Peripheral apolipoprotein E and its emerging role in neurodegenerative disease

The human apolipoprotein E gene (APOE) is polymorphic and coding for three common alleles; ε2, ε3, and ε4. Carriers of the ε4 allele are at a higher risk of developing sporadic late-onset Alzheimer’s Disease (AD). This association appears to be influenced by inherited traits, race, and sex. The connection between APOE genotype and AD-related pathological processes has been studied excessively, however, less attention has been devoted to the apolipoprotein E (apoE) protein levels per se, most possibly due to the inconsistent results presented in studies assessing potential links between cerebrospinal fluid (CSF) apoE levels and AD, and because systemic apoE cannot cross the blood-brain barrier (BBB). Nevertheless, low plasma apoE levels were found to enhance AD and dementia risk, with the APOE ε4 genotype influencing this risk by promoting a reduction of the plasma apoE levels in some populations. In this thesis, we speculate that peripheral apoE-dependent mechanisms are linked to neurodegeneration. To address this hypothesis, we performed a set of clinical and experimental studies. In the first three studies, we aimed to determine whether hepatic APOE ε4 genotype, plasma apoE levels, and diet are linked to brain function and cognition by utilizing a mouse model with humanized-livers that are characterized by the presence of human apoE only in the plasma. A comparison between the brains of APOE ε4/ε4 and APOE ε2/ε3 humanized-liver mice revealed altered endogenous murine apoe levels as well as altered levels of synaptic, neuroinflammatory, and insulin signaling-related markers in the cortex and hippocampus. Higher plasma apoE4 levels were also linked to these associations, mainly in the hippocampus of the humanized APOE ε4/ε4 liver mice. A similar, though less pronounced, effect was observed in the brains of APOE ε3/ε3 humanized-liver mice that were fed a high-fat, versus a normal diet. Utilizing the plasma from APOE ε3/ε3 humanized-liver mice, we further observed that the distribution of apoE3 in plasma lipoparticles differed between sexes. In addition, higher total plasma apoE3 levels were beneficial to the activity levels but appeared to have a negative impact on cognition in these mice. In the remaining studies, we aimed to determine how plasma apoE levels, apoE isoform distribution, and the formation of monomers and disulfide-linked homodimers and heterodimers with apolipoprotein A-II (apoA-II) are influenced by APOE genotype and AD. For that purpose, we studied two cohorts of Norwegian, Black/African-American (B/AA), and non-Hispanic white (NHW) cognitively healthy subjects and patients with AD or mild cognitive impairment (MCI). Only in Norwegian individuals, we observed lower levels of plasma apoE due to AD and APOE ε4 genotype. In addition, in these subjects, the apoE monomer/dimer profile seemed to be influenced by AD status. In the cohort of B/AAs and NHWs, these associations were absent. In both cohorts, we observed associations between plasma apoE levels and CSF AD biomarker levels. Lastly, B/AA subjects presented the highest plasma apoE levels with APOE ε4/ε4-carrying subjects exhibiting significantly higher plasma apoE levels than NHW APOE ε4/ε4 subjects. Overall, our studies suggest that hepatic APOE genotype and plasma apoE levels are associated with AD-related neuropathological changes which seem to be influenced by other factors like race and diet. Whether this influence is due to differences in apoE levels or apoE function (i.e. different distribution between lipoparticles) remains to be investigated in future studies

