Aβ Uptake Research Articles

Clinical application of sparse canonical correlation analysis to detect genetic associations with cortical thickness in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cerebral cortex atrophy. In this study, we used sparse canonical correlation analysis (SCCA) to identify associations between single nucleotide polymorphisms (SNPs) and cortical thickness in the Korean population. We also investigated the role of the SNPs in neurological outcomes, including neurodegeneration and cognitive dysfunction. We recruited 1125 Korean participants who underwent neuropsychological testing, brain magnetic resonance imaging, positron emission tomography, and microarray genotyping. We performed group-wise SCCA in Aβ negative (-) and Aβ positive (+) groups. In addition, we performed mediation, expression quantitative trait loci, and pathway analyses to determine the functional role of the SNPs. We identified SNPs related to cortical thickness using SCCA in Aβ negative and positive groups and identified SNPs that improve the prediction performance of cognitive impairments. Among them, rs9270580 was associated with cortical thickness by mediating Aβ uptake, and three SNPs (rs2271920, rs6859, rs9270580) were associated with the regulation of CHRNA2, NECTIN2, and HLA genes. Our findings suggest that SNPs potentially contribute to cortical thickness in AD, which in turn leads to worse clinical outcomes. Our findings contribute to the understanding of the genetic architecture underlying cortical atrophy and its relationship with AD.

Loss of Insulin Signaling in Microglia Impairs Cellular Uptake of Aβ and Neuroinflammatory Response Exacerbating Alzheimer-like Neuropathology.

Insulin receptors are present on cells throughout the body, including the brain. Dysregulation of insulin signaling in neurons and astrocytes has been implicated in altered mood, cognition, and the pathogenesis of Alzheimers disease (AD). To define the role of insulin signaling in microglia, the primary phagocytes in brain critical for maintenance and damage repair, we created mice with an inducible microglia-specific insulin receptor knockout (MG-IRKO). RiboTag profiling of microglial mRNAs revealed that loss of insulin signaling results in alterations of gene expression in pathways related to innate immunity and cellular metabolism. In vitro, loss of insulin signaling in microglia results in metabolic reprograming with an increase in glycolysis and impaired uptake of Aβ. In vivo, MG-IRKO mice exhibit alterations in mood and social behavior, and when crossed with the 5xFAD mouse model of AD, the resultant mice exhibit increased levels of Aβ; plaque and elevated neuroinflammation. Thus, insulin signaling in microglia plays a key role in microglial cellular metabolism, neuroinflammation and the ability of the cells to take up Aβ; such that reduced insulin signaling in microglia alters mood and social behavior and accelerates AD pathogenesis. Together these data indicate key roles of insulin action in microglia and the potential of targeting insulin signaling in microglia in treatment of AD.

Chemiexcitation-Triggered Photosensitizer Activation for Photooxidation of Aβ1-42 Aggregates.

Oxidative degradation of the pathogenic amyloid-β-peptide (Aβ) aggregation is an effective and promising method to treat Alzheimer's disease under light irradiation. However, the limited penetration of external light sources into deep tissues has hindered the development of this treatment. Therefore, we have designed an unprecedented chemiluminescence-initiated photodynamic therapy system to replace external laser irradiation, primarily composed of d-glucose-based polyoxalate (G-poly(oxalate)), the novel photosensitizer (BD-Se-QM), and bis [2,4,5-trichloro-6-(pentoxy-carbonyl) phenyl] ester. BD-Se-QM possesses excellent singlet oxygen (1O2) generation efficiency and the ability to photooxidize Aβ1-42 aggregates under white light. G-poly(oxalate) not only helps the nanosystem to cross the blood-brain barrier but also has sufficient oxalate ester groups to significantly enhance the efficiency of chemiluminescence resonance energy transfer. The oxalate ester groups in BD-Se-QM/NPs can chemically react with H2O2 to produce high-energy intermediates that activate BD-Se-QM, which can generate 1O2 to inhibit Aβ1-42 aggregates and also promote microglial uptake of Aβ1-42, reducing the Aβ1-42-induced neurotoxicity. The chemically stimulated nanoplatform not only solves the drug delivery problem but also eliminates the need for external light sources. We anticipate that this chemically excited nanosystem could also be used for targeted delivery of other small molecule drugs.

Single-Cell RNA Sequencing Reveals Immunomodulatory Effects of Stem Cell Factor and Granulocyte Colony-Stimulating Factor Treatment in the Brains of Aged APP/PS1 Mice.

