Decreased expression of sirtuin 1 (SirT1) has been implicated in Alzheimer’s disease (AD), and as we previously reported, is related to transcriptional repression by the major risk factor for sporadic AD, apolipoprotein E4 (ApoE4). Herein we describe the discovery of an orally brain-permeable small-molecule, DDL-218, that enhanced SirT1 in ApoE4-expressing neuronal cells and a murine AD model. DDL-218 increased the transcription factor NFYb resulting in upregulation of PRMT5. Mechanistic and modeling studies show that binding of ApoE4 to the SirT1 gene promoter can be displaced by PRMT5 leading to increased SirT1 transcription. DDL-218 treatment elicited improvement in memory in the AD model, suggesting that DDL-218 enhancement of neurotrophic SirT1 in the brain has potential to modulate neuronal activity that may clinically provide an improvement in cognitive function and complement the current anti-Aβ antibody monotherapy. Our findings support further development of DDL-218 as a novel ApoE4-targeted therapeutic candidate for AD.

Alzheimer’s disease (AD) is characterized by amyloid plaques and tau tangles, with apolipoprotein E4 (ApoE4) recognized as a strong genetic risk factor for sporadic AD. ApoE4 has been shown to repress the expression of Sirtuin 1 (SirT1), a key neuroprotective protein, contributing to disease progression. This study describes the discovery and preclinical evaluation of DDL-218, a small-molecule SirT1 enhancer targeting ApoE4 to mitigate its repressive effects on SirT1. A high-throughput screening in N2a-apoE4 cell line identified SirT1 enhancer candidate compounds. Lead compounds were identified through medicinal chemistry and in silico modeling. In vitro testing in ApoE4-expressing neuronal cells and in vivo acute and chronic studies using ApoE4(TR):5xFAD transgenic AD mouse models were conducted. Protein interactions were examined through affinity purification, proteomics, and chromatin immunoprecipitation. Gene expression changes in SirT1, NYFB and PRMT5 were measured by qRT-PCR. Memory improvement was assessed using Barnes Maze test. Hippocampal tissue from DDL-218- and vehicle-treated mice underwent global proteomics and thermal proteome profiling (TPP) to identify differentially expressed proteins and elucidate the mechanism of action of DDL-218. DDL-218 significantly increased SirT1 protein and mRNA levels in neuronal cells by upregulating transcription factor NFYB and enzyme PRMT5. Drug treatment led to displacement of ApoE4 from the SirT1 promoter, allowing enhanced SirT1 expression. In AD model mice, DDL-218 treatment improved memory performance observed in the Barnes Maze test, and enhanced SirT1, NFYB, and PRMT5 mRNA in the brain. Proteomics revealed that DDL-218 upregulated proteins associated with neuronal function, including PTprn2, which plays a role in synaptic plasticity. Additionally, DDL-218 showed favorable brain penetration and no observable adverse effects. DDL-218 successfully enhanced SirT1 expression by counteracting ApoE4’s repressive effects, demonstrating potential as a therapeutic strategy for AD. DDL-218 improved memory and key neural pathways, showing promise in preclinical models. The observed cognitive improvements in the mouse model support further investigation of DDL-218 as a novel treatment for Alzheimer’s, targeting ApoE4-driven disease mechanisms. Future studies will establish protein targets of DDL-218 and clarify its mechanism in increasing NFYB mRNA levels. Additionally, we will evaluate its safety profile and efficacy in other ApoE4-expressing in vivo models.

