Abstract Body

Objectives

A growing body of evidence supports the notion that processes in the periphery contribute to the risk and development of neurodegenerative diseases like Alzheimer's disease (AD). We have previously documented that carriers of the APOE4 allele, the strongest genetic risk factor for AD, exhibit lower plasma levels of apolipoprotein E (apoE), that presence of APOE4 in the liver contributes to pathological changes in the brain, and results from our ongoing studies further suggest that the APOE genotype dictates specific hepatic lipidome signatures. Due to the proposed immunomodulatory role of APOE, for instance in antiviral immunity, we aimed to assess the occurrence of IgG reactivities in plasma from cognitively healthy individuals as well as patients with mild cognitive impairment (MCI) and AD, and potential influence of APOE genotype and sex on the outcome.

Methods

IgG reactivities were assessed in plasma from a total of n=276 subjects by use of a custom antigen bead array including 63 protein fragments, produced within the Human Protein Atlas project, and 7 additional proteins of relevance to AD. The antigen bead array was designed based on results from an initial proteome-wide exploration of autoantibody repertoires of plasma pools using 42k protein fragment arrays at the SciLifeLab national facility Autoimmunity Profiling.

Results

Initial results suggest that antigens with the most plasma reactivity i.e number of reactivities per antigen include UBE2QI, CEP112, IGF1R, INAVA, EPHA1, CCDC85C, POC5 and APOE3. Ongoing data analyses will clarify whether plasma IgG reactivities differ between diagnostic groups and between subjects of different sex and APOE genotypes.

Conclusions

Plasma IgG reactivity screening may provide novel insights into the peripheral immunity of patients with MCI and AD with potential influence of sex and APOE.

Aims

Epidemiological data show that most Alzheimer's disease (AD) patients have evidence of cerebrovascular damage that precedes other neuropathological hallmarks of AD. High-density lipoprotein (HDL) possesses vasoprotective functions while HDL-cholesterol levels are negatively associated with the risk of AD. Several studies suggest that HDL reduces AD risk by decreasing vascular beta-amyloid deposition and inflammation. We previously found that HDL enriched in apolipoprotein (apo)E (HDLE⁺) are more effective in this regard than those lacking apoE (HDLE^-). However, plasma HDL must first interact with brain endothelial cells (ECs) to display its anti-AD properties, a process poorly understood. To this end, our main objectives are to define the HDL trafficking pathway(s) in brain EC (1) and determine whether they are altered in HDLE+ subpopulation (2).

Methods

HDL isolated by ultracentrifugation from plasma of healthy donors was fractionated into HDLE⁺ and HDLE^- using apoE immunoaffinity chromatography. Fluorescent- or ¹²⁵I-radio-labeled HDL were used to measure binding (4^oC), association, internalization or transport (37^oC) through primary human brain EC or the cell line hCMEC/D3.

Results

We confirmed that HDLE+ and HDLE- bind to, are internalized and transported through brain ECs. Notably, HDLE+ particles showed more interaction and transport across brain ECs while they only partially co-localize with HDLE- suggesting independent trafficking routes. Using RNA interference, we showed that HDL transport through brain ECs was limited by both the scavenger receptor BI (SR-BI) and the low-density lipoprotein receptor (LDLR) with the later specifically regulating HDLE+ transport.

Conclusions

Our findings suggest distinct trafficking pathways for HDLE+ and HDLE- through brain ECs and imply different cerebrovascular functions relevant to AD. Moreover, ApoE-containing reconstituted HDL could be a beneficial tool for targeted drug transport across the blood-brain barrier while promoting the health of the cerebrovasculature.

