Blood Bioenergetic Biomarkers in Alzheimer’s Disease APOE ε4-Carriers

Background: This study examined the impact of APOE ε4 on blood cell bioenergetics. Prior studies have shown systemic alterations in blood cells from APOE ε4 carriers. Methods: Platelet mitochondria cytochrome oxidase (COX) and citrate synthase (CS) Vmax activities were measured in APOE ε4 carrier and non-carrier Alzheimer’s disease (AD) subjects, and lymphocyte mitochondria and bioenergetics-relevant protein and viability endpoints were measured using fresh and expanded cultures. Statistical analysis was completed using Student’s T-Test. Results: The mean platelet COX Vmax activity, normalized to protein content, was lower in APOE ε4 carriers and lymphocyte Annexin V, a marker of apoptosis, was signicantly higher. PINK1, a protein involved in mitophagy, was higher in APOE ε4 carrier lymphocytes. mTOR and SIRT1, which play a role in energy sensing, were different between the groups; mTOR phosphorylation decreased while SIRT1 phosphorylation increased in APOE ε4 carrier lymphocytes. The lipid synthesis pathway differed, as AceCSI and ATP CL increased in APOE ε4 carrier lymphocytes, and ACC phosphorylation also increased.


Background
Apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic Alzheimer's Disease (AD). APOE exists as three alleles; ε2 (lowers AD risk), ε3 (neutral AD risk), and ε4 (increases AD risk). Individuals who carry an APOE ε4 allele are 3-4-fold more likely to develop AD, while homozygotes are 10-15-fold more likely (1). About 15-25% of the population carries an APOE ε4 allele and 2-3% are homozygous. The exact mechanism underlying the link between AD risk and APOE is unknown (1).
The APOE gene product, apolipoprotein E (APOE), is a lipid binding protein (i.e. lipoprotein) which functions in cholesterol metabolism. Within the brain, APOE is the main cholesterol carrying protein where it is mostly expressed by astrocytes and transports cholesterol to neurons (1). Allele variants lead to one or two amino acid substitutions in the protein product (APOE ε2, cys112, cys158; APOE ε3, cys112, arg158; and APOE ε4, arg112, arg158). The presence of arg112 in APOE ε4 causes an alternative folding of the peptide that gives rise tos a cleavage site from which a mitochondrial-toxic C-terminal fragment is formed (1)(2)(3). APOE also functions to modulate in ammation and amyloid beta clearance (4)(5)(6).
A goal of this study was to expand and extend our prior study to include male AD subjects (16). We also sought to further our ndings by measuring a range of metabolic outcomes in blood cells and compared some endpoints to post-mortem brain samples. These blood-based mitochondrial biomarkers were used as primary and target engagement outcomes in recent clinical trials (17)(18)(19). This report summarizes ndings from human platelets and lymphocytes. The platelet analysis was conducted as part of the Sequol in AD 2 (SEAD2) clinical trial (ClinicalTrials.gov Identi er: NCT03101085), and the lymphocyte analysis is part of the "White Blood Cell Endpoints in AD" (WEAD) study we designed as an add-on to the SEAD2 study.

Approvals and Human Subjects
The Kansas University Medical Center Human Subjects Committee (KUMC HSC) approved all human subject participation and all participants provided informed consent prior to enrolling. This study was conducted in accordance with the Code of Ethics of the World Medical Association (the Declaration of Helsinki). We enrolled participants who met McKhann et al. AD diagnostic criteria (20). Participants were excluded if they reported any potentially confounding, serious medical risks such as type 1 diabetes, cancer, or a recent cardiac event such as a heart attack or angioplasty. At the beginning of the study the trial participants underwent a 40 ml phlebotomy. Autopsy brain samples were obtained from the KU Alzheimer's Disease Research Center (KU ADRC) Neuropathology Core. The KU ADRC maintains a clinical cohort and collects brains from consenting cohort decedents. The autopsy consent process is approved by the KU HSC.
Phlebotomy and Blood Cell Separation Forty ml of blood was collected in tubes containing acid-citrate-dextrose anticoagulant. One ml of whole blood was removed and stored at -80°C for genotyping; the rest was used for platelet and lymphocyte harvesting.
Fifteen ml of Histopaque 1077 was centrifuged in an AccuSpin tube for 1 minute at 1700 x g. Blood was layered on top of the AccuSpin tube frit and centrifuged for 15 minutes at 400 x g. Platelet-rich plasma and the buffy coat were collected in separate tubes and pelleted by centrifugation for 15 minutes at 1700 x g. The pellets were washed with phosphate buffered saline (PBS) and re-centrifuged. Platelets were used for mitochondrial isolation (see below). Lymphocytes were used for ow cytometry biomarkers and tissue culture expansion (see below). Blood samples were processed on the same day as the blood draw.

