Welcome to the AD/PD™ 2022 Interactive Program
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ALZHEIMER’S MOUSE BRAIN NEURAL PRECURSORS FAIL TO SUPPORT MICROGLIAL-MEDIATED AMYLOID-BETA CLEARANCE
Abstract
Aims
Microglia remove Amyloid beta, but fail to reduce Amyloid burden in Alzheimer’s disease (AD). We previously demonstrated loss of immune-modulatory properties of resident AD brain neural precursor cells (NPC). We hypothesized that AD-brain NPC fail in supporting microglial-mediated Amyloid beta clearance, enabling its accumulation.
Methods
CD11b+ microglia were extracted from 7 months old 5xFAD mice and activated with LPS to mimic AD brain inflammatory environment. Microglial Amyloid-beta content was measured by image analysis. NPC were isolated from newborn wild type (wt) brain, and from transgenic mice expressing GFP under the Nestin promoter, crossed with 5xFAD and wt littermates.
Results
Double-staining for Iba1+ microglia and Amyloid beta in 7-months old 5xFAD brain sections, and feeding freshly isolated CD11b+ brain cells with fluorescent-labeled Amyloid beta, demonstrated a microglial subpopulation exhibiting high Amyloid-beta phagocytic activity. LPS activation increased latex beads uptake but reduced the fraction of high Amyloid-phagocytic microglia. Time-lapse microscopy showed that co-culturing with newborn NPC did not affect the total amount of Amyloid-beta uptake by microglia. Rather, NPC significantly increased the fraction of microglia with high Amyloid-phagocytic activity. NPC-enriched Subventricular zone extracts, and freshly isolated NPC from wt 7-months old mice, induced significant increase in the fraction of microglia with high Amyloid-phagocytic activity. SVZ extracts and freshly-isolated NPC from 5xFAD mice failed to increase high Amyloid-phagocytic microglial fraction. Finally, wt NPC induced a mildly stronger effect than 5xFAD NPC on the degradation of phagocytosed Amyloid-beta in microglia.
Conclusions
Resident AD brain NPC fail to support Amyloid beta clearance by microglia, leading to accelerated disease pathogenesis.
THE BRAIN-SPECIFIC SIGNAL PEPTIDE PEPTIDASE-LIKE 2B (SPPL2B): A NOVEL THERAPEUTIC TARGET TO PREVENT AND COUNTERACT NEUROINFLAMMATION AND AMYLOID-Β PATHOLOGY IN ALZHEIMER'S DISEASE.
Abstract
Aims
Alzheimer´s disease (AD) is an age-related disease characterized by the accumulation of amyloid β-peptide (Aβ) in plaques and neurofibrillary tangles. Identifying new mechanisms and proteins involved in AD pathogenesis is a crucial step to find new therapeutic targets. The intramembrane enzyme Signal Peptide Peptidase Like 2b (SPPL2b) is involved in the proteolysis of two AD-related proteins: TNF-alpha and BRI2 involved in the inflammatory response and Aβ production respectively. Here we have investigated for the first time the expression levels and the pathogenic role of SPPL2b in AD.
Methods
The role of SPPL2b in Aβ metabolism was evaluated in vitro by using human cell lines SH-SY5Y and HEK293 stably expressing APP, in acute brain slices from WT mice, in brains of a new APP knock-in AD mouse model (AppNL-G-F) and humans post mortem AD brain tissues.
Results
APP over-expression in SH-SY5Y cells increased the levels of SPPL2b, which orrelated with a reducion of BRI2. In addition, a decrise in Aβ40 production was observed in SH-SY5Y cells when treated with a SPPLs enzyme inhibitor. In brain slices-maintained ex vivo, Aβ42 exposure induced a strong up-regulation of SPPL2b. Increased levels of SPPL2b were also observed in the cortex of AD human brains. Interestingly, similar results were obtained in 3 months old AppNL-G-F mice. Furthermore, immunofluorescence staining showed that SPPL2b is in microglia deposited in the amyloid plaque, indicating a direct role of SPPL2b in Aβ plaques
Conclusions
These results strongly support the involvement of SPPL2b in AD pathology. Most important, Aβ42 seems directly involved in SPPL2b expression.