Abstract Body

APOE is the strongest and most reproducible genetic risk factor for late-onset AD (LOAD). Recently, it was reported that APOEe4 drives LOAD-risk through a gain of abnormal function. Moreover, 50% reduction in APOE levels showed beneficial effects in Alzheimer’s cellular and mouse models. Thus, reducing APOEe4 expression holds promise as a potential therapeutic target for LOAD. We developed an epigenome therapy platform to reduce APOEe4 expression by targeted modification of the epigenome landscape across APOE locus. Our platform is based on all-in-one AAV vector comprises of CRISPR/deactivated(d)Cas9 and a synthetic repressor molecule. In vitro studies using human (h)iPSC-derived cholinergic neurons, cerebral organoids, and microglia-like cells from a LOAD patient APOEe4 homozygote showed a robust decrease, ~50%, in APOE-mRNA levels. APOEe4 reduction in the e4/4 cerebral organoids led to significant decrease in phosphorylated-Tau level. APOEe4 repression in the hiPSC-derived microglia resulted in a significant increase in Aβ-uptake and rescued inflammation. Next, we moved onto animal studies using humanized-APOE mouse models. Stereotactic injection of the AAV/dCas-repressor vector into the mice hippocampus resulted in a significant decrease, ~70%, in APOE expression, validating the efficient repression effect. No changes in weights or welfare criterions were observed, and the mice displayed normal grooming and eating/drinking behaviors indicating no safety issues. Collectively, we provide in vitro and in vivo proof-of-concept for the utility, efficacy, and specificity of the APOE-targeted epigenome therapy product and demonstrated its ability to fine-tune APOEe4 expression. Our platform has been refined for gene-specific and even allele- and cell-type- specific therapies, and by that enables the advancement towards precision medicine in LOAD. Our epigenome therapy is translational into a novel therapeutics approach to prevent, delay onset and/or halt the progression of LOAD.

Aims

Carrying the ε4 allele of the apolipoprotein E (APOE) gene is the strongest risk factor for Alzheimer’s disease (AD) besides age itself. As one potential mechanism, apoE, and especially apoE4, binds to amyloid-β (Aβ) with high affinity and acts as a catalyst to accelerate Aβ oligomer and fibril formation, increase their stability, and promote their neurotoxicity. Here, we test the hypothesis that inhibiting this early step in the amyloid cascade may thereby reduce or prevent neurodegeneration and AD.

Methods

We developed a high-throughput screening assay to identify inhibitors of the apoE4-Aβ interaction and used it to screen more than 3,000 compounds from small molecule drug repurposing libraries. Changes in cognition (MMSE scores) and clinical diagnoses of National Alzheimer’s Coordinating Center (NACC) participants were modeled using time slopes and Cox proportional hazards, respectively, adjusted for age and sex.

Results

We identified two FDA-approved drugs, imipramine and olanzapine, as novel inhibitors of the apoE4-Aβ interaction. We confirmed that both drugs exhibited low neurotoxicity and blocked apoE-induced intracellular Aβ aggregation, tau hyperphosphorylation, and apoptosis in primary neuronal cultures. When taken by AD patients for their normal clinical indications, imipramine or olanzapine use was associated with improved cognition and increased incidence of receiving a better clinical diagnosis, especially among APOE4 carriers, relative to all other antidepressant or antipsychotic medications.

Conclusions

The critical test of any proposed AD mechanism is whether it leads to effective treatments. Our screening approach identified imipramine and olanzapine, which have no structural, functional, or clinical similarities other than their shared ability to inhibit the apoE4-Aβ interaction, both of which showed evidence of improving AD cognition. These findings validate an apoE-centric approach to developing new AD therapeutics.

Aims

Cerebral amyloid angiopathy (CAA) consists of amyloid-β (Aβ) accumulation in cerebral vasculature eventually leading to lobar symptomatic intracerebral hemorrhages (ICH) and microbleeds (CMB). Since apolipoprotein J (ApoJ) is a versatile protein involved in Aβ aggregation and clearance, we aimed to investigate the effects of chronic recombinant human ApoJ (rhApoJ) treatment in a CAA transgenic mouse model, focusing on vascular damage assessed by the analysis of spontaneous CMBs.

Methods

Twenty-month-old APP23 C57BL/6 mice received 25 doses of rhApoJ (1 mg/kg) (n=9) or saline (n=8) intraperitoneally over 13 weeks, while wild-type mice received saline (n=13). Postmortem brains were examined using T2*-weighted magnetic resonance imaging (MRI) to detect hypointensities indicative of hemorrhagic lesions. Aβ distribution, microglia activation, plasma matrix metalloproteinases and inflammatory markers were analyzed after treatments. Additionally, plasma samples from 40 acute ICH patients and 17 controls were analyzed to explore the clinical relevance of the findings.

