Oxaloacetate Enhances Neuronal Cell Bioenergetic Fluxes and Infrastructure

"This is the peer reviewed version of the following article: Wilkins, Heather M. et al. “Oxaloacetate Enhances Neuronal Cell Bioenergetic Fluxes and Infrastructure.” Journal of neurochemistry 137.1 (2016): 76–87., which has been published in final form at 10.1111/jnc.13545. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving."We tested how the addition of oxaloacetate (OAA) to SH-SY5Y cells affected bioenergetic fluxes and infrastructure, and compared the effects of OAA to malate, pyruvate, and glucose deprivation. OAA displayed pro-glycolysis and pro-respiration effects. OAA pro-glycolysis effects were not a consequence of decarboxylation to pyruvate because unlike OAA, pyruvate lowered the glycolysis flux. Malate did not alter glycolysis flux and reduced mitochondrial respiration. Glucose deprivation essentially eliminated glycolysis and increased mitochondrial respiration. OAA increased, while malate decreased, the cell NAD+/NADH ratio. Cytosolic malate dehydrogenase 1 (MDH1) protein increased with OAA treatment, but not with malate or glucose deprivation. Glucose deprivation increased protein levels of ATP citrate lyase, an enzyme which produces cytosolic OAA, while OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased COX2, PGC1α, PGC1β, and PRC protein levels. OAA increased total and phosphorylated SIRT1 protein. We conclude that adding OAA to SH-SY5Y cells can support or enhance both glycolysis and respiration fluxes. These effects appear to depend, at least partly, on OAA causing a shift in the cell redox balance to a more oxidized state, that it is not a glycolysis pathway intermediate, and possibly its ability to act in an anaplerotic fashion

Mitochondrial DNA Manipulations Affect Tau Oligomerization

Background:Mitochondrial dysfunction and tau aggregation occur in Alzheimer’s disease (AD), and exposing cells or rodents to mitochondrial toxins alters their tau. Objective:To further explore how mitochondria influence tau, we measured tau oligomer levels in human neuronal SH-SY5Y cells with different mitochondrial DNA (mtDNA) manipulations. Methods:Specifically, we analyzed cells undergoing ethidium bromide-induced acute mtDNA depletion, ρ0 cells with chronic mtDNA depletion, and cytoplasmic hybrid (cybrid) cell lines containing mtDNA from AD subjects. Results:We found cytochrome oxidase activity was particularly sensitive to acute mtDNA depletion, evidence of metabolic re-programming in the ρ0 cells, and a relatively reduced mtDNA content in cybrids generated through AD subject mitochondrial transfer. In each case tau oligomer levels increased, and acutely depleted and AD cybrid cells also showed a monomer to oligomer shift. Conclusion:We conclude a cell’s mtDNA affects tau oligomerization. Overlapping tau changes across three mtDNA-manipulated models establishes the reproducibility of the phenomenon, and its presence in AD cybrids supports its AD-relevance

Respiratory chain deficiency alters cellular proteostasis and triggers Alzheimer’s disease-like tau alterations