Background The gene for apolipoprotein E4 (ApoE4 E4) confers an increased risk for development and lowers the age of onset of Alzheimer’s disease (AD), and is a highly suitable target for CRISPR-based editing because ApoE4 differs from ApoE3 by a single nucleotide polymorphism in the codon for residue 112 that codes for arginine (CGC) in E4 and cysteine (TGC) in E3. Editing of E4 to E3 could lower the risk of AD or ameliorate E4-related AD phenotypes. For AD, in order to deliver CRISPR components across the blood-brain barrier to the brain, we have developed a delivery platform termed ‘Synthetic Exosomes’ (SEs) – microfluidically-synthesized deformable nanovesicles approximately the size of natural exosomes that have the ability to cross the BBB and deliver cargo to the brain. Here, we describe our use of SEs carrying CRISPR to successfully edit E4 to E3 in brain tissue of an E4-expressing mouse model.Methods Several CRISPR guide RNAs (gRNA) and Cytosine Base Editor (CBE) mRNAs were synthesized by chemical and in vitro transcription syntheses, respectively. Four combinations of gRNA and CBE mRNA were tested in vitro for their relative activity to edit the E4 (cytosine) to E3 (thymine) in E4-expressing neuroblastoma (E4-N2A) and human ‘Kelly’ neuroblastoma cells, to assess which combination produced the highest E4 to E3 base editing efficiency. The CRISPR RNA combination with the highest efficiency was encapsulated in SEs and injected intravenously (IV) via the tail vein into an AD model E4-expressing (E4-5XFAD) transgenic mouse; as a negative control, an E4-5XFAD mouse was injected with empty SEs. Five days after injection, mice were euthanized and brain, liver, and buffy coat (white blood cells (WBC)) collected to determine the editing of E4 to E3 measured by Next Generation Sequencing. In addition, E3 mRNA was measured in the brain and liver and compared to the %E3 gene editing.Results The highest gRNA+CBE mRNA editing efficiency was ∼50% in E4-N2A cells and the same gRNA+CBE combination delivered in SEs to Kelly neuroblastoma cells showed 6.5% editing efficiency. In the E4-5XFAD mouse in vivo, five days after IV delivery of a single dose of the highest-activity SE-CRISPR gRNA+CBE mRNA, the percent of E4 edited to E3 was 0.14% in brain, 0.8% in liver, and 0.36% in WBCs. As evidence of functional editing, SE-CRISPR-treated mice had 0.03% E3 mRNA in brain and 0.09% E3 mRNA in liver.Conclusions While this level of ApoE4 to E3 editing achieved five days after a single IV injection of SE-CRISPR is small, it provides initial in vivo proof-of-concept that the ApoE4 gene can be successfully edited, and editing results in functional expression of ApoE3 mRNA. The findings presented herein supports further optimization of the SE-CRISPR approach to increase the level of editing in brain as part of clinical development of SE-CRISPR as a powerful novel therapeutic approach for AD.### Competing Interest StatementThe authors have declared no competing interest.

Fatigue and other deleterious mood alterations resulting from prolonged efforts such as a long work shift can lead to a decrease in vigilance and cognitive performance, increasing the likelihood of errors during the execution of attention-demanding activities such as piloting an aircraft or performing medical procedures. Thus, a method to rapidly and objectively assess the risk for such cognitive fatigue would be of value. The objective of the study was the identification in saliva-borne exosomes of molecular signals associated with changes in mood and fatigue that may increase the risk of reduced cognitive performance. Using integrated multiomics analysis of exosomes from the saliva of medical residents before and after a 12 h work shift, we observed changes in the abundances of several proteins and miRNAs that were associated with various mood states, and specifically fatigue, as determined by a Profile of Mood States questionnaire. The findings herein point to a promising protein biomarker, phosphoglycerate kinase 1 (PGK1), that was associated with fatigue and displayed changes in abundance in saliva, and we suggest a possible biological mechanism whereby the expression of the PGK1 gene is regulated by miR3185 in response to fatigue. Overall, these data suggest that multiomics analysis of salivary exosomes has merit for identifying novel biomarkers associated with changes in mood states and fatigue. The promising biomarker protein presents an opportunity for the development of a rapid saliva-based test for the assessment of these changes.

