Here we reaffirm 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 finding to men with AD and an APOE ε4 allele. We did not replicate the smaller study’s finding of lower platelet CS Vmax activities in the APOE ε4 carriers.
COX Vmax is lower in AD subjects, a finding replicated in brain, fibroblasts, and blood cells (14, 16, 21-29). Mechanistic studies show that COX is assembled differently in AD cohorts, which may contribute to Vmax deficits (30). Additional studies associate the COX deficit with changes to its mRNA expression within brain (31, 32). It is apparent that the COX deficit is attributed to at least some extent to mitochondrial DNA, either through inheritance or somatic mutations (21, 33). Deficits 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 (35-39). Bioenergetic stress, as a consequence of reduced glucose metabolism, may play a role in lymphocyte apoptosis (37, 38, 40). Our overall findings suggest increased lymphocyte apoptosis may reflect a consequence of bioenergetic stress.
A previous study claimed SIRT1 phosphorylation at the site we interrogated reflects 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 flux, lipid homeostasis, insulin secretion, and inflammation. Energy stress activates SIRT1, which essentially serves as a stress response master regulator (42-46).
mTOR promotes cell growth. It activates under anabolic conditions that coincide with energy-sufficient 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 flux (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 finding 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 specific, 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 fluorodeoxyglucose 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, 53-56). Speculated causes of WMHs include microvascular disease, defective myelin, gliosis, inflammation, 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 (57-59). 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 influences 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).