Alzheimer's disease (AD) leads to progressive neurodegeneration and dementia. AD primarily affects older adults with neuropathological changes including amyloid-beta (Aβ) deposition, neuroinflammation, and neurodegeneration. We have previously demonstrated that systemic treatment with combined stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) reduces the Aβ load, increases Aβ uptake by activated microglia and macrophages, reduces neuroinflammation, and restores dendrites and synapses in the brains of aged APPswe/PS1dE9 (APP/PS1) mice. However, the mechanisms underlying SCF+G-CSF-enhanced brain repair in aged APP/PS1 mice remain unclear. This study used a transcriptomic approach to identify the potential mechanisms by which SCF+G-CSF treatment modulates microglia and peripheral myeloid cells to mitigate AD pathology in the aged brain. After injections of SCF+G-CSF for 5 consecutive days, single-cell RNA sequencing was performed on CD11b+ cells isolated from the brains of 28-month-old APP/PS1 mice. The vast majority of cell clusters aligned with transcriptional profiles of microglia in various activation states. However, SCF+G-CSF treatment dramatically increased a cell population showing upregulation of marker genes related to peripheral myeloid cells. Flow cytometry data also revealed an SCF+G-CSF-induced increase of cerebral CD45high/CD11b+ active phagocytes. SCF+G-CSF treatment robustly increased the transcription of genes implicated in immune cell activation, including gene sets that regulate inflammatory processes and cell migration. The expression of S100a8 and S100a9 was robustly enhanced following SCF+G-CSF treatment in all CD11b+ cell clusters. Moreover, the topmost genes differentially expressed with SCF+G-CSF treatment were largely upregulated in S100a8/9-positive cells, suggesting a well-conserved transcriptional profile related to SCF+G-CSF treatment in resident and peripherally derived CD11b+ immune cells. This S100a8/9-associated transcriptional profile contained notable genes related to pro-inflammatory and anti-inflammatory responses, neuroprotection, and Aβ plaque inhibition or clearance. Altogether, this study reveals the immunomodulatory effects of SCF+G-CSF treatment in the aged brain with AD pathology, which will guide future studies to further uncover the therapeutic mechanisms.

Inhibition of Sphingosine Kinase 1 Reduces Sphingosine-1-Phosphate and Exacerbates Amyloid-Beta-Induced Neuronal Cell Death in Mixed-Glial-Cell Culture.

In Alzheimer's disease (AD) pathology, the accumulation of amyloid-beta (Aβ), a main component of senile plaques, activates glial cells and causes neuroinflammation. Excessive neuroinflammation results in neuronal dropouts and finally produces the symptoms of AD. Recent studies suggest that disorder in sphingosine-1-phosphate (S1P) metabolism, especially the decreased expression of sphingosine kinase (SK)1, followed by the reduction in the amount of S1P, can be a promotive factor in AD onset. Thus, we explored the possibility that dysregulated S1P metabolism affects AD through the altered function in glial cells. We evaluated the effect of PF-543, a pharmacological inhibitor of SK1, on the inflammatory responses by lipopolysaccharide (LPS)-activated glial cells, microglia, and astrocytes. The treatment with PF-543 decreased the intracellular S1P content in glial cells. The PF-543 treatment enhanced the nitric oxide (NO) production in the LPS-treated neuron/glia mixed culture. Furthermore, we found that the augmented production of NO and reactive oxygen species (ROS) in the PF-543-treated astrocytes affected the microglial inflammatory responses through humoral factors in the experiment using an astrocyte-conditioned medium. The PF-543 treatment also decreased the microglial Aβ uptake and increased the number of injured neurons in the Aβ-treated neuron/glia mixed culture. These results suggest that a decrease in the glial S1P content can exacerbate neuroinflammation and neurodegeneration through altered glial cell functions.

Relationships between PET and blood plasma biomarkers in corticobasal syndrome.

Corticobasal syndrome (CBS) can result from underlying Alzheimer's disease (AD) pathologies. Little is known about the utility of blood plasma metrics to predict positron emission tomography (PET) biomarker-confirmed AD in CBS. A cohort of eighteen CBS patients (8amyloid beta [Aβ]+; 10Aβ-) and 8 cognitively unimpaired (CU) individuals underwent PET imaging and plasma analysis. Plasma concentrations were compared using a Kruskal-Wallis test. Spearman correlations assessed relationships between plasma concentrations and PET uptake. CBS Aβ+ group showed a reduced Aβ42/40 ratio, with elevated phosphorylated tau (p-tau)181, glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) concentrations, while CBS Aβ- group only showed elevated NfL concentration compared to CU. Both p-tau181 and GFAP were able to differentiate CBS Aβ- from CBS Aβ+ and showed positive associations with Aβ and tau PET uptake. This study supports use of plasma p-tau181 and GFAP to detect AD in CBS. NfL shows potential as a non-specific disease biomarker of CBS regardless of underlying pathology. Plasma phosphorylated tau (p-tau)181 and glial fibrillary acidic protein (GFAP) concentrations differentiate corticobasal syndrome (CBS) amyloid beta (Aβ)- from CBS Aβ+. Plasma neurofilament light concentrations are elevated in CBS Aβ- and Aβ+ compared to controls. Plasma p-tau181 and GFAP concentrations were associated with Aβ and tau positron emission tomography (PET) uptake. Aβ42/40 ratio showed a negative correlation with Aβ PET uptake.