Alzheimer’s disease (AD) is the most common cause of dementia, yet there is no cure or diagnostics available prior to the onset of clinical symptoms. Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are released from almost all types of cell. Genome-wide association studies have linked multiple AD genetic risk factors to microglia-specific pathways. It is plausible that microglia-derived EVs may play a role in the progression of AD by contributing to the dissemination of insoluble pathogenic proteins, such as tau and Aβ. Despite the potential utility of EVs as a diagnostic tool, our knowledge of human brain EV subpopulations is limited. Here we present a method for isolating microglial CD11b-positive small EVs from cryopreserved human brain tissue, as well as an integrated multiomics analysis of microglial EVs enriched from the parietal cortex of four late-stage AD (Braak V-VI) and three age-matched normal/low pathology (NL) cases. This integrated analysis revealed 1,000 proteins, 594 lipids, and 105 miRNAs using shotgun proteomics, targeted lipidomics, and NanoString nCounter technology, respectively. The results showed a significant reduction in the abundance of homeostatic microglia markers P2RY12 and TMEM119, and increased levels of disease-associated microglia markers FTH1 and TREM2, in CD11b-positive EVs from AD brain compared to NL cases. Tau abundance was significantly higher in AD brain-derived microglial EVs. These changes were accompanied by the upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free cholesterol were elevated in microglial EVs from the AD brain. Lipidomic analysis also revealed a proinflammatory lipid profile, endolysosomal dysfunction, and a significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids, suggesting a potential defect in acyl-chain remodeling. Additionally, four miRNAs associated with immune and cellular senescence signaling pathways were significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may be accompanied by endolysosomal impairment and the release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. We suggest that the analysis of microglia-derived EVs has merit for identifying novel EV-associated biomarkers and providing a framework for future larger-scale multiomics studies on patient-derived cell-type-specific EVs.

Subcortical white matter ischemic lesions are increasingly recognized to have pathologic overlap in individuals with Alzheimer’s disease (AD). The interaction of white matter ischemic lesions with amyloid pathology seen in AD is poorly characterized. We designed a novel mouse model of subcortical white matter ischemic stroke and AD that can inform our understanding of the cellular and molecular mechanisms of mixed vascular and AD dementia. Subcortical white matter ischemic stroke underlying forelimb motor cortex was induced by local stereotactic injection of an irreversible eNOS inhibitor. Subcortical white matter ischemic stroke or sham procedures were performed on human ApoE4-targeted-replacement (TR):5XFAD mice at 8 weeks of age. Behavioral tests were done at 7, 10, 15, and 20 weeks. A subset of animals underwent 18 FDG-PET/CT. At 20 weeks of age, brain tissue was examined for amyloid plaque accumulation and cellular changes. Compared with sham E4-TR:5XFAD mice, those with an early subcortical ischemic stroke showed a significant reduction in amyloid plaque burden in the region of cortex overlying the subcortical stroke. Cognitive performance was improved in E4-TR:5XFAD mice with stroke compared with sham E4-TR:5XFAD animals. Iba-1+ microglial cells in the region of cortex overlying the subcortical stroke were increased in number and morphologic complexity compared with sham E4-TR:5XFAD mice, suggesting that amyloid clearance may be promoted by an interaction between activated microglia and cortical neurons in response to subcortical stroke. This novel approach to modeling mixed vascular and AD dementia provides a valuable tool for dissecting the molecular interactions between these two common pathologies.