HIGH-DIMENSIONAL IMMUNE PROFILING OF ALZHEIMER’S DISEASE AND DEMENTIA WITH LEWY BODIES
Abstract
Aims
Alzheimer’s disease (AD) and Lewy body dementia (DLB) are the most frequent types of dementia in the elderly. Recent evidence suggests a crucial role of peripheral immune cells in both diseases, however, underlying mechanisms remain unclear. We here aimed to comprehensively map systemic immune changes in both dementias using high-dimensional single-cell characterization of the immune system.
Methods
Peripheral blood mononuclear cells (PBMCs) were isolated from 166 subjects of which 38 controls (age 61.3y±4.9; 44.7% female), 42 mild-cognitive impaired (MCI; age 65.8y±6.6; 45.2% female), 59 AD (age 64.0y±4.6; 52.5% female) and 27 DLB (age 69.9y±5.0; 18.5% female) patients from the Amsterdam Dementia Cohort. Singe-cell protein expression was measured with cytometry by time-of-flight (CyTOF) and data was analyzed using unsupervised clustering algorithms.
Results
The fraction of CD4+ T cells declined among PBMCs derived from MCI, AD and DLB patients compared to controls. AD patients had higher levels of circulating B cells while DLB patients had more circulating CD14+ classical monocytes. Circulating CD8+ T cells were higher in MCI patients and specifically a subset of CD8+ effector memory T cells re-expressing CD45RA (termed TEMRA) increased almost 2-fold in both MCI and AD patients.
Conclusions
We demonstrate immune alterations in AD and DLB patients, including the accumulation of TEMRA CD8+ T cells in MCI and AD and of classical monocytes in DLB patients. These findings highlight the role of innate and adaptive immunity in both dementias and signify the demand for a greater understanding of how these cells contribute to underlying brain pathology in AD and DLB.
SIALIC ACID-DRIVEN UNTIMELY IMMUNE AGING IN OBESITY-ALZHEIMER’S DISEASE COMORBIDITY ACCELERATES COGNITIVE DECLINE
Abstract
Aims
Mid-life obesity is an established risk factor for Alzheimer’s disease (AD), and systemic immune dysfunction is an important common player in both morbidities. We investigated whether obesity exacerbates AD, and if so, whether this is linked to metabolite-induced systemic immune dysfunction.
Methods
We fed 2-month-old 5xFAD mice, a model of AD-like amyloidosis, with a high-fat obesogenic diet for 24-28 weeks. Cognitive state was longitudinally assessed via novel object recognition task. We used flow cytometry and cytometry by time-of-flight to analyze the immune landscape in the spleen, and histo-/immunohistochemistry and ELISA to examine brain pathology. To identify the molecular signatures specific to the comorbid state as compared to AD or obesity alone, we performed untargeted metabolomic analysis on plasma, and single-nucleus RNA sequencing of the hippocampus and visceral adipose tissue.
Results
We found that high-fat diet accelerated cognitive decline in 5xFAD mice, and that this was associated with untimely immune aging phenotypes, including increased splenic frequencies of FOXP3+ Tregs and exhausted T cells. Non-targeted plasma metabolomics revealed that N-acetylneuraminic acid (NANA), the predominant sialic acid, was specifically elevated in obese-AD comorbid mice, and was inversely correlated with cognitive performance and positively correlated with splenic Tregs. Single-nucleus RNA-sequencing identified macrophages in the visceral fat as one potential source of NANA. The potential ability of NANA to induce immune exhaustion was confirmed both in vivo in middle-aged wild-type mice, and in human T cells exposed to NANA.
Conclusions
Our study identified sialic acid-driven immune dysfunction as the missing link between AD and obesity, and potentially other AD-predisposing conditions.
REGIONAL ASSOCIATIONS BETWEEN WHITE MATTER HYPERINTENSITIES (WMH) AND AMYLOID PATHOLOGY IN PRECLINICAL ALZHEIMER’S DISEASE
Abstract
Aims
To assess the regional relationship between WMH and amyloid burden in a non-demented population, and evaluate their joint contribution to cognitive impairment.