Distinct effects of blood pressure parameters on Alzheimer’s and vascular markers in 1,952 Asian individuals without dementia

BackgroundRisk factors for cardiovascular disease, including elevated blood pressure, are known to increase risk of Alzheimer’s disease. There has been increasing awareness of the relationship between long-term blood pressure (BP) patterns and their effects on the brain. We aimed to investigate the association of repeated BP measurements with Alzheimer’s and vascular disease markers.MethodsWe recruited 1,952 participants without dementia between August 2015 and February 2022. During serial clinic visits, we assessed both systolic BP (SBP) and diastolic BP (DBP), and visit-to-visit BP variability (BPV) was quantified from repeated measurements. In order to investigate the relationship of mean SBP (or DBP) with Alzheimer’s and vascular markers and cognition, we performed multiple linear and logistic regression analyses after controlling for potential confounders (Model 1). Next, we investigated the relationship of with variation of SBP (or DBP) with the aforementioned variables by adding it into Model 1 (Model 2). In addition, mediation analyses were conducted to determine mediation effects of Alzheimer’s and vascular makers on the relationship between BP parameters and cognitive impairment.ResultsHigh Aβ uptake was associated with greater mean SBP (β = 1.049, 95% confidence interval 1.016–1.083). High vascular burden was positively associated with mean SBP (odds ratio = 1.293, 95% CI 1.015–1.647) and mean DBP (1.390, 1.098–1.757). High tau uptake was related to greater systolic BPV (0.094, 0.001–0.187) and diastolic BPV (0.096, 0.007–0.184). High Aβ uptake partially mediated the relationship between mean SBP and the Mini-Mental State Examination (MMSE) scores. Hippocampal atrophy mediated the relationship between diastolic BPV and MMSE scores.ConclusionsEach BP parameter affects Alzheimer’s and vascular disease markers differently, which in turn leads to cognitive impairment. Therefore, it is necessary to appropriately control specific BP parameters to prevent the development of dementia. Furthermore, a better understanding of pathways from specific BP parameters to cognitive impairments might enable us to select the managements targeting the specific BP parameters to prevent dementia effectively.

Intermittent hypoxia training enhances Aβ endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer’s disease

BackgroundBeta-amyloid (Aβ) deposition in the brain parenchyma is a crucial initiating step in the amyloid cascade hypothesis of Alzheimer’s disease (AD) pathology. Furthermore, dysfunction of plaque-associated microglia, also known as disease-associated microglia (DAM) has been reported to accelerate Aβ deposition and cognitive impairment. Our previous research demonstrated that intermittent hypoxia training (IHT) improved AD pathology by upregulating autophagy in DAM, thereby enhancing oligomeric Aβ (oAβ) clearance. Considering that oAβ internalization is the initial stage of oAβ clearance, this study focused on the IHT mechanism involved in upregulating Aβ uptake by DAM.MethodsIHT was administered to 8-month-old APP/PS1 mice or 6-month-old microglial vacuolar protein sorting 35 (VPS35) knockout mice in APP/PS1 background (MG VPS35 KO: APP/PS1) for 28 days. After the IHT, the spatial learning-memory capacity of the mice was assessed. Additionally, AD pathology was determined by estimating the nerve fiber and synapse density, Aβ plaque deposition, and Aβ load in the brain. A model of Aβ-exposed microglia was constructed and treated with IHT to explore the related mechanism. Finally, triggering receptor expressed on myeloid cells 2 (TREM2) intracellular recycling and Aβ internalization were measured using a fluorescence tracing technique.ResultsOur results showed that IHT ameliorated cognitive function and Aβ pathology. In particular, IHT enhanced Aβ endocytosis by augmenting the intracellular transport function of microglial TREM2, thereby contributing to Aβ clearance. Furthermore, IHT specifically upregulated VPS35 in DAM, the primary cause for the enhanced intracellular recycling of TREM2. IHT lost ameliorative effect on Aβ pathology in MG VPS35 KO: APP/PS1 mice brain. Lastly, the IHT mechanism of VPS35 upregulation in DAM was mediated by the transcriptional regulation of VPS35 by transcription factor EB (TFEB).ConclusionIHT enhances Aβ endocytosis in DAM by upregulating VPS35-dependent TREM2 recycling, thereby facilitating oAβ clearance and mitigation of Aβ pathology. Moreover, the transcriptional regulation of VPS35 by TFEB demonstrates a close link between endocytosis and autophagy in microglia. Our study further elucidates the IHT mechanism in improving AD pathology and provides evidence supporting the potential application of IHT as a complementary therapy for AD.Graphical abstract

