ß-Hydroxybutyrate preferentially enhances neuron over astrocyte respiration while signaling cellular quiescence.

While ketone bodies support overall brain energy metabolism, it is increasingly clear specific brain cell types respond differently to ketone body availability. Here, we characterized how SH-SY5Y neuroblastoma cell, primary neuron, and primary astrocyte bioenergetics and nutrient sensing pathways respond to ß-hydroxybutyrate (ßOHB). SH-SY5Y cells and primary neurons, but not astrocytes, exposed to ßOHB increased respiration and decreased PI3K-Akt-mTOR signaling. Despite increased carbon availability and respiration, SH-SY5Y cells treated with ßOHB reduced their overall metabolic activity and cell cycling rate. Levels of the quiescence-regulating Yamanaka factors increased to a broader extent in SH-SY5Y cells and primary neurons. We propose a ßOHB-induced increase in neuron respiration, accompanied by activation of quiescence associated pathways, could alleviate bioenergetic stress and limit cell senescence. This in turn could potentially benefit conditions, including brain aging and neurodegenerative diseases, that feature bioenergetic decline and cell senescence.

Bioenergetic and inflammatory systemic phenotypes in Alzheimer's disease APOE ε4-carriers.

We examined the impact of an APOE ε4 genotype on Alzheimer's disease (AD) subject platelet and lymphocyte metabolism. Mean platelet mitochondrial cytochrome oxidase Vmax activity was lower in APOE ε4 carriers and lymphocyte Annexin V, a marker of apoptosis, was significantly higher. Proteins that mediate mitophagy and energy sensing were higher in APOE ε4 lymphocytes which could represent compensatory changes and recapitulate phenomena observed in post-mortem AD brains. Analysis of the lipid synthesis pathway found higher AceCSI, ATP CL, and phosphorylated ACC levels in APOE ε4 lymphocytes. Lymphocyte ACC changes were also observed in post-mortem brain tissue. Lymphocyte RNAseq showed lower APOE ε4 carrier sphingolipid Transporter 3 (SPNS3) and integrin Subunit Alpha 1 (ITGA1) expression. RNAseq pathway analysis revealed APOE ε4 alleles activated inflammatory pathways and modulated bioenergetic signaling. These findings support a relationship between APOE genotype and bioenergetic pathways and indicate platelets and lymphocytes from APOE ε4 carriers exist in a state of bioenergetic stress. Neither medication use nor brain-localized AD histopathology can account for these findings, which define an APOE ε4-determined molecular and systemic phenotype that informs AD etiology.

A ketogenic diet differentially affects neuron and astrocyte transcription.

Ketogenic diets (KDs) alter brain metabolism. Multiple mechanisms may account for their effects, and different brain regions may variably respond. Here, we considered how a KD affects brain neuron and astrocyte transcription. We placed male C57Bl6/N mice on either a 3-month KD or chow diet, generated enriched neuron and astrocyte fractions, and used RNA-Seq to assess transcription. Neurons from KD-treated mice generally showed transcriptional pathway activation while their astrocytes showed a mix of transcriptional pathway suppression and activation. The KD especially affected pathways implicated in mitochondrial and endoplasmic reticulum function, insulin signaling, and inflammation. An unbiased analysis of KD-associated expression changes strongly implicated transcriptional pathways altered in AD, which prompted us to explore in more detail the potential molecular relevance of a KD to AD. Our results indicate a KD differently affects neurons and astrocytes, and provide unbiased evidence that KD-induced brain effects are potentially relevant to neurodegenerative diseases such as AD.

Detection of mitochondria-pertinent components in exosomes.