Sporadic Alzheimer’s disease (AD) is defined clinically as a progressive brain disorder resulting in memory loss and, eventually, an inability to perform simple tasks. Pathologically, AD brains accumulate insoluble protein aggregates known as neurofibrillary tangles (NFTs) and amyloid plaques. Definitive diagnosis of AD requires the presence of NFTs and amyloid plaques. Furthermore, variants in genes coding for amyloid associate with early onset forms of the disease. For these reasons and more, removing amyloid plaques from sporadic AD patient brains has been the field’s major therapeutic target for decades. Unfortunately, clinical trials focused on treating AD through amyloid reduction continue to fail. The current standard of care for AD typically extends patient lifespan for months rather than years. The field needs new therapeutic targets and rescuing brain energy production represents a reasonable strategy. Reduced glucose utilization occurs early in AD brains and correlates fairly well with disease progression. Widespread mitochondrial dysfunction accompanies decreased glucose consumption. AD mitochondria display changes in number, ultrastructure, and enzyme activity. The evidence for mitochondrial dysfunction in AD is clear, however, the notion that defective mitochondria could initiate pathological cascades remains controversial. Thus, therapies aimed at mitochondrial function have been slow to reach clinical trials. The following studies examine the relationship between mitochondrial defects and AD pathology and provide evidence that mitochondrial dysfunction leads to AD-relevant retrograde responses. Retrograde responses maintain cellular homeostasis by adapting nuclear gene expression and cytosolic signaling pathways to changes in mitochondrial function. AD mitochondrial dysfunction likely initiates numerous retrograde responses, yet few studies examine defective mitochondria’s influence on AD pathology. Here, we provide evidence that reduced mitochondrial respiratory flux leads to AD-like tau alterations, including changes in splicing, conformation and oligomerization. Alzheimer’s disease cybrids recapitulate disease relevant tau alterations. Further experiments suggest mitochondrial function affects cellular proteostasis pathways including the mitochondrial unfolded protein response (mtUPR), integrated stress response (ISR), autophagy/mitophagy, and proteasome function. Although initial studies in C. elegans and rat hepatoma cells established a link between mtDNA depletion and mtUPR activation, we find mammalian cells downregulate the mtUPR upon mtDNA depletion. Instead, mtDNA depleted human cells activate the ISR, a pathway which alters cellular metabolism and halts general protein translation to preserve proteostasis during stress. Finally, we examine how mtDNA depletion affects cytochrome oxidase (COX) and complex I activity. AD tissue displays decreased COX activity, while complex I activity does not change. The reason for specific reductions in COX activity remain unclear. We found COX activity decreases proportionally to declines in mtDNA levels. Whether complex I activity follows the same pattern will give insight into potential mechanisms for reduced COX activity in AD

Mitochondrial Dysfunction and Stress Responses in Alzheimer’s Disease

Alzheimer’s disease (AD) patients display widespread mitochondrial defects. Brain hypometabolism occurs alongside mitochondrial defects, and correlates well with cognitive decline. Numerous theories attempt to explain AD mitochondrial dysfunction. Groups propose AD mitochondrial defects stem from: (1) mitochondrial-nuclear DNA interactions/variations; (2) amyloid and neurofibrillary tangle interactions with mitochondria, and (3) mitochondrial quality control defects and oxidative damage. Cells respond to mitochondrial dysfunction through numerous retrograde responses including the Integrated Stress Response (ISR) involving eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). AD brains activate the ISR and we hypothesize mitochondrial defects may contribute to ISR activation. Here we review current recognized contributions of the mitochondria to AD, with an emphasis on their potential contribution to brain stress responses

Mitochondria-Derived Damage-Associated Molecular Patterns in Neurodegeneration

Inflammation is increasingly implicated in neurodegenerative disease pathology. As no acquired pathogen appears to drive this inflammation, the question of what does remains. Recent advances indicate damage-associated molecular pattern (DAMP) molecules, which are released by injured and dying cells, can cause specific inflammatory cascades. Inflammation, therefore, can be endogenously induced. Mitochondrial components induce inflammatory responses in several pathological conditions. Due to evidence such as this, a number of mitochondrial components, including mitochondrial DNA, have been labeled as DAMP molecules. In this review, we consider the contributions of mitochondrial-derived DAMPs to inflammation observed in neurodegenerative diseases

A Bioenergetics Systems Evaluation of Ketogenic Diet Liver Effects

Ketogenic diets induce hepatocyte fatty acid oxidation and ketone body production. To further evaluate how ketogenic diets affect hepatocyte bioenergetic infrastructure, we analyzed livers from C57Bl/6J male mice maintained for one month on a ketogenic or standard chow diet. Compared to the standard diet, the ketogenic diet increased cytosolic and mitochondrial protein acetylation and also altered protein succinylation patterns. SIRT3 protein decreased while SIRT5 protein increased, and gluconeogenesis, oxidative phosphorylation, and mitochondrial biogenesis pathway proteins were variably and likely strategically altered. The pattern of changes observed can be used to inform a broader systems overview of how ketogenic diets affect liver bioenergetics.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author