Understanding the Liver’s Role in the Clearance of Aβ40

The clearance of peripheral beta amyloid (Aβ) is a potential target for the treatment of Alzheimer’s disease (AD). The liver has been implicated in the elimination of Aβ from the peripheral circulation. Here, the single-pass uptake of Aβ40 in perfused livers from young and old rats (6 to 10 rats per group) was investigated with the multiple indicator dilution technique. Aβ40 had volumes of distribution between those of the vascular marker Evans Blue and the extracellular marker sucrose. The hepatic extraction of Aβ40 was negligible, explained in part by the small permeability surface area products consistent with a high endothelial barrier to liver uptake. There were no substantial effects of age on any of these results. In vitro experiments with isolated hepatocytes and liver sinusoidal endothelial cells showed only very small amounts of Aβ uptake consistent with low intrinsic clearance. These results indicate that the hepatic clearance of Aβ is capacity-limited, explained by the low-permeability surface area products and hepatocyte uptake. However, this does not preclude an effect of aging in longer-term in vivo studies where age-related changes in liver blood flow and protein binding influence liver clearance.

Bacteroidota inhibit microglia clearance of amyloid-beta and promote plaque deposition in Alzheimer’s disease mouse models

The gut microbiota and microglia play critical roles in Alzheimer’s disease (AD), and elevated Bacteroides is correlated with cerebrospinal fluid amyloid-β (Aβ) and tau levels in AD. We hypothesize that Bacteroides contributes to AD by modulating microglia. Here we show that administering Bacteroides fragilis to APP/PS1-21 mice increases Aβ plaques in females, modulates cortical amyloid processing gene expression, and down regulates phagocytosis and protein degradation microglial gene expression. We further show that administering Bacteroides fragilis to aged wild-type male and female mice suppresses microglial uptake of Aβ1-42 injected into the hippocampus. Depleting murine Bacteroidota with metronidazole decreases amyloid load in aged 5xFAD mice, and activates microglial pathways related to phagocytosis, cytokine signaling, and lysosomal degradation. Taken together, our study demonstrates that members of the Bacteroidota phylum contribute to AD pathogenesis by suppressing microglia phagocytic function, which leads to impaired Aβ clearance and accumulation of amyloid plaques.

Association of Basal Forebrain Volume with Amyloid, Tau, and Cognition in Alzheimer's Disease.

Degeneration of cholinergic basal forebrain (BF) neurons characterizes Alzheimer's disease (AD). However, what role the BF plays in the dynamics of AD pathophysiology has not been investigated precisely. To investigate the baseline and longitudinal roles of BF along with core neuropathologies in AD. In this retrospective cohort study, we enrolled 113 subjects (38 amyloid [Aβ]-negative cognitively unimpaired, 6 Aβ-positive cognitively unimpaired, 39 with prodromal AD, and 30 with AD dementia) who performed brain MRI for BF volume and cortical thickness, 18F-florbetaben PET for Aβ, 18F-flortaucipir PET for tau, and detailed cognitive testing longitudinally. We investigated the baseline and longitudinal association of BF volume with Aβ and tau standardized uptake value ratio and cognition. Cross-sectionally, lower BF volume was not independently associated with higher cortical Aβ, but it was associated with tau burden. Tau burden in the orbitofrontal, insular, lateral temporal, inferior temporo-occipital, and anterior cingulate cortices were associated with progressive BF atrophy. Lower BF volume was associated with faster Aβ accumulation, mainly in the prefrontal, anterior temporal, cingulate, and medial occipital cortices. BF volume was associated with progressive decline in language and memory functions regardless of baseline Aβ and tau burden. Tau deposition affected progressive BF atrophy, which in turn accelerated amyloid deposition, leading to a vicious cycle. Also, lower baseline BF volume independently predicted deterioration in cognitive function.