We screened cell line and plasma-derived exosomes for molecules that localize to mitochondria or that reflect mitochondrial integrity. SH-SY5Y cell-derived exosomes contained humanin, citrate synthase, and fibroblast growth factor 21 protein, and plasma-derived exosomes contained humanin, voltage-dependent anion-selective channel 1, and transcription factor A protein. Nuclear mitochondrial (NUMT) DNA complicated analyses of mitochondrial DNA (mtDNA), which otherwise suggested exosomes contain at most very low amounts of extended mtDNA sequences but likely contain degraded pieces of mtDNA. Cell and plasma-derived exosomes contained several mtDNA-derived mRNA sequences, including those for ND2, CO2, and humanin. These results can guide exosome-focused, mitochondria-pertinent biomarker development.

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.

Effects of Microglial Cytokines on Alzheimer's Disease-Related Phenomena.

Recent association studies indicate several genes highly expressed by microglia influence Alzheimer's disease (AD) risk, which suggests microglial function contributes to this disease. Here, we evaluated how one component of microglial function, cytokine release, affects AD-related phenomena. First, we used a 3-hour lipopolysaccharide (LPS) treatment to activate mouse BV2 microglial cells. Next, we removed the LPS-containing medium, added LPS-free medium, and after 6 hours collected the medium conditioned by the activated BV2 microglial cells. We then exposed human neuronal SH-SY5Y cells to the conditioned medium for 24 hours. At the end of the 24-hour exposure, we assessed amyloid-ß protein precursor (AßPP), tau, apolipoprotein E (ApoE), and lipid status. The amount of AßPP was unaffected, although a slight decrease in soluble AßPPα suggested a subtle reduction in AßPP non-amyloidogenic processing occurred. Tau mRNA increased, but total and phosphorylated tau levels were unchanged. ApoE mRNA increased, while ApoE protein levels were lower. Per cell lipid droplet number decreased and lipid oxidation increased. These results show cytokine release by activated microglial cells can influence specific AD-relevant physiologies and pathologies.

Neuroketotherapeutics: A modern review of a century-old therapy.

Neuroketotherapeutics represent a class of bioenergetic medicine therapies that feature the induction of ketosis. These therapies include medium-chain triglyceride supplements, ketone esters, fasting, strenuous exercise, the modified Atkins diet, and the classic ketogenic diet. Extended experience reveals persons with epilepsy, especially pediatric epilepsy, benefit from ketogenic diets although the mechanisms that underlie its effects remain unclear. Data indicate ketotherapeutics enhance mitochondrial respiration, promote neuronal long-term potentiation, increase BDNF expression, increase GPR signaling, attenuate oxidative stress, reduce inflammation, and alter protein post-translational modifications via lysine acetylation and ß-hydroxybutyrylation. These properties have further downstream implications involving Akt, PLCγ, CREB, Sirtuin, and mTORC pathways. Further studies of neuroketotherapeutics will enhance our understanding of ketone body molecular biology, and reveal novel central nervous system therapeutic applications.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism.

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with ß-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

A Mitochondrial Biomarker-Based Study of S-Equol in Alzheimer's Disease Subjects: Results of a Single-Arm, Pilot Trial.

Reductions in bioenergetic fluxes, mitochondrial enzyme activities, and mitochondrial number are observed in Alzheimer's disease (AD). Preclinical work indicates estrogen pathway signaling by either estrogen or selective ß estrogen receptor (ERß) agonists benefits these parameters. To assess whether an ERß agonist could improve mitochondrial function in actual AD subjects, we administered S-equol (10 mg twice daily) to 15 women with AD and determined the platelet mitochondria cytochrome oxidase (COX) activity before initiating S-equol (lead-in), after two weeks of S-equol (active treatment), and two weeks after stopping S-equol (wash-out). Because the intra-individual variation of this enzyme across samples taken at different times was unknown we used a nonparametric, single-arm, dichotomous endpoint that classified subjects whose active treatment COX activity exceeded the average of their lead-in and wash-out measures as positive responders. Eleven positive responses were observed (p < 0.06). The implications of this finding on our null hypothesis (that S-equol does not influence platelet mitochondria COX activity) are discussed. To our knowledge, this is the first time a direct mitochondrial target engagement biomarker has been utilized in an AD clinical study.