AbstractBackgroundExosomes are small extracellular vesicles (EVs) originated from multivesicular endosomes which carry biochemical information about their cell‐of‐origin and signals for surrounding cells. EVs may also play a role in spread of proteopathic tau and amyloid beta seeds in Alzheimer’s disease (AD). Thus, brain exosomes may be a valuable therapeutic target for AD treatment. Ability of brain exosomes to cross the blood brain barrier make them a potential diagnostic and monitoring tool. Despite recent progress in development of EV isolation methods, our knowledge of human brain exosomal subpopulations and their roles in disease progression is very limited.MethodCryopreserved parietal cortex from 5 late stage AD (Braak V‐VI) and 3 control (NL) cases were used for the experiment. Brain exosomes/EVs were purified by sucrose density gradient ultracentrifugation after gentle enzymatic and mechanical dissociation of the brain tissue. Microglia‐derived EVs were isolated from the brain EV fraction by immunoprecipitation with anti‐CD11b antibodies. We performed analysis of miRNA transcriptome and proteome using NanoString and quantitative proteomics respectively.ResultWe identified 105 miRNAs which are present in more than 80% of analyzed human cases. The pathways controlled by five significantly upregulated miRNAs can be converged to control microglia activation (TGFβ, TLRs, and chemokine signaling pathways) and neuronal survival and function (LTD and LTP, NGF and neurotrophin signaling pathways). One of the upregulated exosomal miRNAs (miR‐188‐5p) is known to be downregulated in human AD brain and it can restore synaptic and cognitive deficits in 5xFAD mice. MiR‐381‐3p can promote recovery of spinal cord injury in rats. On the protein level we found a moderate increase (around 50%) in exosomal markers CD9 and CD81 and decrease in CHMP4B (ESCRT‐III) protein in the AD group. Levels of mitochondrial proteins and annexins were around 2 times higher in AD group compared to NL controls. ApoE protein was also significantly upregulated in AD microglial EVs.ConclusionOur data revealed the presence of anti‐inflammatory and neuroprotective miRNA signature in CD11b‐positive microglia‐derived exosomes. Proteomics data suggests upregulation of ESCRT‐independent pathway of exosome biogenesis in AD microglia. Increased association of microglial EVs with ApoE maybe related to amyloid packaging and release by exosome‐mediated mechanism.

A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

Neuroinflammation plays a crucial role in the development and progression of Alzheimer's disease (AD), in which activated microglia are found to be associated with neurodegeneration. However, there is limited evidence showing how neuroinflammation and activated microglia are directly linked to neurodegeneration in vivo. Besides, there are currently no effective anti-inflammatory drugs for AD. In this study, we report on an effective anti-inflammatory lipid, linoleic acid (LA) metabolite docosapentaenoic acid (DPAn-6) treatment of aged humanized EFAD mice with advanced AD pathology. We also report the associations of neuroinflammatory and/or activated microglial markers with neurodegeneration in vivo. First, we found that dietary LA reduced proinflammatory cytokines of IL1-β, IL-6, as well as mRNA expression of COX2 toward resolving neuroinflammation with an increase of IL-10 in adult AD models E3FAD and E4FAD mice. Brain fatty acid assays showed a five to six-fold increase in DPAn-6 by dietary LA, especially more in E4FAD mice, when compared to standard diet. Thus, we tested DPAn-6 in aged E4FAD mice. After DPAn-6 was administered to the E4FAD mice by oral gavage for three weeks, we found that DPAn-6 reduced microgliosis and mRNA expressions of inflammatory, microglial, and caspase markers. Further, DPAn-6 increased mRNA expressions of ADCYAP1, VGF, and neuronal pentraxin 2 in parallel, all of which were inversely correlated with inflammatory and microglial markers. Finally, both LA and DPAn-6 directly reduced mRNA expression of COX2 in amyloid-beta42 oligomer-challenged BV2 microglial cells. Together, these data indicated that DPAn-6 modulated neuroinflammatory responses toward resolution and improvement of neurodegeneration in the late stages of AD models.