Peripheral apolipoprotein E and its emerging role in neurodegenerative disease

The human apolipoprotein E gene (APOE) is polymorphic and coding for three common alleles; ε2, ε3, and ε4. Carriers of the ε4 allele are at a higher risk of developing sporadic late-onset Alzheimer’s Disease (AD). This association appears to be influenced by inherited traits, race, and sex. The connection between APOE genotype and AD-related pathological processes has been studied excessively, however, less attention has been devoted to the apolipoprotein E (apoE) protein levels per se, most possibly due to the inconsistent results presented in studies assessing potential links between cerebrospinal fluid (CSF) apoE levels and AD, and because systemic apoE cannot cross the blood-brain barrier (BBB). Nevertheless, low plasma apoE levels were found to enhance AD and dementia risk, with the APOE ε4 genotype influencing this risk by promoting a reduction of the plasma apoE levels in some populations. In this thesis, we speculate that peripheral apoE-dependent mechanisms are linked to neurodegeneration. To address this hypothesis, we performed a set of clinical and experimental studies. In the first three studies, we aimed to determine whether hepatic APOE ε4 genotype, plasma apoE levels, and diet are linked to brain function and cognition by utilizing a mouse model with humanized-livers that are characterized by the presence of human apoE only in the plasma. A comparison between the brains of APOE ε4/ε4 and APOE ε2/ε3 humanized-liver mice revealed altered endogenous murine apoe levels as well as altered levels of synaptic, neuroinflammatory, and insulin signaling-related markers in the cortex and hippocampus. Higher plasma apoE4 levels were also linked to these associations, mainly in the hippocampus of the humanized APOE ε4/ε4 liver mice. A similar, though less pronounced, effect was observed in the brains of APOE ε3/ε3 humanized-liver mice that were fed a high-fat, versus a normal diet. Utilizing the plasma from APOE ε3/ε3 humanized-liver mice, we further observed that the distribution of apoE3 in plasma lipoparticles differed between sexes. In addition, higher total plasma apoE3 levels were beneficial to the activity levels but appeared to have a negative impact on cognition in these mice. In the remaining studies, we aimed to determine how plasma apoE levels, apoE isoform distribution, and the formation of monomers and disulfide-linked homodimers and heterodimers with apolipoprotein A-II (apoA-II) are influenced by APOE genotype and AD. For that purpose, we studied two cohorts of Norwegian, Black/African-American (B/AA), and non-Hispanic white (NHW) cognitively healthy subjects and patients with AD or mild cognitive impairment (MCI). Only in Norwegian individuals, we observed lower levels of plasma apoE due to AD and APOE ε4 genotype. In addition, in these subjects, the apoE monomer/dimer profile seemed to be influenced by AD status. In the cohort of B/AAs and NHWs, these associations were absent. In both cohorts, we observed associations between plasma apoE levels and CSF AD biomarker levels. Lastly, B/AA subjects presented the highest plasma apoE levels with APOE ε4/ε4-carrying subjects exhibiting significantly higher plasma apoE levels than NHW APOE ε4/ε4 subjects. Overall, our studies suggest that hepatic APOE genotype and plasma apoE levels are associated with AD-related neuropathological changes which seem to be influenced by other factors like race and diet. Whether this influence is due to differences in apoE levels or apoE function (i.e. different distribution between lipoparticles) remains to be investigated in future studies

Behavioral and cognitive performance of humanized APOEε3/ε3 liver mice in relation to plasma apolipoprotein E levels

Abstract Plasma apolipoprotein E levels were previously associated with the risk of developing Alzheimer’s disease (AD), levels of cerebrospinal fluid AD biomarkers, cognition and imaging brain measures. Outside the brain, the liver is the primary source of apoE and liver transplantation studies have demonstrated that liver-derived apoE does not cross the blood–brain-barrier. How hepatic apoE may be implicated in behavioral and cognitive performance is not clear. In the current study, we behaviorally tested FRGN mice with humanized liver harboring the ε3/ε3 genotype (E3-human liver (HL)) and compared their behavioral and cognitive performance with that of age-matched ε3/ε3 targeted replacement (E3-TR) mice, the latter produces human apoE3 throughout the body whereas the E3-HL mice endogenously produce human apoE3 only in the liver. We also compared the liver weights and plasma apoE levels, and assessed whether plasma apoE levels were correlated with behavioral or cognitive measures in both models. E3-HL were more active but performed cognitively worse than E3-TR mice. E3-HL mice moved more in the open field containing objects, showed higher activity levels in the Y maze, showed higher activity levels during the baseline period in the fear conditioning test than E3-TR mice, and swam faster than E3-TR mice during training to locate the visible platform in the water maze. However, E3-HL mice showed reduced spatial memory retention in the water maze and reduced fear learning and contextual and cued fear memory than E3-TR mice. Liver weights were greater in E3-HL than E3-TR mice and sex-dependent only in the latter model. Plasma apoE3 levels were similar to those found in humans and comparable in female and male E3-TR mice but higher in female E3-HL mice. Finally, we found correlations between plasma apoE levels and behavioral and cognitive measures which were predominantly model-dependent. Our study demonstrates mouse-model dependent associations between plasma apoE levels, behavior and cognition in an ‘AD-neutral’ setting and suggests that a humanized liver might be sufficient to induce mouse behavioral and cognitive phenotypes