Mitochondrial Isolation
Platelets were resuspended in MSHE buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.4) and disrupted by nitrogen cavitation, at 1200 psi, for 20 minutes. The ruptured platelets were centrifuged at 1000 x g for 15 minutes, 4°C. The supernatant was transferred to a new tube, while the pellet (intact platelets) was resuspended in MSHE buffer and subjected to nitrogen cavitation for a second time (1200 psi for 20 minutes). Both supernatants were combined and centrifuged at 12,000 x g for 10 minutes, 4°C. The resulting mitochondrial pellet was resuspended in MSHE buffer.

COX and CS Vmax Assays
We added aliquots of the enriched platelet mitochondrial suspensions to cuvettes and spectrophotometrically determined each suspension's COX and CS Vmax activities. For the COX Vmax, we followed the conversion of reduced cytochrome c to oxidized cytochrome c and calculated the pseudo-rst order rate constant (msec -1 ). For the CS Vmax, we followed the formation of 5-thio-2nitrobenzoate (nmol/min) (21). The COX rate was normalized to mg protein (msec -1 /mg protein) or to the CS rate (yielding a value with units of msec -1 /nmol/min), which we herein refer to simply as COX/CS. The CS rate was normalized to mg protein to yield a nal activity with units of nmol/min/mg protein.

Dye-Based Assays and Flow Cytometry
Fresh lymphocytes were suspended in HBSS (with Ca 2+ /Mg 2+ ) at 1x10^6/mL. Four mL of lymphocytes were used for negative (no stain), JC1, MitoSox, and Mitotracker/Annexin V staining. For staining we used 10 μL of 200 μM JC1, 4 μL of 10 μM Mitotracker Red, 2 μL of 5 mM MitoSox, or no dye. Cells were incubated at 37°C with 5% CO 2 for 30 minutes. All samples were centrifuged to pellet cells (1700 x g for 5 minutes). Cells were washed with HBSS and centrifuged again (1700 x g for 5 minutes). The supernatants were removed and 500 μL of HBSS was added to each tube (for all samples except the MitoTracker/Annexin V samples). For MitoTracker/Annexin V samples, 100 μL of 1X Annexin V binding buffer was added with 5 μL of Annexin V dye. MitoTracker/Annexin V samples were incubated at room temperature for 15 minutes, following which 400 μL of 1X Annexin V binding buffer was added. All samples were placed on ice and immediately analyzed using an LSRII ow cytometer (BD Bioscience).

Lymphocyte Culture
Lymphocytes were resuspended in complete RPMI medium (RPMI, 10% FBS, Pen/Strep, 20 U/mL IL-2, and 20 ng/mL CD3) at 1x10^6 cells/mL in a T75 culture ask. Cells were fed every other day and split as needed to keep cell concentrations at 1x10^6 cells/mL. Cells were used for immunochemistry as described below after seven days of culture.

Immunochemistry
For expanded lymphocyte cultures and autopsied human brain superior frontal gyrus tissue sections, protein was collected in RIPA buffer with protease/phosphatase inhibitors (ThermoFisher). Equal protein amounts were resolved by SDS-PAGE (Criterion TGX gels, BioRad) and proteins were transferred to PVDF membranes (ThermoFisher). Immunoblots were completed and antibodies are listed in Table 1. To visualize bands, we used WestFemto Super Signal HRP Substrate (ThermoFisher) and a ChemiDoc XRS imaging platform.

APOE Genotyping
We used a single nucleotide polymorphism (SNP) allelic discrimination assay to determine APOE genotypes. This involved adding 5 ul of blood to a Taqman Sample-to-SNP kit (ThermoFisher). Taqman probes to the two APOE-de ning SNPs, rs429358 (C_3084793_20) and rs7412 (C _904973_10) (ThermoFisher), were used to identify APOE ε2, ε3, and ε4 alleles.

Data and Statistical Analyses
We organized the data from the participants into two groups, one in which participants had at least one APOE ε4 allele, and one in which they did not. We compared means by two-way Student's T-tests with signi cance de ned as p<0.05. Table 2 summarizes subject demographics. As we found in our prior study, APOE ε4 carriers have lower platelet mitochondrial COX Vmax activities when compared to non-carriers (ε3/ε3) ( Figure 1). Unlike our prior study, we found no change in CS Vmax with APOE genotype. Supplemental data table 1 (Table S1) summarizes enzyme Vmax ndings by individual genotypes (heterozygotes versus homozygotes).