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 1 month on a ketogenic or standard chow diet. Compared with 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.

Platelet cytochrome oxidase and citrate synthase activities in APOE ε4 carrier and non-carrier Alzheimer's disease patients.

A degradation product of APOE ε4-encoded apolipoprotein E protein targets mitochondria and inhibits cytochrome oxidase (COX), and autopsy brains from young adult APOE ε4 carriers show reduced COX activity. To further explore relationships between APOE alleles and COX, we measured platelet mitochondria COX activity in AD subjects with (n=8) and without (n=7) an APOE ε4 allele and found the mean COX activity, when normalized to sample total protein, was lower in the APOE ε4 carriers (p<0.05). Normalizing COX activity to citrate synthase (CS) activity eliminated this difference, but notably the mean CS activity was itself lower in the APOE ε4 carriers (p<0.05). COX and CS protein levels did not appear to cause the lower APOE ε4 carrier COX and CS Vmax activities. If confirmed in larger studies, these data could suggest mitochondria at least partly mediate the well-recognized association between APOE alleles and AD risk.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aß1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Oxaloacetate enhances neuronal cell bioenergetic fluxes and infrastructure.

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 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, whereas OAA altered neither ATP citrate lyase mRNA nor protein levels. OAA, but not glucose deprivation, increased cytochrome oxidase subunit 2, PGC1α, PGC1ß, and PGC1 related co-activator 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. We examined how oxaloacetate (OAA) affects bioenergetic fluxes. To advance the understanding of how OAA mediates these changes, we compared the effects of OAA to malate, pyruvate, and glucose deprivation. We further examined how OAA affects levels of enzymes that facilitate its cytosolic metabolism, and found OAA increased the expression of malate dehydrogenase 1 (MDH1-cytosolic). We propose the following: OAA supports both glycolysis and respiration fluxes, shifts the cell redox balance toward a more oxidized state, and acts in an anaplerotic fashion. Abbreviations not defined in the text: MDH2, malate dehydrogenase 2 (mitochondrial).

Effect of one month duration ketogenic and non-ketogenic high fat diets on mouse brain bioenergetic infrastructure.

Diet composition may affect energy metabolism in a tissue-specific manner. Using C57Bl/6J mice, we tested the effect of ketosis-inducing and non-inducing high fat diets on genes relevant to brain bioenergetic infrastructures, and on proteins that constitute and regulate that infrastructure. At the end of a one-month study period the two high fat diets appeared to differentially affect peripheral insulin signaling, but brain insulin signaling was not obviously altered. Some bioenergetic infrastructure parameters were similarly impacted by both high fat diets, while other parameters were only impacted by the ketogenic diet. For both diets, mRNA levels for CREB, PGC1α, and NRF2 increased while NRF1, TFAM, and COX4I1 mRNA levels decreased. PGC1ß mRNA increased and TNFα mRNA decreased only with the ketogenic diet. Brain mtDNA levels fell in both the ketogenic and non-ketogenic high fat diet groups, although TOMM20 and COX4I1 protein levels were maintained, and mRNA and protein levels of the mtDNA-encoded COX2 subunit were also preserved. Overall, the pattern of changes observed in mice fed ketogenic and non-ketogenic high fat diets over a one month time period suggests these interventions enhance some aspects of the brain's aerobic infrastructure, and may enhance mtDNA transcription efficiency. Further studies to determine which diet effects are due to changes in brain ketone body levels, fatty acid levels, glucose levels, altered brain insulin signaling, or other factors such as adipose tissue-associated hormones are indicated.

Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis.