Peripheral amyloid-β clearance mediates cognitive impairment in non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a prevalent risk factor for cognitive impairment. Cerebral amyloid-β (Aβ) accumulation, as an important pathology of cognitive impairment, can be caused by impaired Aβ clearance in the periphery. The liver is the primary organ for peripheral Aβ clearance, but the role of peripheral Aβ clearance in NAFLD-induced cognitive impairment remains unclear. MethodsWe examined correlations between NAFLD severity, Aβ accumulation, and cognitive performance in female Sprague-Dawley rats. The impact of NAFLD on hepatic Aβ clearance and the involvement of low-density lipoprotein receptor-related protein 1 (LRP-1) were assessed in rat livers and cultured hepatocytes. Additionally, a case-control study, including 549 NAFLD cases and 549 controls (782 males, 316 females), investigated the interaction between NAFLD and LRP-1 rs1799986 polymorphism on plasma Aβ levels. FindingsThe severity of hepatic steatosis and dysfunction closely correlated with plasma and cerebral Aβ accumulations and cognitive deficits in rats. The rats with NAFLD manifested diminished levels of LRP-1 and Aβ in liver tissue, with these reductions inversely proportional to plasma and cerebral Aβ concentrations and cognitive performance. In vitro, exposure of HepG2 cells to palmitic acid inhibited LRP-1 expression and Aβ uptake, which was subsequently reversed by a peroxisome proliferator-activated receptor α (PPARα) agonist. The case-control study revealed NAFLD to be associated with an increment of 8.24% and 10.51% in plasma Aβ40 and Aβ42 levels, respectively (both P <0.0001). Moreover, the positive associations between NAFLD and plasma Aβ40 and Aβ42 levels were modified by the LRP-1 rs1799986 polymorphism (P for interaction = 0.0017 and 0.0015, respectively). InterpretationLRP-1 mediates the adverse effect of NAFLD on peripheral Aβ clearance, thereby contributing to cerebral Aβ accumulation and cognitive impairment in NAFLD. FundingMajor International (Regional) Joint Research Project, National Key Research and Development Program of China, National Natural Science Foundation of China, and the Angel Nutrition Research Fund.

HuM195 and its single-chain variable fragment increase Aβ phagocytosis in microglia via elimination of CD33 inhibitory signaling.

CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of β-amyloid 42 (Aβ42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aβ42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.

Association of Glycemic Variability With Imaging Markers of Vascular Burden, β-Amyloid, Brain Atrophy, and Cognitive Impairment.

We aimed to investigate the association between glycemic variability (GV) and neuroimaging markers of white matter hyperintensities (WMH), beta-amyloid (Aβ), brain atrophy, and cognitive impairment. This was a retrospective cohort study that included participants without dementia from a memory clinic. They all had Aβ PET, brain MRI, and standardized neuropsychological tests and had fasting glucose (FG) levels tested more than twice during the study period. We defined GV as the intraindividual visit-to-visit variability in FG levels. Multivariable linear regression and logistic regression were used to identify whether GV was associated with the presence of severe WMH and Aβ uptake with DM, mean FG levels, age, sex, hypertension, and presence of APOE4 allele as covariates. Mediation analyses were used to investigate the mediating effect of WMH and Aβ uptake on the relationship between GV and brain atrophy and cognition. Among the 688 participants, the mean age was 72.2 years, and the proportion of female participants was 51.9%. Increase in GV was predictive of the presence of severe WMH (coefficient [95% CI] 1.032 [1.012-1.054]; p = 0.002) and increased Aβ uptake (1.005 [1.001-1.008]; p = 0.007). Both WMH and increased Aβ uptake partially mediated the relationship between GV and frontal-executive dysfunction (GV → WMH → frontal-executive; direct effect, -0.319 [-0.557 to -0.080]; indirect effect, -0.050 [-0.091 to -0.008]) and memory dysfunction (GV → Aβ → memory; direct effect, -0.182 [-0.338 to -0.026]; indirect effect, -0.067 [-0.119 to -0.015]), respectively. In addition, increased Aβ uptake completely mediated the relationship between GV and hippocampal volume (indirect effect, -1.091 [-2.078 to -0.103]) and partially mediated the relationship between GV and parietal thickness (direct effect, -0.00101 [-0.00185 to -0.00016]; indirect effect, -0.00016 [-0.00032 to -0.000002]). Our findings suggest that increased GV is related to vascular and Alzheimer risk factors and neurodegenerative markers, which in turn leads to subsequent cognitive impairment. Furthermore, GV can be considered a potentially modifiable risk factor for dementia prevention.

