White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer's Disease

Abstract

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer's. Age remains the greatest risk factor for Alzheimer's and the prevalence of age-related late onset Alzheimer's is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer's risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer's and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

Keywords: ABAD, Aβ-binding alcohol dehydrogenase; ABAD, Aβ-binding-alcohol-dehydrogenase; ACER3, alkaline ceramidase; AD, Alzheimer's disease; APO-ε4, apolipoprotein ε4; APP, amyloid precursor protein; Aging oxidative stress; Alzheimer's disease; BACE1, beta-secretase 1; BBB, blood brain barrier; CC, corpus callosum; CMRglu, cerebral glucose metabolic rate; COX, complex IV cytochrome c oxidase; CPT1, carnitine palmitoyltransferase 1; Cldn11, claudin 11; Cyp2j6, arachidonic acid epoxygenase; Cytosolic phospholipase A2; DHA, docosahexaesnoic acid; Erbb3, Erb-B2 receptor tyrosine kinase 3; FDG-PET, 2-[18F]fluoro-2-deoxy-d-glucose; GFAP, glial fibrillary acidic protein; H2O2, hydrogen peroxide; HADHA, hydroxyacyl-CoA dehydrogenase; HK, hexokinase; Ketone bodies; LC MS, liquid chromatography mass spectrometer; MAG, myelin associated glycoprotein; MBP, myelin basic protein; MCT1, monocarboxylate transporter 1; MIB, mitochondrial isolation buffer; MOG, myelin oligodendrocyte glycoprotein; MTL, medial temporal lobe; Mitochondria; NEFA, nonesterified fatty acids; Neurodegeneration; OCR, oxygen consumption rate; Olig2, oligodendrocyte transcription factor; PB, phosphate buffer; PCC, posterior cingulate; PCR, polymerase chain reaction; PDH, pyruvate dehydrogenase; PEI, polyethyleneimine; RCR, respiratory control ratio; ROS, reactive oxygen species; S1P, sphingosine; TLDA, TaqMan low density array; WM, white matter; WT, wild type; White matter; cPLA2, cytosolic phospholipase A2.

Fig. 1

Mitochondrial function and cytoplasmic phospholipase A₂ activity in reproductively aging female mice: A. Respiratory control ratio (RCR = OCR_{state 4}/OCR_{state 3}) and B. H₂O₂ production in isolated whole brain mitochondria from reproductively aging female mice. C. cPLA₂ enzyme activity in whole brain tissue homogenate obtained during mitochondrial isolation preparation. (A. N = 6–8; B. N = 6–7; C. N = 5–7.) (*p < 0.05, **p < 0.005 and ***p < 0.0005).

Fig. 2

Cytoplasmic phospholipase A₂-sphingomyelinase pathway activation during reproductive senescence: A. cPLA₂ enzyme activity in hippocampal tissue homogenate. B. Arachidonic acid production determined by GC-MS in tissue homogenate obtained during mitochondrial isolation preparation. B. Acid sphingomyelinase enzyme activity in hippocampal tissue homogenate. (A. N = 4–11; B. N = 3,-4; C. N = 7–12.) (*p < 0.05, ***p < 0.0005, ****p < 0.00005).

Fig. 3

Co-localization of immunoreactivity of reactive astrocytes and cytoplasmic phospholipase A₂ in brain slices from the aging female mouse model: white matter tracts were co-labeled with GFAP (FITC) and cPLA₂ (CY3) antibodies, and assessed for astrocyte reactivity and cPLA₂ cellular localization in the A. fimbria, B. cingulum, C. the Schaffer collateral pathway, and D. anterior commissure during reproductive aging. E. Representative images depicting GFAP (FITC) and cPLA₂ (CY3) labeling in the cingulum of reproductively incompetent female mice. (A, B, C, D. N = 4 per group) (*p < 0.05, and ***p < 0.0005).