Results

Mice chronically treated with rhApoJ showed fewer cortical CMBs (50-300 µm diameter) (p=0.012) and fewer larger hemorrhages (>300µm) (p=0.002), compared to APP23 saline-treated mice, without showing differences in parenchymal or cerebrovascular Aβ levels. After the administration, rhApoJ led to an increase in plasma levels of Gro-α (p=0.035) and MIP-1α (p=0.035) and a reduction in circulating MMP-12 (p=0.046). In patients with lobar ICH, plasma MMP-12 levels correlated with a larger hemorrhage volume (p=0.040) and a more irregular ICH shape (p=0.036) after adjusted linear regressions.

Conclusions

Chronic rhApoJ treatment in aged APP233 mice mitigated CAA-associated neurovascular damage by reducing cortical microbleeds. The study suggests that rhApoJ treatment may prevent blood-brain barrier leakage and the appearance of CMBs associated with CAA, partially through modulating circulating MMP-12 levels. These findings could have implications for potential therapeutic strategies targeting CAA-related complications.

Abstract Body

Aims

Apolipoprotein E4 (APOE4) dysregulates brain cholesterol metabolism, is associated with neuroinflammation, and increases the risk of Alzheimer’s disease (AD). The ATP-binding cassette transporter A1 (ABCA1) plays an important role in cholesterol transport but increased lysosomal ABCA1 can drive cellular senescence. We have previously shown that APOE4 intracellular aggregation traps ABCA1 into lysosomes. The aim of this study is to examine whether cholesterol accumulation and lysosomal ABCA1 in APOE4 drive cellular senescence.

Methods

Analysis of bulk and single-nuclei RNA sequencing (seq) data in postmortem brain tissues from ROSMAP, together with cellular and APOE-targeted replacement animal models were employed.

Results

Analysis of bulk RNA seq data in ROSMAP revealed a higher Senescence-Associated Secretory Phenotype (SASP) score in APOE4 carriers than in APOE3 carriers with AD (n=151, p=0.025). ABCA1 expression in postmortem brain tissues was positively correlated with the SASP pathway score (n=396, p<0.001). In agreement, single-nuclei RNA seq analysis from human postmortem tissues confirmed a greater SASP score in APOE4 astrocytes compared with APOE3 (n=36). Using cellular and mouse models, we identified that APOE4 promoted cholesterol accumulation and ABCA1 sequestration in lysosomes, activating mTORC1 and SASP. Reducing cellular cholesterol by treatment with cyclodextrin in APOE4-TR mice reduced lysosomal ABCA1 by increasing its recycling to the plasma membrane, exporting cholesterol to small HDL particles, and reducing mTORC1 activation and SASP profiles.

Conclusions

This work provides novel insights into how cholesterol accumulation in APOE4 accelerates senescence through dysregulation of ABCA1 trafficking into lysosomes and underscores the importance of reducing lysosomal cholesterol to mitigate the effects of APOE4.

Abstract Body

A resistant case to autosomal dominant Alzheimer’s Disease (AD) in a PSEN1 mutation carrier was reported in a woman homozygous for the APOE3 Christchurch (APOE3ch) mutation. Whether APOE3ch is the reason for the protective effect remains unclear. We generated a humanized APOE3ch knockin mouse and crossed it to an amyloid-ß (Aß) plaque depositing model. We injected human AD-Tau brain extract to investigate tau seeding and spreading in the presence or absence of amyloid. We demonstrate that APOE3ch has similar effects as were seen in the PSEN1 carrier. There was a dyslipidemia phenotype in the periphery, a reduction in Aß deposition, a marked reduction in plaque-associated tau pathology, enhanced microglial response around plaques, and increased myeloid cell phagocytosis specifically to tau aggregates. Thus, APOE3ch influences the microglial response to Aβ plaques, which suppresses Aβ-induced tau seeding and spreading. The results reveal new possibilities to target Ab-induced tauopathy for AD therapeutic development.