Pathogenic characteristics of genetically modified cells with TREM2 T44A mutation

AbstractBackgroundTriggering Receptor Expressed on Myeloid Cells‐2 (TREM2) is known as a risk gene for Alzheimer’s disease (AD). and affect AD pathogenesis through beta‐amyloid (Aβ)‐related neuroinflammations and phagocytosis. TREM2 R47H mutation was the first reported from an AD patient in 2013. Although the TREM2 R47H variant was revealed to increase the risk of AD by three times, further mutations still need to study. Here, we found TREM2 T44A mutation from a patient of a 65‐year‐old AD patient. In order to confirm the pathogenicity of TREM2 T44A, the genetically modified cells were produced and investigated the association of the TREM2 gene with Aβ‐related pathology.MethodTREM2 Wild type (WT) and T44A were transfected onto the HEK 293, SHSY5Y, U87 cells by using a plasmid transfection system and selected using geneticin (G418). Gene expression and TREM2 protein was measured. For measuring Aβ uptakes, Aβ was measured after incubation with cells. Cell phagocytosis was conducted with treating pHrodo labeled with Aβ and obtained cell images and video.ResultTREM2 gene was overexpressed in transfected cells and TREM2 protein was elevated, but TREM2 T44A lowered compared to WT. TREM2 T44A showed higher Aβ42/40 ratio, with increasing extracellular Aβ42. The Aβ42 uptake were relatively lowered than TREM2 WT. Through images and video, TREM2 T44A showed lower red fluorescence than WT, but similar to MOCK.ConclusionIn this study, we transfected TREM2 WT and T44A in a variety of cell lines (HEK 293, SHSY5Y, U87) to investigate the pathogenicity of TREM2 T44A. TREM2 T44A was shown to increase extracellular Aβ42 and decrease Aβ42 uptake. This suggests that the Aβ uptake and elimination function of TREM2 was loosed by the mutation, and therefore Aβ was not completely removed, resulting in a slight increase in extracellular Aβ in the mutant cell. The loss of the Aβ uptake correlated with confocal and video results, the red fluorescence signals were decreased in TREM2 T44A, suggesting relatively lower Aβ phagocytosis. Thus, Aβ phagocytosis was inhibited by TREM2 mutation, and the lowered Aβ uptake could not clear it, suggesting that Aβ growth and accumulation may influence the pathogenicity of AD.

Distinct effects of cholesterol profile components on amyloid and vascular burdens

AbstractBackgroundCholesterol plays important roles in β‐amyloid (Aβ) metabolism and atherosclerosis. However, the relationships of plasma cholesterol levels with Aβ and cerebral small vessel disease (CSVD) burdens are not fully understood in Asians. Herein, we investigated the relationships between plasma cholesterol profile components and Aβ and CSVD burdens in a large, non‐demented Korean cohort.MethodWe enrolled 1,175 non‐demented participants (456 with unimpaired cognition [CU] and 719 with mild cognitive impairment [MCI]) aged ≥ 45 years who underwent Aβ PET at the Samsung Medical Center in Korea. We performed linear regression analyses with each cholesterol (low‐density lipoprotein cholesterol [LDL‐c], high‐density lipoprotein cholesterol [HDL‐c], and triglyceride) level as a predictor and each image marker (Aβ uptake on PET, white matter hyperintensity [WMH] volume, and hippocampal volume) as an outcome after controlling for potential confounders.ResultIncreased LDL‐c levels (β = 0.014 to 0.115, p = 0.013) were associated with greater Aβ uptake, independent of the APOE e4 allele genotype and lipid‐lowering medication. Decreased HDL‐c levels (β = −0.133 to −0.006, p = 0.032) were predictive of higher WMH volumes. Increased LDL‐c levels were also associated with decreased hippocampal volume (direct effect β = −0.053, p = 0.040), which was partially mediated by Aβ uptake (indirect effect β = −0.018, p = 0.006).ConclusionOur findings highlight that increased LDL‐c and decreased HDL‐c levels are important risk factors for Aβ and CSVD burdens, respectively. Furthermore, considering that plasma cholesterol profile components are potentially modified by diet, exercise, and pharmacological agents, our results provide evidence that regulating LDL‐c and HDL‐c levels is a potential strategy to prevent dementia.

