expression and TCX tau levels. By discovering AD brain endophenotype‐specific DNAm changes, we can identify core components of complex mechanisms revealing important biological insights into AD pathophysiology.