Methods
We included 662 non-demented participants from three AMYPAD-affiliated cohorts: EPAD-LCS(N=176), ALFA+(N=310), and EMIF-AD-Twin60++(N=176). With PET imaging, we assessed cortical amyloid burden regionally - within early-accumulating regions (medial-orbitofrontal, precuneus, and cuneus) - and globally, using the Centiloid method. Regional WMH volume was computed using Bayesian Model Selection (BaMoS). Global associations between WMH, amyloid, and cardiovascular risk-scores (Framingham and CAIDE) were assessed with linear models. Partial least square (PLS) regression was used to identify regional associations. Models were adjusted for age, sex, and APOE-e4 status. Individual PLS scores were then related to cognitive performance in 4 domains (attention, memory, executive-functioning, language) in EMIF-AD only, due to data availability.
Results
While no significant global association was found, the PLS model yielded two components of interest, demonstrating regional associations between amyloid and WMH (Figure 1). In the first component (C1), a fronto-parietal pattern of WMH was associated with frontal and parietal amyloid burden, higher vascular risk scores, and older age. The second component (C2) showed posterior WMH, mainly in the occipital lobe, associated with higher amyloid deposition in parieto-occipital areas, lower age and cardiovascular risk scores. C1 was associated with all cognitive domains and C2 only with memory and attention ( Figure 2).
Conclusions
In a large pre-dementia population, we observed two distinct patterns of regional associations between presumed cerebrovascular pathology and amyloid burden, and demonstrated their joint influence on cognitive processes.
THE ALZHEIMER SUSCEPTIBILITY GENE BIN1 INDUCES ISOFORM-DEPENDENT NEUROTOXICITY THROUGH EARLY ENDOSOME DEFECTS
Abstract
Aims
BIN1 is the most associated susceptibility gene for AD after APOE. The contribution of BIN1 and its isoforms to AD pathogenesis remains unclear. The objective of this work was to assess the neurotoxicity of BIN1 isoforms and understand the underlying mechanisms.
Methods
We used Drosophila to assess in vivo human BIN1 isoforms role on neuronal survival and endolysosomal trafficking. We also generated BIN1 knock-out human induced neurons (hiNs) and cerebral organoids to study BIN1 isoforms in models closer to AD pathology.
Results
We observed in Drosophila that human brain BIN1 isoform1 (BIN1iso1) overexpression, contrary to human muscular BIN1 isoform8 (BIN1iso8) and human ubiquituous BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level was responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs
Conclusions
Our results show that a tight regulation of BIN1iso1 is required in neurons for the endo-lysosomal pathway and neuron survival. Interestingly, increasing evidence point toward a dysregulation of the endo-lysosomal pathway as a potential cause of AD. Our results support a role of BIN1 in these mechanisms and therefore propose a role for BIN1 in AD pathogenesis.
ROLE OF MIDKINE AND PLEIOTROPHIN IN AMYLOID PATHOLOGY – FRIENDS OR FOES
Abstract
Aims
Amyloid cascade hypothesis is long considered a framework for understanding AD pathogenesis and the development of disease modifying therapeutics. However, the relationship between accumulation of aggregated Aβ and neurodegeneration is not fully understood. A number of amyloid associated proteins (AAPs) accumulate in the brain as Aβ deposits. We hypothesize that Aβ aggregation may not be sufficiently toxic to induce downstream neurodegeneration unless accompanied by AAP accumulation.
Methods
Our studies leverage on data from the AMP-AD initiative that has used proteomics to identify candidate AAPs that are increased in AD. Two AAPs’, Midkine (MDK) and Pleiotrophin (PTN) were cloned and packaged in AAV and delivered to newborn CRND8 mice. Amyloid burden and Aβ levels in SDS soluble and SDS-insoluble, Formic Acid soluble fractions were compared between various cohorts.
Results
Anti-MDK and anti-PTN antibodies label Aβ-amyloid structures in TgCRND8 brains. Moreover, both PTN and MDK bound to fibrillar Aβ. Overexpression of both PTN and MDK resulted in significant increase of Amyloid burden and CAA in the mouse brain. Biochemical analysis of frozen hemibrains suggests that both Aβ42 and Aβ40 levels are increased in the MDK and PTN overexpressing cohort.
Conclusions
These studies provide important insights into the biology of AAP in AD pathogenesis. We have shown that overexpression of MDK and PTN can modify Aβ deposition in a mouse model of AD. Moreover, is likely that basic mechanisms of AAP binding to amyloid also apply to proteins implicated in peripheral amyloidosis which makes these studies important in the broad field of neurodegenerative disorders.