Results
Lymphocytes from APOE ε4 carriers and non-carriers showed similar levels of mitochondrial superoxide production (MitoSox), mitochondrial membrane potential (JC1), and mitochondrial number (MitoTracker). These data are summarized in Table S2 (supplemental data). We observed an increase in apoptotic lymphocytes from APOE ε4 carriers versus non-carriers (ε3/ε3) ( Figure 2). Supplemental data table 3 (Table S3) examines the platelet mitochondria and fresh lymphocyte biomarkers by sex in these AD subjects. In addition to women, we can now report biochemical phenotypes extend to men with AD. Comparing women to men revealed only one difference, which was women had a signi cantly higher lymphocyte mitochondrial membrane potential.
To facilitate lymphocyte protein expression assays we expanded them in culture. Levels of PINK1, a protein involved in mitophagy, was higher in APOE ε4 carrier lymphocytes. mTOR and SIRT1, which play a role in energy sensing, were different between groups. Speci cally, mTOR phosphorylation decreased while SIRT1 phosphorylation increased in APOE ε4 carrier lymphocytes. We also observed changes in the lipid synthesis pathway. AceCSI and ATP CL increased, and ACC phosphorylation increased (Figure 3-4). For data summarizing all proteins examined see supplemental data table 4 (Table S4).
The change in ACC phosphorylation appeared especially robust, which prompted us to examine this parameter in the superior frontal gyrus of post-mortem AD and control human brains. APOE ε4 carrier post-mortem human brain samples showed increased pACC regardless of whether the subject donor carried an AD diagnosis (Figure 4).

Discussion
Here we rea rm a smaller study (16) that found lower platelet COX activity in women with AD and an APOE ε4 allele, versus those without an APOE ε4 allele. We further extend this nding to men with AD and an APOE ε4 allele. We did not replicate the smaller study's nding of lower platelet CS Vmax activities in the APOE ε4 carriers.
COX Vmax is lower in AD subjects, a nding replicated in brain, broblasts, and blood cells (14,16,(21)(22)(23)(24)(25)(26)(27)(28)(29). Mechanistic studies show that COX is assembled differently in AD cohorts, which may contribute to Vmax de cits (30). Additional studies associate the COX de cit with changes to its mRNA expression within brain (31,32). It is apparent that the COX de cit is attributed to at least some extent to mitochondrial DNA, either through inheritance or somatic mutations (21,33). De cits in COX functionality will lead to bioenergetic stress including changes to redox balance and ATP production (24,33). As a systemic biomarker, reduced platelet COX Vmax correlates strongly with brain glucose metabolism (34). Future studies should leverage this systemic biomarker to understand the origins of bioenergetic stress observed in AD.
To extend our biomarker observations, we utilized lymphocytes from the same blood draw. APOE ε4 carriers showed an increase in pSIRT1, PINK1, AceCS1, ATP CL, and pACC levels; a decrease in pmTOR; and an increase in Annexin V staining. We found no changes in lymphocyte mitochondrial mass, mitochondrial membrane potential, or mitochondrial superoxide using our methodologies. We did see a sex difference in the lymphocyte mitochondrial membrane potential, in which female AD subjects had higher mitochondrial membrane potentials compared to male AD subjects.
Lymphocyte apoptosis can be attributed to "neglect" or loss of extrinsic signals, a process that occurs through mitochondrial energy failure and the loss of anapleurosis (35)(36)(37)(38)(39). Bioenergetic stress, as a consequence of reduced glucose metabolism, may play a role in lymphocyte apoptosis (37,38,40). Our overall ndings suggest increased lymphocyte apoptosis may re ect a consequence of bioenergetic stress.
A previous study claimed SIRT1 phosphorylation at the site we interrogated re ects SIRT1 activation (41). SIRT1 regulates chromatin remodeling, allowing for gene expression changes that adapt to stress (42). SIRT1 functions to alter cell metabolism including glycolysis ux, lipid homeostasis, insulin secretion, and in ammation. Energy stress activates SIRT1, which essentially serves as a stress response master regulator (42)(43)(44)(45)(46). mTOR promotes cell growth. It activates under anabolic conditions that coincide with energy-su cient states and deactivates under catabolic conditions of energy stress. Serine 2448 phosphorylation levels positively correlate with mTOR activity, and suggest a downstream stimulation of mTORC2 and mTORC1, protein complexes implicated in cell metabolic regulation (47,48). Decreased mTOR 2448 phosphorylation in APOE ε4 carrier lymphocytes suggests that allele shifts the anabolic-catabolic balance to a more catabolic setting.
Cells experiencing catabolic shifts typically increase autophagy, a process of internal digestion that replenishes raw molecular materials. PINK1 helps mediate autophagy and increased PINK1 suggests increased mitophagy/autophagy ux (49). Furthermore, mTOR modulates autophagy through ULK1 (unc-51 like kinase 1 or ATG1), as inhibition of mTOR during nutrient starvation leads to activation of ULK1 and autophagy (50). In this case, elevated PINK1 and reduced mTOR activation in AD APOE ε4 carrier lymphocytes suggests some degree of cell-level energy stress in the APOE ε4 carriers.
The most robust nding in lymphocytes was the increase in pACC and ACC expression by APOE genotype. ACC is an enzyme which converts acetyl coA into malonyl coA through carboxylation, which represents an early integral step in fatty acid synthesis. ACC phosphorylation inhibits its activity and turns off lipid biosynthesis. To understand if this change was speci c, we also examined ACC expression and phosphorylation in human post-mortem brain samples. We found that pACC levels also increased in brains from AD and APOE ε4 carriers.
AceCS1 is a cytosolic enzyme that catalyzes the conversion of acetate and CoA to acetyl-CoA, where it enters lipid synthesis. SIRT1 reportedly regulates its activity (46). ATP CL converts citrate to acetyl CoA and oxaloacetate, and links carbohydrate and fatty acid metabolism. Both ATP CL and AceCS1 expression are higher in APOE ε4 carriers, which could represent a cause or consequence of the observed ACC changes. Increased AceCS1 and ATP CL could potentially increase if an ACC-mediated reduction in lipid biosynthesis leads to a secondary increase in acetyl coA. Based on this pattern of observations, investigating acetyl CoA and its up/downstream metabolites in APOE ε4 carriers could prove informative.
Our data indicate that compared to AD APOE ε4 non-carriers, AD APOE ε4 carrier lymphocytes exhibit a relative state of bioenergetic stress and catabolic shift. We already know from uorodeoxyglucose positron emission tomography (FDG PET) studies that brains from cognitively normal, middle-aged APOE ε4 carriers show reduced glucose utilization (11,13,51,52), but clearly this APOE-dependent metabolic phenotype extends beyond the brain.
The APOE ε4 allele also associates with an increased burden of brain white matter hyperintensity (WMH) (7,(53)(54)(55)(56). Speculated causes of WMHs include microvascular disease, defective myelin, gliosis, in ammation, neurodegeneration, or a combination of these factors. Neuroimaging studies suggest APOE ε4 carriers have increased myelin breakdown and these effects can be found in infants during brain development (57)(58)(59). Myelin represents a sizeable brain lipid depository, and in a state of bioenergetic stress or starvation the brain may switch towards a more catabolic state that features myelin consumption over synthesis (60).
The literature emphasizes hepatocytes, astrocytes, and macrophages, but not lymphocytes and platelets, express the APOE gene. The presence of an APOE-associated molecular phenotype in lymphocytes, which others also report (61), and platelets warrants consideration (16). Perhaps circulating APOE protein in uences these cells. Under stress conditions neurons will increase APOE expression, which raises the question of whether other stressed cell types demonstrate this behavior. Maybe even low levels of expression can impact a cell. Myeloid APOE can alter lymphocyte biology through non-cell autonomous signaling events (62). Other genes with variants in linkage disequilibrium with the APOE isoforms, such as TOMM40, could also play a role (63).

