Abstract Body

INTRODUCTION: Lipid dyshomeostasis impacts age-related neurodegenerative disorders. Dysfunction of human APOE and lysosomal glucocerebrosidase (GBA1) is co-dependent with age of onset and cognitive decline in PD and LBD. Exploring functional lipid interactions between human APOE, GBA1 and aging sheds light on the pathogenesis of AD and LBD/PDD.

IN VIVO RESULTS: In vivo GBA1 inhibition as animal models of PD/LBD results in α-synucleinopathy, neuroinflammation, and lipid fluctuations in neurons, microglia, and astrocytes (Rocha et al., 2015; Brekk et al., 2020). Experiments using GBA1-inhibited young (2-3 month) (Connolly et al 2023), and old (16-18 month) WT or APOE-KO mice revealed both compensatory and potential pathogenic glial and inflammatory changes in the brain. There were pronounced microglial and astrocytic morphological and biochemical responses, intensified by the APOE-KO and aging, especially in the hippocampus and cerebral cortex.

IN VITRO RESULTS: To investigate cholesterol transport and lipid exchange in human APOE2, 3, and 4 variants, a lipid stress system was developed. Using human fibroblasts or iPSC-derived neurons and astrocytes (APOE3/3 and APOE4/4), APOE isoform regulation of neuron-glia mechanisms under glycolipid, cholesterol, and inflammation stress was determined. The results show superior lipid and cholesterol transport capacities of APOE2 and 3, compared to APOE4. Synthetic APOE4 also failed to protect cells under lipid accumulation. APOE4 human neurons released higher A-beta 1-42 and 1-40 peptides than APOE3 neurons. Pharmacological reversal of APOE4's lipid transport limitations improved cell survival.

CONCLUSIONS: Human APOE4 carries less cholesterol and lipids. When this is corrected, the APOE4 variant functions similarly to APOE2 and 3 and reduces cell death. In AD, PD, LBD, and aging of the brain, the GBA1-APOE lipid biological networks are potentially critical to these pathologies.

Aims

The apolipoprotein E (ApoE) Ɛ4 allele is associated with a significant risk for both late-onset Alzheimer’s Disease (AD) development as well as cerebral amyloidosis, but the degree to which cerebrospinal fluid (CSF) ApoE glycosylation affects disease progression is unclear. Here, we investigate CSF ApoE glycosylation in relation to and CSF tau and phosphorylated tau (pTau) and cognitive performance measured cross-sectionally in ADNI.

Methods

ApoE glycosylation was measured in CSF and plasma (n=119) obtained for baseline visits in ADNI cohorts and modeled as a function of CSF tau/ptau levels and longitudinal cognitive changes across domains. Participants consisted of predominantly white older individuals ranging in age from 55-89. ApoE glycosylation was assayed in CSF using Mass Spectrometric Immunoassay (MSIA). We assessed the whether % ApoE glycosylated predicted the performance on harmonized cognitive assessments and CSF tau measures. In structural equation modeling, effects of total CSF apoE glycosylation upon cognitive measures and AD biomarkers were estimated.

Results

Total CSF ApoE glycosylation demonstrated highly significant correlations with decreased levels of both Tau (p<0.0001; regression estimate -416.759) and pTau (p<0.0001; regression estimate -51.799). Total CSF ApoE glycosylation also was predictive of better performance on harmonized composite memory scores (p<0.007), executive function scores (p<0.019), and further demonstrated a significant correlation with language scores (p<0.0001).

Conclusions

The degree of CSF ApoE glycosylation predicts performance on memory and executive function cross-sectionally and associated with the concentrations of CSF tau and pTau181. We conclude that apoE glycosylation is a novel AD biomarker of function significance. These novel discoveries underscore the importance of understanding the mechanisms that glycosylate apoE in the brain in order to develop therapeutic approaches.

Aims

We aim at determining the relationship between APOE ε4 effects on endolysosome dysfunction and intracellular lipid accumulation in human astrocytes in Alzheimer’s disease (AD) brains.

Methods

Global transcriptomic analyses were performed on astrocytes derived from isogenic APOE isoforms from human induced pluripotent stem cells (hiPSCs) by bulk RNA sequencing and post-mortem AD human brains of APOE ε4 carriers compared to non-carriers by single-nucleus RNA sequencing. Fluorescent reporters, immunocytochemistry, and Western blot were used to quantify lysosome function and autophagy in hiPSC-astrocytes.

Results

Transcriptome data reveals APOE ε4 drives lysosome and autophagy dysregulation in astrocytes derived from hiPSCs and dorsolateral prefrontal cortex (dlPFC) of AD patients. APOE ɛ4/ɛ4 astrocytes exhibit reduced lysosomal proteolysis, impaired acidification, loss of membrane protein LAMP1, and macroautophagy dysfunction. Stimulation of lysosome and autophagy function via mTORC1 inhibition rescued neutral lipid accumulation and increased translation in APOE ɛ4/ɛ4 astrocytes.