Around 95% of Alzheimer's disease (AD) cases are sporadic AD that occurs through the interaction of genetic and environmental risk factors. APOE4 genetic risk for AD is modulated by dietary fatty acids such that saturated and trans-unsaturated fat increase AD risk, while monounsaturated and polyunsaturated fatty acids (MUFA, PUFA) reduce AD risk. Among PUFAs, omega-3 (n-3) fatty acids like docosohexaenoic acid (DHA) have been extensively studied and associate with positive effects in humans and AD models, but the role of n-6 fatty acids like linoleic acid (LA) is largely considered to be negative and pathogenic because the arachidonic acid is a substrate for cyclooxygenase 2 (COX2) production of prostaglandins that cause inflammation. However, epidemiological studies report that n-6 LA enriched diets are associated with less memory decline in human cohorts. Therefore, we investigated the impact of n-6 LA or a candidate protective metabolite for protective mechanisms in AD models. n-6 LA-enriched diet or its long chain metabolite docosapentaenoic acid (DPAn-6) were tested in three experiments in AD models, including aged Tg2576 mice, young and aged E4FAD (APOE4 with 5FAD) mice. The aged E4FAD mice develop advanced AD pathology including amyloid deposits, neurodegeneration and behavioral deficits. We found that n-6 LA-enriched diet or oral DPAn-6 boosted adaptive and innate immune function. These include promoting the production of anti-Aβ autoantibodies and microglia phagocytosis of amyloid-beta (Aβ) along with reduced Aβ deposits, limited brain CT4+ T cell invasion and improved cognitive deficits in AD models. N-6 LA-enriched diet or DPAn-6 also inhibited expression of cyclooxygenase-2 (COX2) that is implicated in inflammation and 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGCR) that is a rate-limiting enzyme for cholesterol synthesis, a target of statins. Furthermore, DPAn-6 also increased brain hippocampal volume in E4FAD mice. We speculate that the n-6 LA-enriched diet's beneficial effects might be through DPAn-6. We found n-6 Fatty Acids may be a novel Immunotherapeutic approach for preventing or treating AD through multiple targets including boosting immune function while inhibiting neuroinflammation and expression of two key enzymes with strong epidemiological evidence for risk reduction, the NSAID target COX-2 and the statin target HMGCR.

Innate immune genes including TREM2 control AD risk; their transcriptional dysregulation revealed a TREM2 centered hub. Shared microglial genes altered in models for aging, APP/ plaques, and tau/ tauopathy (Kang et al 2018), including Clec7a, Itgax, Ccl3, Ccl4, Cst7, Lilrb4 and others, strongly overlapped an ApoE-TREM2 pathway that drives a miR-155 stimulated transcriptional neurodegenerative microglial phenotype for dysfunctional Clec7a+ “bad” microglia (Krasemann et al 2017). An alternative disease restricting, phagocytic, amyloid-resolving and TREM2 centered, plaque-associated ‘DAM” microglia phenotype has also been proposed (Keren-Shaul et al 2017) which was induced by low dose curcumin in AD models. We tested the impact of an immunomodulatory transcriptional regulator, curcumin, on cortical microglial gene expression in amyloid laden 5xFAD mice expressing human ApoE3 or ApoE4 (EFAD mice, LaDu). Mice were treated from 11 to 13 months with 500 ppm curcumin and cortical gene expression was analyzed using RNAseq. Curcumin intervention suppressed the neurodegenerative microglial transcriptome in the E3FAD mice including suppression of Clec7a and the listed and other signature “neurodegenerative” microglial genes but not homeostatic genes like TMEM119. This correction was not seen with the ApoE4 carriers. Real time qPCR proved a robust reduction of miR-155 with curcumin intervention in the aging E3FAD but not E4FAD mice. Conventional markers of neuroinflammation including GFAP message and protein were selectively reduced by treatment only in E3FAD. Principle component analysis demonstrated sex-dependent clustering, but no clear sex-dependent treatment effect. Baseline RNA expression in the untreated EFAD mice revealed many significant differences in inflammatory gene expression defining an altered ApoE4-related milieu that provides context and candidate explanations for this differential response to treatment. Our studies indicate that low dose oral curcumin can favor a potential disease restricting good DAM microglia phenotype while reducing traditional pro-inflammatory cytokines. However, the success of curcumin in suppressing the microglial neurodegenerative phenotype and its driver miR-155 with late intervention in E3FAD was blunted by ApoE4. Potential ApoE4 pharmacogenomic interactions with other immunodulatory prevention or treatment approaches are likely to occur and impact immunomodulatory clinical trial outcomes, a prospect that we are currently examining.