Fig. 4

Cytoplasmic phospholipase A₂ enzyme activity in cultured astrocytes and neurons following H₂O₂ exposure: embryonic hippocampal neurons and astrocytes in culture were treated with H₂O₂ and assessed for cPLA₂ activation using an enzyme activity assay. cPLA₂ enzyme activity normalized to untreated cells in cultured A. astrocytes and B. neurons. C. cPLA₂ enzyme activity normalized to untreated cells in cultured astrocytes following exposure to physiologically relevant concentrations of H₂O₂. H₂O₂ concentrations were determined by the levels of H₂O₂ produced by whole brain mitochondria isolated from reproductively incompetent female mice. D. Levels of cPLA₂ enzyme activation in astrocytes and neurons (nmol/min/ml/mg of protein).

Fig. 5

Myelin and fatty acid metabolism, myelin generation and repair, and inflammation related gene expression in reproductively aging female mice: A. Table depicting the fold change and p-value differences in expression of white matter related genes between reproductively irregular and reproductively incompetent female mice. Red p-value is indicative of upregulation while a green p-value indicates down regulation. Boxes outlined in red reached statistical significance. B. Heatmaps of gene expression organized by function: myelin and fatty acid metabolism, myelin generation and repair, and inflammation. Each gene was normalized and colored based on its relative expression level across 4 reproductive aging groups. This method allows genes that have different magnitude of signal intensity but which belong to the same functional group and share similar expression patterns to be displayed on the same heatmap. (N = 5 per group.)

Fig. 6

Immunohistochemical and Western blot analysis of white matter integrity in the aging female mouse model: A. Immunohistochemical mapping of myelin basic protein area in the corpus callosum of the aging female mouse model. B. Representative immunohistochemical images mapping myelin basic protein area in the anterior commissure and corpus callosum. Mask of corpus callosum generated via slide book software is highlighted in purple. C. Myelin basic protein expression during reproductive aging in the female mouse. (C. N = 4–10 per group). (*p < 0.05).

Fig. 7

Electron microscopic analysis of the structural integrity of white matter in reproductively aging female mice: representative electron microscopy images of myelinated axons in the anterior commissure of A. reproductively irregular, B. reproductively incompetent and C. aged female mice. Quantitative analysis of the percentage of compromised axons in the D. Schaffer collateral pathway, E. anterior commissure and F. corpus callosum in reproductively aging female mice. (A, B, C. N = 3–4; D. N = 4; E. N = 4; F. N = 3). (*p < 0.05) and **p < 0.005).

Fig. 8

Electron microscopic analysis of lipid droplet accumulation in reproductively aging female mice: representative electron microscopy images of lipid droplet accumulation in the anterior commissure of A. reproductively irregular, B. reproductively incompetent and C. aged female mice. Average number of lipid droplets per cell body in the D. anterior commissure and E. corpus callosum. (A, B. N = 3–4; C. N = 4; D. N = 4; E. N = 3). (*p < 0.05).

Fig. 9

Lipid profile of female brain during reproductive aging: A. Sequence of catabolic events that occur during myelin breakdown. B. Levels of brain lipids in reproductively irregular, reproductively incompetent and aged female mice. Lipids were separated into three categorical panels: ceramides, non-esterified fatty acids and TCA metabolites. Red values are indicative of the peak level of a particular lipid and green values indicate statistical significance. (N = 4–6 per group.)

Fig. 10

Expression of proteins involved in fatty acid transport and metabolism in the aging female mouse model: A. CPT1 protein expression in isolated whole brain mitochondria. B. HADHA protein expression in isolated whole brain mitochondria. C. ABAD protein expression in isolated whole brain mitochondria (A, B, C. N = 3–8). (**p < 0.005 and ***p < 0.0005).

Fig. 11

Hippocampal, cortical and plasma ketone body levels in the aging female mouse: A. Hippocampal levels of ketone bodies. B. Cortical levels of ketone bodies. C. Plasma levels of ketone bodies. (A. N = 4 per group; B. N = 5–6; C. N = 3–7). (*p < 0.05 and **p < 0.005).

Fig. 12

Schematic model of mitochondrial H₂O₂ activation of cPLA₂-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA₂-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H₂O₂, in the prodromal/preclinical phase of Alzheimer's with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA₂-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.