Phospholipase D3 drives microglial response to amyloid pathology in Alzheimer’s disease

AbstractBackgroundAmyloid‐beta (Aβ) plaque formation is a well‐established hallmark for Alzheimer’s disease (AD). However, the processes behind plaque formation are not understood. Previous work from our group identified rare coding variants for PLD3, with one variant in particular, p.A442A, disrupting a splicing enhancer binding site within the mRNA transcript. In this study, we present findings that PLD3 regulates Aβ clearance in the brain through both cell autonomous and non‐autonomous means.MethodTo replicate the splicing effect observed in patient brain samples in vitro, PLD3 p.A442A expressing induced pluripotent stem cells (iPSCs) were differentiated into neurons or microglia and evaluated for extracellular Aβ and Aβ uptake, respectively. In an APP/PSEN1xPld3KO mouse model, Aβ dynamics were assessed with in vivo microdialysis and plaque pathology was assessed with immunohistochemistry. Frozen cortical tissue from these mice was analyzed through bulk RNAseq to identify transcriptomic and molecular changes that are attributed to Pld3 deficiency. PLD3 expression was assessed in an independent single‐cell RNAseq (scRNAseq) dataset from iPSC‐derived microglia to assess a potential cell‐autonomous role for PLD3 in microglia.ResultTranscriptomics from human samples reveal a significant decrease of PLD3 expression in AD brains versus controls, particularly in the brain regions most susceptible to amyloid pathology. Similar to PLD3‐variant brains, iPSC‐derived neurons carrying PLD3 p.A442A exhibited alternative splicing and displayed elevated Aβ levels in the conditioned media. Loss of Pld3 in APP/PSEN1 mice led to increased interstitial levels of Aβ and reduced Aβ clearance. Immunohistochemistry revealed an increase in diffuse, nonfibrillar plaque area and reduced microglial recruitment in APP/PSEN1xPld3KO. Transcriptomics data from brain tissue in these mice revealed altered expression of genes that define the disease associated microglia signatures. To begin to understand the role of PLD3 in microglia, we analyzed scRNAseq from CRISPRi screen in iPSC‐derived microglia. We found that PLD3 expression is enriched in interferon‐associated microglia. Finally, we demonstrate that iPSC‐derived microglia carrying PLD3 p.A442A exhibit reduced internalization of Aβ and myelin, suggesting a defect in phagocytic capacity.ConclusionTogether, we show that PLD3 plays a critical role in the brain by facilitating the microglial response to Aβ plaque pathology, modulating plaque composition.

Uncovering the Role of an ABCA7 Frameshift Deletion in African American Alzheimer’s Disease Cellular Pathology

AbstractBackgroundThe ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene is a risk factor for Alzheimer’s disease (AD). While ABCA7 has been implicated as a genetic determinant of AD across populations, the risk effect is the strongest in African Americans (AAs). We previously identified a common 44 base pair deletion in ABCA7 (p.Arg578Alafs) that is predicted to truncate the protein and is significantly associated with AD in AAs (frequency in cases = 15.2%, cognitively unimpaired (CU) = 9.74%, p = 1.41×10−5). Clinically, deletion homozygotes are similar to deletion heterozygotes, supporting dominant gain‐of‐function as a putative disease‐causing mechanism. We sought to determine if the deletion transcript is stable and if the truncated protein is expressed as steps to understanding the mechanism leading to AD risk.MethodRT‐PCR analysis of heterozygotes was conducted to assess the stability of the deletion transcript. FLAG‐tagged Arg578Alafs and wildtype ABCA7 vectors were constructed then overexpressed in HEK293APPsw cells. Western blot analysis of FLAG‐Arg578Alafs was performed to determine if the deletion produces a truncated protein. Induced pluripotent stem cell (iPSC) lines from six AA individuals with AD bearing the Arg578Alafs mutation (three heterozygous; three homozygous) were created in pair with isogenic CRISPR‐corrected control lines. These iPSC lines were validated for pluripotency, genomic stability, and lack of off‐target editing. The isogenic ABCA7 deletion and CRISPR‐corrected control iPSC lines will be differentiated into microglia and neurons and functionally assessed for cell type‐specific AD phenotypes.ResultInitial results comparing AD patients to CU individuals show that patient‐derived microglia have normal rates of phagocytosis but are impaired in the uptake and clearance of fibrillar Aβ. RT‐PCR analysis of the deletion in heterozygotes demonstrates that a stable RNA transcript is expressed from the ABCA7 deletion allele. Western blot analysis of overexpressed FLAG‐Arg578Alafs shows that the truncated protein is expressed, albeit at low levels compared to the wildtype protein.ConclusionThis ABCA7 deletion produces a stable transcript which appears to be translated into low levels of protein. This supports a potential gain of function as the disease risk mechanism. Studies in isogenic lines will further elucidate the functional effects of this ABCA7 deletion on AD pathology in AAs.