Brain bioenergetic function declines in some neurodegenerative diseases, this may influence other pathologies and administering bioenergetic intermediates could have therapeutic value. To test how one intermediate, oxaloacetate (OAA) affects brain bioenergetics, insulin signaling, inflammation and neurogenesis, we administered intraperitoneal OAA, 1-2 g/kg once per day for 1-2 weeks, to C57Bl/6 mice. OAA altered levels, distributions or post-translational modifications of mRNA and proteins (proliferator-activated receptor-gamma coactivator 1α, PGC1 related co-activator, nuclear respiratory factor 1, transcription factor A of the mitochondria, cytochrome oxidase subunit 4 isoform 1, cAMP-response element binding, p38 MAPK and adenosine monophosphate-activated protein kinase) in ways that should promote mitochondrial biogenesis. OAA increased Akt, mammalian target of rapamycin and P70S6K phosphorylation. OAA lowered nuclear factor κB nucleus-to-cytoplasm ratios and CCL11 mRNA. Hippocampal vascular endothelial growth factor mRNA, doublecortin mRNA, doublecortin protein, doublecortin-positive neuron counts and neurite length increased in OAA-treated mice. (1)H-MRS showed OAA increased brain lactate, GABA and glutathione thereby demonstrating metabolic changes are detectable in vivo. In mice, OAA promotes brain mitochondrial biogenesis, activates the insulin signaling pathway, reduces neuroinflammation and activates hippocampal neurogenesis.

Astrocyte-secreted factors modulate the developmental distribution of inhibitory synapses in nucleus laminaris of the avian auditory brainstem.

Nucleus laminaris (NL) neurons in the avian auditory brainstem are coincidence detectors necessary for the computation of interaural time differences used in sound localization. In addition to their excitatory inputs from nucleus magnocellularis, NL neurons receive inhibitory inputs from the superior olivary nucleus (SON) that greatly improve coincidence detection in mature animals. The mechanisms that establish mature distributions of inhibitory inputs to NL are not known. We used the vesicular GABA transporter (VGAT) as a marker for inhibitory presynaptic terminals to study the development of inhibitory inputs to NL between embryonic day 9 (E9) and E17. VGAT immunofluorescent puncta were first seen sparsely in NL at E9. The density of VGAT puncta increased with development, first within the ventral NL neuropil region and subsequently throughout both the ventral and dorsal dendritic neuropil, with significantly fewer terminals in the cell body region. A large increase in density occurred between E13­15 and E16­17, at a developmental stage when astrocytes that express glial fibrillary acidic protein (GFAP) become mature. We cultured E13 brainstem slices together with astrocyte-conditioned medium (ACM) obtained from E16 brainstems and found that ACM, but not control medium, increased the density of VGAT puncta. This increase was similar to that observed during normal development. Astrocyte-secreted factors interact with the terminal ends of SON axons to increase the number of GABAergic terminals. These data suggest that factors secreted from GFAP-positive astrocytes promote maturation of inhibitory pathways in the auditory brainstem.

Astrocyte-secreted factors modulate a gradient of primary dendritic arbors in nucleus laminaris of the avian auditory brainstem.

Neurons in nucleus laminaris (NL) receive binaural, tonotopically matched input from nucleus magnocelluaris (NM) onto bitufted dendrites that display a gradient of dendritic arbor size. These features improve computation of interaural time differences, which are used to determine the locations of sound sources. The dendritic gradient emerges following a period of significant reorganization at embryonic day 15 (E15), which coincides with the emergence of astrocytes that express glial fibrillary acidic protein (GFAP) in the auditory brainstem. The major changes include a loss of total dendritic length, a systematic loss of primary dendrites along the tonotopic axis, and lengthening of primary dendrites on caudolateral NL neurons. Here we have tested whether astrocyte-derived molecules contribute to these changes in dendritic morphology. We used an organotypic brainstem slice preparation to perform repeated imaging of individual dye-filled NL neurons to determine the effects of astrocyte-conditioned medium (ACM) on dendritic morphology. We found that treatment with ACM induced a decrease in the number of primary dendrites in a tonotopically graded manner similar to that observed during normal development. Our data introduce a new interaction between astrocytes and neurons in the auditory brainstem and suggest that these astrocytes influence multiple aspects of auditory brainstem maturation.