APOE genotype dictates lipidomic signatures in primary human hepatocytes

Apolipoprotein E (APOE) genetic variants are most notably known for their divergent impact on the risk of developing Alzheimer’s disease. While APOE genotype has been consistently shown to modulate lipid metabolism in a variety of cellular contexts, the effect of APOE alleles on the lipidome in hepatocytes is unknown. In this study, we investigated the contribution of APOE alleles to lipidomic profiles of donor-derived primary human hepatocytes from 77 subjects. Lipidomic data obtained by liquid chromatography-mass spectrometry were analyzed across ε2/ε3, ε3/ε3, and ε3/ε4 genotypes to reveal how APOE modulates lipid relative levels over age and between groups. Hepatic APOE concentration, measured by ELISA, was assessed for correlation with lipid abundance in subjects grouped as per APOE genotype and sex. APOE genotype-specific differential lipidomic signatures associated with age for multiple lipid classes but did not differ between sexes. Compared to ε2/ε3, ε3/ε4 hepatocytes had higher abundance of acylcarnitines (AC) and acylphosphatidylglycerol (AcylPG) as a class, as well as higher medium and long-chain ACs, AcylPG, phosphatidylglycerol (PG), bis(monoacylglycerol)phosphate (BMP), monoacylglycerol (MG) and diacylglycerol (DG) species. The ε3/ε4 hepatocytes also exhibited a higher abundance of medium and long-chain ACs compared to the ε3/ε3 hepatocytes. Only in the ε3/ε4 hepatocytes, APOE concentration was lower and showed a negative correlation with BMP levels, specifically in females. APOE genotype dictates a differential lipidome in primary human hepatocytes. The lipids involved suggest mitochondrial dysfunction with accompanying alterations in neutral lipid storage, reflective of a general disturbance of free fatty acid metabolism in human hepatocytes with the ε4 allele

Increased plasma and brain immunoglobulin A in Alzheimer’s disease is lost in apolipoprotein E ε4 carriers

Background: Alzheimer’s disease (AD) is foremost characterized by β-amyloid (Aβ)-extracellular plaques, tau-intraneuronal fibrillary tangles (NFT), and neuroinflammation, but over the last years it has become evident that peripheral inflammation might also contribute to the disease. AD patients often demonstrate increased levels of circulating proinflammatory mediators and altered antibody levels in the blood. In our study, we investigated the plasma Immunoglobulin A (IgA) levels in association with apolipoprotein E (APOE) ε4 status and Aβ pathology. Methods: IgA levels in antemortem-collected (cohort I) and postmortem-collected (cohort II) plasma samples from AD patients (n = 30 in cohort I and n = 16 in cohort II) and non-demented age-matched controls (NC) (n = 42 in cohort I and n = 7 in cohort II) were measured using ELISA. Hippocampal sections from cohort II were immunostained against IgA, and the IgA area fraction as well as the number of IgA positive (IgA+) cells in the cornu ammonis region were analysed using ImageJ. The relationship between plasma IgA levels and cognition, C-reactive protein (CRP), and cerebrospinal fluid (CSF) AD biomarkers in cohort I as well as neuropathology, IgA+ cell number, and IgA area fraction in cohort II was analysed before and after grouping the cohorts into APOEε4 carriers and APOEε4 non-carriers. Results: Plasma IgA levels were higher in AD patients compared to NC in both cohorts. Also, AD patients demonstrated higher IgA area fraction and IgA+ cell number compared to NC. When APOEε4 status was considered, higher plasma IgA levels in AD patients were only seen in APOEε4 non-carriers. Finally, plasma IgA levels, exclusively in APOEε4 non-carriers, were associated with cognition, CRP, and CSF Aβ levels in cohort I as well as with IgA area fraction, IgA+ cell number, and Aβ, Lewy body, and NFT neuropathology in cohort II. Conclusions: Our study suggests that AD pathology and cognitive decline are associated with increased plasma IgA levels in an APOE allele-dependent manner, where the associations are lost in APOEε4 carriers

Plasma apolipoprotein E levels, isoform composition, and dimer profile in relation to plasma lipids in racially diverse patients with Alzheimer’s disease and mild cognitive impairment