Conclusions

APOE ε4 impairs regulation of lysosome and autophagy function in human astrocytes. Abnormal mTORC1/TFEB signaling may underlie reduced catabolism but increased lipid synthesis and translation in APOE ɛ4 astrocytes.

Aims

Compared to the ‘neutral’ E3, the E4 allele of Apolipoprotein E (APOE) confers up to a 15-fold increase in Alzheimer’s Disease (AD) risk. Conversely, the neuroprotective E2 allele decreases AD risk by a similar degree. Here, we aimed to assess the therapeutic potential of allelic ‘switching’ by investigating the physiological changes associated with an inducible, in vivo APOE4 to APOE2 transition in a novel transgenic mouse model.

Methods

The APOE “switch mouse” (APOE4s2) uses the Cre-loxP system to allow for inducible APOE allele switching from E4 to E2. These mice express a floxed human APOE4 coding region followed by the human APOE2 coding region. Allelic discrimination (RT-PCR) and mass spec-based proteomic analyses were employed to validate the transition. scRNAseq was used to measure physiological changes following the E4 to E2 allele switch. Behavioral measures and neuropathological analyses were applied to assess the effects of cell-selective (astrocytes or microglia) allelic switching on AD pathology.

Results

mRNA and protein analyses confirm that tamoxifen induces an efficient recombination and expression of human APOE2 in target tissues. scRNAseq revealed that whole-body replacement of APOE4 with APOE2 results in distinct alterations to glial cell transcriptomes affecting pathways involved with metabolism, inflammation, and amyloid beta. scRNAseq implicated astrocytes as the most affected cell type post-switch, while astrocyte-selective E4 to E2 ‘switching’ drove transcriptomic changes that almost completely overlapped with global switching. Importantly, astrocyte-selective E4 to E2 switching was sufficient to significantly decrease amyloid-associated astro- and micro-gliosis and improve cognition.

Conclusions

Together, these data suggest that a successful transition from E4 to E2 has broad impact on the cerebral transcriptome and that an astrocyte-specific E4 to E2 ‘switch’ improves AD associated pathologies.

Aims

The presence of cardiovascular risk factors (CVRF) drives the risk of cerebrovascular disease. However, mechanisms driving cerebrovascular disease in populations with low presence of CVRF are still unknown. This study aims to elucidate biological mechanisms underlying the presence of cerebrovascular disease in middle-age cognitively unimpaired (CU) individuals, at low risk for late-life dementia.

Methods

A total of 1,139 CU participants from the ALFA study were included. Global and regional WMH volumes were measured through magnetic resonance imaging. Genetic predisposition to WMH was estimated using polygenic risk scores (PRSWMH). Covariate adjusted Spearman’s rank correlation tests were performed to evaluate the association between the PRSWMH and WMH volumes. Analyses were stratified by the cardiovascular risk for late-life dementia (CAIDE) [Figure 1] (Low risk; CAIDE≤9) and adjusted for age and sex. Genetic variants included in the PRSWMH were annotated to the nearest genes, and an enrichment analysis was performed to explore the biological mechanisms underlying WMH. Differential effects on individuals based on the presence of dementia-related cardiovascular risk factors were additionally analyzed.

Results

Genetic predisposition to WMH was associated with larger WMH volumes in individuals with a low CAIDE score [Figure 2, Figure 3]. PRSWMH-related genetic variants were annotated to genes involved in biological processes interconnected through lipid-regulatory mechanisms [Figure 4]. Post-hoc analyses revealed that higher genetic predisposition to WMH was associated with larger WMH volumes in individuals at low risk who had hypercholesterolemia, were older or with lower years of education [Figure 5, Figure 6].

Conclusions

In middle-aged individuals at low risk for late-life dementia, preventable lipid-related mechanisms drive the development of WMH, an early marker of cerebrovascular disease. These findings highlight the relevant role of genetics in absence of traditional CVRF for WMH.

Aims

Extracellular vesicles (EV) play crucial roles in pathological protein transfer between cell-to-cell in Alzheimer’s disease (AD). While apolipoprotein E (APOE) ε4 allele is a genetic risk factor strongly associated with late-onset AD, its impact on the cargo composition of EV in AD remains largely unknown. The purpose of this study is to perform comprehensive lipidomic and proteomic profiling of brain-derived extracellular vesicles (BDEV), stratified by APOE genotype (APOE3/3 vs APOE4/4).

Methods

Frozen samples of frontal cortex of AD patients with APOE3/3 (N=20) or APOE4/4 (N=20) were obtained from Mayo Clinic Brain Bank. EV were isolated from brain tissue with discontinuous sucrose gradient ultracentrifugation and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). We further conducted multidimensional mass spectrometry for shotgun lipidomic analysis and mass spectrometry-based data-independent acquisition (DIA) for proteomic analysis.