Alzheimer's disease (AD) genetics implies a causal role for innate immune genes, TREM2 and CD33, products that oppose each other in the downstream Syk tyrosine kinase pathway, activating microglial phagocytosis of amyloid (Aβ). We report effects of low (Curc-lo) and high (Curc-hi) doses of curcumin on neuroinflammation in APPsw transgenic mice. Results showed that Curc-lo decreased CD33 and increased TREM2 expression (predicted to decrease AD risk) and also increased TyroBP, which controls a neuroinflammatory gene network implicated in AD as well as phagocytosis markers CD68 and Arg1. Curc-lo coordinately restored tightly correlated relationships between these genes' expression levels, and decreased expression of genes characteristic of toxic pro-inflammatory M1 microglia (CD11b, iNOS, COX-2, IL1β). In contrast, very high dose curcumin did not show these effects, failed to clear amyloid plaques, and dysregulated gene expression relationships. Curc-lo stimulated microglial migration to and phagocytosis of amyloid plaques both in vivo and in ex vivo assays of sections of human AD brain and of mouse brain. Curcumin also reduced levels of miR-155, a micro-RNA reported to drive a neurodegenerative microglial phenotype. In conditions without amyloid (human microglial cells in vitro, aged wild-type mice), Curc-lo similarly decreased CD33 and increased TREM2. Like curcumin, anti-Aβ antibody (also reported to engage the Syk pathway, increase CD68, and decrease amyloid burden in human and mouse brain) increased TREM2 in APPsw mice and decreased amyloid in human AD sections ex vivo. We conclude that curcumin is an immunomodulatory treatment capable of emulating anti-Aβ vaccine in stimulating phagocytic clearance of amyloid by reducing CD33 and increasing TREM2 and TyroBP, while restoring neuroinflammatory networks implicated in neurodegenerative diseases.

Alzheimer’s disease (AD) and mixed dementia (MxD) comprise the majority of dementia cases in the growing global aging population. MxD describes the coexistence of AD pathology with vascular pathology, including cerebral small vessel disease (SVD). Cardiovascular disease increases risk for AD and MxD, but mechanistic synergisms between the coexisting pathologies affecting dementia risk, progression and the ultimate clinical manifestations remain elusive. To explore the additive or synergistic interactions between AD and chronic hypertension, we developed a rat model of MxD, produced by breeding APPswe/PS1ΔE9 transgenes into the stroke-prone spontaneously hypertensive rat (SHRSP) background, resulting in the SHRSP/FAD model and three control groups (FAD, SHRSP and non-hypertensive WKY rats, n = 8–11, both sexes, 16–18 months of age). After behavioral testing, rats were euthanized, and tissue assessed for vascular, neuroinflammatory and AD pathology. Hypertension was preserved in the SHRSP/FAD cross. Results showed that SHRSP increased FAD-dependent neuroinflammation (microglia and astrocytes) and tau pathology, but plaque pathology changes were subtle, including fewer plaques with compact cores and slightly reduced plaque burden. Evidence for vascular pathology included a change in the distribution of astrocytic end-foot protein aquaporin-4, normally distributed in microvessels, but in SHRSP/FAD rats largely dissociated from vessels, appearing disorganized or redistributed into neuropil. Other evidence of SVD-like pathology included increased collagen IV staining in cerebral vessels and PECAM1 levels. We identified a plasma biomarker in SHRSP/FAD rats that was the only group to show increased Aqp-4 in plasma exosomes. Evidence of neuron damage in SHRSP/FAD rats included increased caspase-cleaved actin, loss of myelin and reduced calbindin staining in neurons. Further, there were mitochondrial deficits specific to SHRSP/FAD, notably the loss of complex II, accompanying FAD-dependent loss of mitochondrial complex I. Cognitive deficits exhibited by FAD rats were not exacerbated by the introduction of the SHRSP phenotype, nor was the hyperactivity phenotype associated with SHRSP altered by the FAD transgene. This novel rat model of MxD, encompassing an amyloidogenic transgene with a hypertensive phenotype, exhibits several features associated with human vascular or “mixed” dementia and may be a useful tool in delineating the pathophysiology of MxD and development of therapeutics.

