Abstract Body

The APOE gene in humans is polymorphic with gene variant frequencies differing between ethnicities. Low levels of plasma apoE, mainly derived from the liver, increase the risk of AD but do not per se differ between AD patients and controls when accounting for APOE genotype. Plasma apoE does not cross the blood-brain-barrier hence it remains to be elucidated why low plasma apoE is related to higher AD risk. Our studies show that low plasma apoE was unfavorably linked to CSF AD biomarkers and cognition. Low plasma apoE in cognitively healthy APOE3/E4 carriers was associated with higher plasma glucose which in turn was linked to reduced brain glucose metabolism in several brain areas. Furthermore, patients with MCI and who remained stable over a period of 24 months exhibited higher plasma apoE compared to those progressing to an AD diagnosis, even in the absence of the APOE4 allele. Carriers of the APOE4-allele exhibited higher plasma triglycerides, known to cross the blood-brain-barrier and to cause insulin resistance in the brain, but unaltered levels of cholesterol. Interestingly, specifically plasma apoE3 isoform levels were inversely correlated to plasma triglycerides whereas higher plasma apoE4 isoform levels were paralleled by higher plasma triglycerides. We also found that dimerization of the apoE3 isoform in plasma differs between AD patients and controls, the functional relevance of these findings remain to be investigated. We conclude that plasma apoE levels are associated with plasma glucose and triglyceride levels which when pathological may be the driver of the increased risk of AD.

Abstract Body

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer disease (AD). In addition to effects of ApoE on amyloid-β, results have shown that ApoE markedly influences pathological forms of tau and tau-mediated neurodegeneration with apoE4 having a strong deleterious effect on both parameters. In the brain, ApoE is produced and secreted primarily by astrocytes and also by activated microglia though the cell-specific role of each form of ApoE in the setting of neurodegeneration has not been determined. We utilized a well characterized mouse model of tauopathy, P301S Tau transgenic mice expressing floxed APOE-ε4 or APOE-ε3 alleles. We crossed these mice with Aldh1l1-CreERT2 mice and at 5.5 months of age, after the onset of tau pathology, administered tamoxifen or vehicle. We then compared mice with or without inducible knockout of astrocyte APOE at 9 months of age. Removing astrocytic APOE4 but not APOE3 markedly reduced tau-mediated neurodegeneration, decreased p-tau pathology, and improved nesting behavior. Single nucleus RNA sequencing analysis revealed striking gene expression changes in all cell types in P301S mice with astrocytic APOE4 playing a role in modulating neuron and astrocyte gene expression with little effect on microglial gene expression. Importantly, removal of astrocytic APOE4 decreased tau-induced synaptic loss and microglial phagocytosis of synaptic elements suggesting a key role for astrocytic APOE in synaptic degeneration.

Aims

The presence of the APOE4 allele is the major genetic risk factor for Alzheimer’s Disease (AD). In the brain, ApoE is mainly produced by astrocytes, which play a crucial role in AD pathogenesis. Here, we aim to uncover the role of different APOE genotypes (E2, E3, E4 and knockout) for the physiological (dys-) function of human astrocytes.

Methods

APOE isogenic human induced pluripotent stem cells (iPSCs) are differentiated to functional astrocytes and analysed for glutamate uptake, inflammatory signalling, beta-amyloid uptake as well as cholesterol metabolism. Further, full proteomic analysis was carried out in astrocytes at baseline and after activation.

Results

Astrocyte differentiation potential and proliferation was comparable among lines. However, we observed genotype-dependent differences in ApoE levels, glutamate uptake, cholesterol metabolism and receptor-mediated beta-amyloid uptake. Further, astrocytes activated with IL-1β or TNF-α showed genotype-dependent release of pro-inflammatory cytokines with APOE4>E3>E2>KO. Liquid chromatography-mass spectrometry-based proteomic screen combined with gene set enrichment analysis revealed genotype-dependent differences in cellular pathways including cytokine production, energy metabolism, ECM organization and energy metabolism.

Conclusions

The APOE genotype strongly influences disease-relevant physiological properties of human astrocytes. Our preliminary data suggest that a combination of loss-of-function and gain-of-toxic-function in APOE4 astrocytes may mediate AD pathology.

Abstract Body

Apolipoprotein E4 is the most important risk factor for late-onset AD. ApoE is a ligand for the LDL receptor gene family which regulates the formation of the brain, neurotransmission and network homeostasis. Several ApoE receptors also participate in ApoE mediated amyloid turnover in the brain. We have previously shown that ApoE4 impairs endolysosomal transport by delaying the maturation of early endosomes. This results in endosomal enlargement, a typical cellular feature of AD. ApoE4 differs from ApoE3 by one amino acid. This raises its isoelectric point to ~6.4, which closely matches the luminal pH of the early endosomes. We hypothesized that the resulting charge neutralization of ApoE4 would reduce its solubility, favor ApoE particle interaction and thus decrease the rate at which ApoE4 dissociates from its receptors. We further reasoned that lowering the pH in the early endosome would aid in maintaining ApoE particle solubility and promote its dissociation from ApoE receptors, thus restoring normal endolysosomal trafficking. Selective lowering of early endosomal pH can be achieved by disrupting the function of NHE6, the early endosome specific proton leak channel. We have shown that this intervention completely normalizes ApoE4 trafficking and relieves the endosomal retention of ApoE receptors and associated glutamate receptors. We further show that pharmacological or genetic inhibition of NHE6 prevents the ApoE4 mediated accumulation of amyloid plaques in hAPP knockin mice and restores the ApoE4 mediated resistance of homeostatic synaptic plasticity. We propose NHE6 inhibition as a rational therapeutic approach to negate the ApoE4 risk for late-onset AD.