Results

There were no significant differences in size distribution and morphology between APOE4/4 and APOE3/3 BDEV by TEM and NTA. We identified 17 lipid classes and 173 distinct lipid molecules in the BDEV lipidome. APOE4/4 EV exhibited increased levels of Linoleic acid (18:2) and Dihomo-γ-linolenic acid (20:3), precursors of proinflammatory mediator arachidonic acid (20:4), which are significantly associated with Braak stages over APOE3/3 EV. Among 4000 unique identified proteins, differentially expressed proteins in APOE4/4 over APOE3/3 showed enrichment in abnormal fatty acid or lipoprotein transport and cell type specificity in microglia and astrocytes in female while neurotransmitter regulation and astrocytes and neuron in male.

Conclusions

Both proteomic and lipidomic analyses of BDEV identified metabolic dysfunction influenced by APOE genotype. These molecular alterations may indicate enhanced potency of tau seeding activity or transfer in APOE4/4 EVs over APOE3/3 EVs, therefore warrants further investigation.

Aims

Mitochondrial dysfunction is implicated in Parkinson’s pathogenesis, but detailed understanding of the cause and effect of α-synuclein aggregation is lacking. As part of the PD-MitoQUANT project (www.pdmitoquant.eu), we characterised in vitro neuronal models of pre-formed fibril (p91)-induced phosphorylated α-synuclein (p-α-syn) aggregation.

Methods

Mouse primary neurons were exposed to p91 fibrils for 11-14 days and assayed for p-α-syn aggregation (Western blot, immunofluorescence), excitability (multi-electrode arrays, Fluo-4 imaging), mitophagy (mtRosella), and mitochondrial energetics (Seahorse, NAD(P)H auto-fluorescence). Whole cell and mitochondrial lysates were extracted for transcriptomics, proteomics and metabolomics. Target knockdown was performed via siRNA (primary cultures) and RNAi (C. elegans).

Results

Cellular dysfunctions mediated by p91 included reduced excitability and spontaneous calcium transients, and increased Parkin-independent mitophagy. Functional bioenergetic studies identified reduced mitochondrial respiratory capacity and NAD(P)H pool, suggesting that while p91-induced p-α-syn aggregation was not directly toxic within this paradigm, the ability of these neurons to adapt to stress may be altered. This was further evidenced by increased sensitivity to excitotoxicity. Integrated transcriptome and proteome analyses further indicated p91-mediated metabolic alterations, and RNAi-mediated knockdown of candidate genes in C. elegans identified targets with a functional impact in vivo. Computational metabolic modelling specifically predicted dysregulation of glycan and lipid metabolism, and metabolomics analysis validated the dysregulation of several lipids. These processes are now under further investigation.

Conclusions

In conclusion, we established robust preformed fibril-mediated in vitro models of p-α-syn aggregation and identified widespread, yet subtle, cellular and mitochondrial dysfunctions. Several target candidates and pathways have been taken forward for further investigation.

Aims

One of the main challenges in understanding the etiology of Alzheimer's disease (AD) is identifying the vital biochemical factors that promote the formation and aggregation of beta-amyloid (Aβ) plaques. Previous studies have identified the role of membrane cholesterol and glycolipids in AD pathogenesis owing to their abnormal interaction with Aβ peptides, leading to conformational changes and further inducing oligomerization [1, 2].

Our study aimed to ascertain AD-associated lipid dysregulation in the brain tissue and link these changes to peripheral lipid profiles in cerebrospinal fluid (CSF).

Methods

Aβ peptide and lipid profiles in the brain tissue sections were characterized by matrix-assisted laser desorption/ionization- time-of-flight based mass spectrometry (MALDI-TOF IMS) followed by fluorescent imaging on the same sections for proper plaque annotation. Lipidomic profiling was performed in CSF derived from the same subject using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) using dynamic multiple reaction monitoring (dyMRM) in a pilot cohort (N=30).

Results

IMS analysis on AD brain sections revealed differential Aβ peptides and associated glycosphingolipid dysregulation. Lipidomic assay in the CSF characterized over 300 lipid species from 15 classes. Significant dysregulation in glycolipid species was evident in the CSF samples, showing increased gangliosides in AD.

Conclusions

The results above suggest an association between lipid metabolism and amyloidogenic protein aggregation. This could shed light on biochemical factors that promote Aβ plaque formation and identify potential drug targets that will be important for developing neuroprotective strategies for treating AD.

References:

[1] Barrett, P. J.et al. Science 336, 1168-1171, (2012).

[2] Fantini, J.et al. Expert Rev. Mol. Med. 12, (2010).