The apolipoprotein E (apoE) isotype apoE4 can increase systemic and central inflammation, independent of amyloid accumulation. ApoE4 dysregulates innate immune toll-like receptor (TLR) signaling in AD by modulating expression of is major regulator, miR146a, a microRNA enriched in the brain. We examined the effect of apoE isotype and age on brain levels of miR146a, its epigenetic expression regulation by histone acetylation (Hac), and the effect of exercise. ApoE3 and apoE4 targeted replacement mice with and without mutant familial AD (5xFAD) transgenes (E-FAD), at 6 month-old and 13 month-old, were exercised with running wheels for the last 10 weeks, and were compared to sedentary controls. In young sedentary mice, apoE4 greatly reduced levels of miR146a compared to apoE3, both in the brain (29%; p<0.0001) and plasma (47%; p<0.05), which correlated with each other (r2=0.74; p<0.05). The presence of 5xFAD transgenes increased brain miR146a in both E3-FAD and E4-FAD young mice; however, miR146a levels in E4-FAD mice remained lower than in E3-FAD mice (62%; p<0.05), despite increased amyloid and inflammation. ApoE4 brains showed increased expression of interleukin receptor associated kinase-1, IRAK1 (that mediates TLR signaling and is normally downregulated by miR146)(160%; p<0.05); IRAK1 mRNA inversely correlated with miR146a levels (r2=0.637; p<0.0001). ApoE4 is known to induce histone deacetylases which could reduce Hac at the miR146a gene, explaining the lower levels of miR146a. In old sedentary mice, 146a levels were not induced by FAD transgenes, possibly reflecting the known age changes in Hac at the miR146a gene. Exercise in a small number of young mice increased miR146a in apoE4 mice (209%; p<0.05) which could be caused by the known induction of Hac by exercise. ApoE4 dysregulated negative feedback of TLR signaling by lower miRNA146a can explain early-life hypersensitivity to innate immune stimuli (including Aβ) in apoE4 carriers, and may be relevant to ApoE4 AD risk. This is a candidate nexus of interaction with factors including exercise, age and Aß pathology.

Synaptic neurodegeneration is thought to be an early event initiated by soluble β-amyloid (Aβ) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aβ aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aβ oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7–8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4+/+/FAD+/−) relative to E4FAD- (non-carrier; APOE4+/+/FAD−/−) mice, suggesting NP1 is modulated by Aβ expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD.

Synaptic neurodegeneration is thought to be an early event initiated by soluble β-amyloid (Aβ) aggregates that closely correlates with cognitive decline in Alzheimer disease (AD). Apolipoprotein ε4 (APOE4) is the most common genetic risk factor for both familial AD (FAD) and sporadic AD; it accelerates Aβ aggregation and selectively impairs glutamate receptor function and synaptic plasticity. However, its molecular mechanisms remain elusive and these synaptic deficits are difficult to monitor. AD- and APOE4-dependent plasma biomarkers have been proposed, but synapse-related plasma biomarkers are lacking. We evaluated neuronal pentraxin 1 (NP1), a potential CNS-derived plasma biomarker of excitatory synaptic pathology. NP1 is preferentially expressed in brain and involved in glutamate receptor internalization. NP1 is secreted presynaptically induced by Aβ oligomers, and implicated in excitatory synaptic and mitochondrial deficits. Levels of NP1 and its fragments were increased in a correlated fashion in both brain and plasma of 7-8 month-old E4FAD mice relative to E3FAD mice. NP1 was also found in exosome preparations and reduced by dietary DHA supplementation. Plasma NP1 was higher in E4FAD+ (APOE4+/+/FAD+/-) relative to E4FAD- (non-carrier; APOE4+/+/FAD-/-) mice, suggesting NP1 is modulated by Aβ expression. Finally, relative to normal elderly, plasma NP1 was also elevated in patients with mild cognitive impairment (MCI) and elevated further in the subset who progressed to early-stage AD. In those patients, there was a trend towards increased NP1 levels in APOE4 carriers relative to non-carriers. These findings indicate that NP1 may represent a potential synapse-derived plasma biomarker relevant to early alterations in excitatory synapses in MCI and early-stage AD.