Cerebellar‐cortical functional connectivity is associated with Alzheimer’s disease pathology and cerebellar atrophy

AbstractBackgroundBrain atrophy has been associated with Alzheimer’s disease (AD) pathology. Previous research also suggests that functional connectivity (FC) among distributed neural networks is disrupted in preclinical AD. The cerebellum has been thought to play a crucial role in motor control, yet recent evidence points to its additional role in non‐motor domains as well as cognitive decline in AD. Cerebellar volume has been shown to decline along the course of AD progression. Moreover, neuroimaging studies have delineated intrinsic cerebellar‐cortical functional networks that presumably support cognitive functions. Despite the accumulating findings on neurodegeneration or FC, how cerebellar FC and atrophy relate to AD pathology in the early stage has not been extensively investigated.MethodData from 83 cognitively normal older adults (mean 71.0 yrs, 66% females) in the Alzheimer’s Disease Neuroimaging Initiative were analyzed. Volumetric data were obtained from FreeSurfer 6.0 segmentation. Resting state fMRI scans were preprocessed using the CONN Toolbox. Aβ and tau standardized uptake value ratio (SUVR) data were obtained from [18F]‐Florbetapir or [18F]‐Florbetaben (Aβ) and [18F]‐Flortaucipir (tau) PET scans. For Aβ, SUVR values were transformed to the Centiloid scale, and a global summary measure was used. For tau, SUVR values in the temporal meta regions of interest were used. Seed‐based functional connectivity metrics were computed using the 7 cerebellar parcellations (Buckner et al., 2011) that are functionally coupled to different cortical networks (Yeo et al., 2011).ResultCerebellar volume showed no significant relationship with amyloid or tau SUVR. By contrast, hippocampal atrophy was positively associated with amyloid or tau SUVR and showed trending significance (ps &lt; 0.1). Increased Aβ burden was associated with increased cerebellar‐cortical FC in the attention, frontoparietal, and default mode networks. Increased tau load was associated with increased cerebellar‐cortical FC in the visual, somatomotor, and attention networks. Cerebellar volume was associated with cerebellar‐cortical FC in the visual, somatomotor, attention networks, but not with the limbic, frontoparietal, or default mode networks.ConclusionOur findings suggest that functional cerebellar‐cortical connectivity varies with amyloid and tau pathology in different networks, and such functional changes may precede cerebellar atrophy in preclinical AD.

Anti‐inflammatory clearance of amyloid beta by a chimeric Gas6 fusion protein

AbstractBackgroundAβ immunotherapy is the most promising approach in treating Alzheimer’s disease (AD). Although Aβ‐antibodies can substantially reduce Aβ burden in the brain, solutions for antibody‐induced inflammation and subsequent ARIA (Amyloid‐related imaging abnormalities) are still in demand. Here we developed a novel chimeric protein platform, GAIA (Gas6‐mediated anti‐inflammatory adaptor). GAIA platform utilizes TAM receptors, which are expressed on glial cells and mediate phagocytosis without associated inflammatory response.MethodWe developed a GAIA‐based novel chimeric protein (αAβ‐Gas6) which is composed of two distinct functional domains; a single‐chain variable fragment of Aducanumab, which selectively binds to Aβ, fused with Gas6, a TAM receptor‐binding domain. Aβ clearance and phagocytosis‐associated inflammatory responses of αAβ‐Gas6 were measured in comparison with Aducanumab.ResultαAβ‐Gas6 showed selective uptake of Aβ through phagocytosis in vitro. While Aducanumab induced phagocytosis only in primary microglia, αAβ‐Gas6 significantly induced phagocytosis in both cultured microglia and astrocyte. In comparison with Aducanumab, αAβ‐Gas6 secreted significantly lower amount of pro‐inflammatory cytokines, such as TNF, IL‐6, and IL‐1β. In vivo application of αAβ‐Gas6 reduced Aβ burden in an extent significantly lower in mice treated with Aducanumab. Finally, αAβ‐Gas6 groups showed significantly reduced excessive synapse elimination levels comparable to non‐AD wild type groups.ConclusionWe developed a novel chimeric fusion protein αAβ‐Gas6 by engineering TAM receptor‐binding domain Gas6. In comparison with antibody drug, αAβ‐Gas6 showed advanced amyloid clearance, significantly reduced neuroinflammation, and neurotoxicity. Thus, our results suggest a novel immunotherapeutic agent which can bifunctionally target Aβ and modulate gilal cells to reduce neuroinflammation.