Abstract Background The APOEε4-promoted risk of Alzheimer’s disease (AD) is lower in Black/African-Americans (B/AAs), compared to non-Hispanic whites (NHWs). Previous studies reported lower plasma apolipoprotein E (apoE) levels in NHW APOEε4-carriers compared to non-carriers, and low plasma apoE levels were directly associated with an increased risk of AD and all dementia. We further showed that APOEε3/ε3 AD patients exhibited reduced plasma apoE dimers compared to corresponding control subjects. Whether plasma apoE levels and apoE dimer formation differ between races/ethnicities and therefore may help explain AD risk racial disparity remains to be elucidated. Methods Using mass spectrometry, we determined total plasma apoE and apoE isoform levels in a cohort of B/AAs (n = 58) and NHWs (n = 67) including subjects with normal cognition (B/AA: n = 25, NHW: n = 28), mild cognitive impairment (MCI) (B/AA: n = 24, NHW: n = 24), or AD dementia (B/AA: n = 9, NHW: n = 15). Additionally, we used non-reducing western blot analysis to assess the distribution of plasma apoE into monomers/disulfide-linked dimers. Plasma total apoE, apoE isoform levels, and % apoE monomers/dimers were assessed for correlations with cognition, cerebrospinal fluid (CSF) AD biomarkers, sTREM2, neurofilament light protein (NfL), and plasma lipids. Results Plasma apoE was predominantly monomeric in both racial groups and the monomer/dimer distribution was not affected by disease status, or correlated with CSF AD biomarkers, but associated with plasma lipids. Plasma total apoE levels were not related to disease status and only in the NHW subjects we observed lower plasma apoE levels in the APOEε4/ε4-carriers. Total plasma apoE levels were 13% higher in B/AA compared to NHW APOEε4/ε4 subjects and associated with plasma high-density lipoprotein (HDL) in NHW subjects but with low-density lipoprotein levels (LDL) in the B/AA subjects. Higher plasma apoE4 levels, exclusively in APOEε3/ε4 B/AA subjects, were linked to higher plasma total cholesterol and LDL levels. In the controls, NHWs and B/AAs exhibited opposite associations between plasma apoE and CSF t-tau. Conclusions The previously reported lower APOEε4-promoted risk of AD in B/AA subjects may be associated with differences in plasma apoE levels and lipoprotein association. Whether differences in plasma apoE levels between races/ethnicities result from altered APOEε4 expression or turnover, needs further elucidation

Plasma apolipoprotein E levels in longitudinally followed patients with mild cognitive impairment and Alzheimer's disease

Background: Low levels of plasma apolipoprotein E (apoE) and presence of the APOE epsilon 4 allele are associated with an increased risk of Alzheimer's disease (AD). Although the increased risk of AD in APOE epsilon 4-carriers is well-established, the protein levels have received limited attention. Methods: We here report the total plasma apoE and apoE isoform levels at baseline from a longitudinally (24 months) followed cohort including controls (n = 3 9), patients with stable amnestic mild cognitive impairment during 24 months follow up (MCI-MCI, n = 3 0) , patients with amnestic MCI (aMCI) that during follow-up were clinically diagnosed with AD with dementia (ADD) (MCI-ADD, n = 28), and patients with AD with dementia (ADD) at baseline (ADD, n = 28). We furthermore assessed associations between plasma apoE levels with cerebrospinal fluid (CSF) AD biomarkers and alpha-synuclein, as well as both CSF and plasma neurofilament light chain (NfL), YKL-40 and kallikrein 6. Results: Irrespective of clinical diagnosis, the highest versus the lowest apoE levels were found in APOE epsilon 2/epsilon 3 versus APOE epsilon 4/epsilon 4 subjects, with the most prominent differences exhibited in females. Total plasma apoE levels were 32% and 21% higher in the controls versus MCI-ADD and ADD patients, respectively. Interestingly, MCI-ADD patients exhibited a 30% reduction in plasma apoE compared to MCI-MCI patients. This decrease appeared to be associated with brain amyloid-beta (A beta(42)) pathology regardless of disease status as assessed using the Amyloid, Tau, and Neurodegeneration (A/T/N) classification. In addition to the association between low plasma apoE and low levels of CSF A beta(42), lower apoE levels were also related to higher levels of CSF total tau (t-tau) and tau phosphorylated at Threonine 181 residue (p-tau) and NfL as well as a worse performance on the mini-mental-state-examination. In MCI-ADD patients, low levels of plasma apoE were associated with higher levels of CSF alpha-synuclein and kallikrein 6. No significant correlations between plasma apoE and the astrocytic inflammatory marker YKL40 were observed. Conclusions: Our results demonstrate important associations between low plasma apoE levels, A beta pathology, and progression from aMCI to a clinical ADD diagnosis