Conclusion
Despite years of research, the mechanisms that underlie the AD-APOE association remain unclear. Our data support the view that bioenergetic metabolism-related stresses may mediate this. As APOE expression occurs outside the brain and is functionally important outside the brain, it seems reasonable to propose a systemic AD phenotype may exist. This could manifest as subtle metabolic shifts of the type we now demonstrate and could perhaps explain other associations including reported connections between dementia and type II diabetes mellitus (64). This study also argues peripheral metabolism biomarkers may re ect brain metabolism. If further validated, peripheral biomarkers like the ones we now show, or those developed by others (65)

Declarations
Ethics approval and consent to participate. The Kansas University Medical Center Human Subjects Committee (KUMC HSC) approved all human subject participation and all participants provided informed consent prior to enrolling. This study was conducted in accordance with the Code of Ethics of the World Medical Association (the Declaration of Helsinki).

Consent for publication. NA
Availability of data and materials. The datasets generated and/or analysed during the current study are not publicly available due to restrictions with our HSC approval but are available from the corresponding author on reasonable request.  Fresh Lymphocyte Apoptotic Biomarker. Lymphocytes were isolated and stained for Annexin V as described in materials and methods. % lymphocytes positive for Annexin V by APOE genotype (ε4 carriers versus ε3/ε3). * indicates p<0.05.

Figure 4
Cultured Lymphocyte and Autopsy Human Brain lipid signaling pathway protein expression. Lymphocytes (or autopsied human brain) were lysed and assayed for protein expression as described in materials and methods. A. Lymphocyte AceCS1/ACTIN densitometry by APOE genotype B. Lymphocyte ATP CL/ACTIN densitometry by APOE genotype C. Lymphocyte ACC densitometry by APOE genotype E. ACC densitometry in autopsied human brain samples by APOE genotype or diagnosis. Red indicates ND subjects when data are separated by APOE genotype. Green indicates APOE ε4 Non-Carriers when data are separated by diagnosis. * indicates p<0.05, ** indicates p<0.01. Data are shown as mean +/-SEM.