Aims

Determine whether APOE splice quantitative trait locus (sQTL) variants affect APOE4-related risk for Alzheimer’s disease (AD), age-at-AD-onset, and APOE protein levels.

Methods

Participants (N=25,120) were ages 60+, non-Hispanic, of European ancestry, had APOE genotype available, and diagnosed as cases or controls. Only rs157580 and rs439401 APOE sQTLs were genotyped in >80% of the available sample and further investigated. Associations of APOE sQTLs with AD risk (mixed model case-control regression) and log-normalized age-at-AD-onset (linear mixed model) were evaluated in APOE*3/4 and APOE*4/4 carriers. Associations with APOE protein levels were evaluated in post-mortem dorsolateral prefrontal cortex (dlPFC) tissue from APOE*3/4 carriers (APOE*4/4 not considered due to sample paucity).

Results

Rs157580 and rs439401 reduced AD risk in APOE*4/4 carriers (Table1). Rs157580 also increased age-at-AD-onset in APOE*4/4 (beta=0.027, P-value=0.023) and APOE*3/4 carriers (beta=0.004, P-value=0.089). Rs157580 increased APOE protein levels in the dlPFC of APOE*3/4 carriers (Figure1).

MAF=minor allele frequency; CN=control, OR=odds ratio; CI=95% confidence interval.

Figure1. Association of rs157580 with APOE protein levels. Error bars show 95-percentile range. X-axis indicates genotypes and sample sizes.

Conclusions

Our results bring new insights into mechanisms that alter APOE expression and how this affects APOE4-related AD risk. This is crucially relevant for the development of AD drug therapies aimed at APOE4.

Aims

To identify apolipoprotein E (APOE)-specific factors that may predict life expectancy after a diagnosis of Alzheimer’s disease (AD).

Methods

The Swedish Alzheimer Treatment Study (SATS) is a prospective, observational, clinical-practice-based, multicenter study that includes 999 participants diagnosed with mild-to-moderate AD at the start of cholinesterase inhibitor (ChEI) therapy (time of diagnosis). Cognitive abilities and activities of daily living (ADL) were evaluated at baseline and semiannually over 3 years, and the date of death was recorded.

Results

After 20 years of follow-up, 309/320 (97%) of the APOE non-ε4-carriers, 497/528 (94%) of the one-ε4-carriers, and 140/151 (93%) of the two-ε4-carriers had died (p=0.154). In the multivariate Cox regression models, risk factors for shorter lifespan in all patients were older age, lower cognitive ability at baseline, and faster cognitive decline/year. Moreover, a higher ChEI dose and/or longer treatment duration predicted increased survival. In APOE non-ε4-carriers, male sex, antidiabetic or antihypertensive/cardiac therapy, worse basic ADL capacity at baseline, and faster instrumental ADL progression were observed to decrease life expectancy. In one-ε4-carriers, male sex, antihypertensive/cardiac therapy, and faster basic ADL deterioration predicted shorter survival. In two-ε4-carriers, antidiabetics and longer AD duration were risk factors for decreased lifespan.

Conclusions

Common risk factors for death, such as male sex, cardiovascular disorders, and functional impairment were demonstrated in non-ε4-carriers and one-ε4-carriers, but not in two-ε4-carriers. The consequences of dementia, such as cognitive decline and duration of AD, may have a greater impact on survival time in APOE two-ε4-carriers. Optimal ChEI treatment had positive effects in all AD patients irrespective of APOE genotype.

Aims

The apolipoprotein E (APOE) ε4 is associated with increased AD risk and no effective treatment exists. The apoE4 protein has impaired interaction with astrocyte’s ATP-binding cassette transporter A1 (ABCA1) resulting in: a) poor cholesterol efflux, and b) build-up of residual cholesterol in lipid rafts, impeding astrocyte function (Rawat 2019) and increasing neuron cell death (Voskuhl 2018). Cynomolgus monkeys (cynos) have arginine in the critical 112 and 158 positions (like hu apoE4) but not in the 61 position, a 93% homology with human apoE, as well as lipid and amyloid metabolism similar to humans, making cynos a representative model for humans. In a dose-range finding toxicology study in 2-year old cynos, we explored effects of CS6253 ABCA1 agonist treatment on CSF and blood amyloid markers.

Methods

CS6253 was administered iv at 0 (control), 75, 150 and 225 mg/kg (n=2/sex/group) 5 times over 10 days. CSF was collected before treatment and 6 hours after the 5^th/last dosing. CSF and plasma Aβ42 and Aβ40 were analyzed by SIMOA, CSF APP and amyloid protein complex2B1 (AP2B1) by mass spectrometry.

Results

CS6253 treatment decreased CSF levels of Aβ42, Aβ40, APP and AP2B1 and increased plasma Aβ42, Aβ40, both following dose-response pattern, see Figures.

Conclusions

The CS6253 ABCA1 agonist reductions of Aβ42, Aβ40, APP and AP2B1 in CSF of juvenile cynos are consistent with previous finding in apoE4 mice models where brain Aβ42 (and P-tau) was reduced, AD pathology was prevented, and cognition improved. Studies in human APOE ε4 carriers are needed to corroborate these findings.