Mitochondria Profoundly Influence Apolipoprotein E Biology.

BACKGROUND: Mitochondria can trigger Alzheimer's disease (AD)-associated molecular phenomena, but how mitochondria impact apolipoprotein E (APOE; apoE) is not well known. OBJECTIVE: Consider whether and how mitochondrial biology influences APOE and apoE biology. METHODS: We measured APOE expression in human SH-SY5Y neuronal cells with different forms of mitochondrial dysfunction including total, chronic mitochondrial DNA (mtDNA) depletion (ρ0 cells); acute, partial mtDNA depletion; and toxin-induced mitochondrial dysfunction. We further assessed intracellular and secreted apoE protein levels in the ρ0 cells and interrogated the impact of transcription factors and stress signaling pathways known to influence APOE expression. RESULTS: SH-SY5Y ρ0 cells exhibited a 65-fold increase in APOE mRNA, an 8-fold increase in secreted apoE protein, and increased intracellular apoE protein. Other models of primary mitochondrial dysfunction including partial mtDNA-depletion, toxin-induced respiratory chain inhibition, and chemical-induced manipulations of the mitochondrial membrane potential similarly increased SH-SY5Y cell APOE mRNA. We explored potential mediators and found in the ρ0 cells knock-down of the C/EBPα and NFE2L2 (Nrf2) transcription factors reduced APOE mRNA. The activity of two mitogen-activated protein kinases, JNK and ERK, also strongly influenced ρ0 cell APOE mRNA levels. CONCLUSION: Primary mitochondrial dysfunction either directly or indirectly activates APOE expression in a neuronal cell model by altering transcription factors and stress signaling pathways. These studies demonstrate mitochondrial biology can influence the biology of the APOE gene and apoE protein, which are implicated in AD.

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.

Pharmacologic enrichment of exosome yields and mitochondrial cargo.

In studies with human participants, exosome-based biospecimens can facilitate unique biomarker assessments. As exosome cargos can include mitochondrial components, there is interest in using exosomes to inform the status of an individual's mitochondria. Here, we evaluated whether targeted pharmacologic manipulations could influence the quantity of exosomes shed by cells, and whether these manipulations could impact their mitochondrial cargos. We treated human SH-SY5Y cells with bafilomycin A1, which interferes with general autophagy and mitophagy by inhibiting lysosome acidification and lysosome-autophagosome fusion; deferiprone (DFP), which enhances receptor-mediated mitophagy; or both. Exosome fractions from treated cells were harvested from the cell medium and analyzed for content including mitochondria-derived components. We found bafilomycin increased particle yields, and a combination of bafilomycin plus DFP consistently increased particle yields and mitochondria-associated content. Specifically, the exosome fractions from the bafilomycin plus DFP-treated cells contained more mitochondrial DNA (mtDNA), mtDNA-derived mRNA transcripts, and citrate synthase protein. Our data suggest pharmacologic manipulations that enhance mitophagy initiation, while inhibiting the lysosomal digestion of autophagosomes and multivesicular bodies, could potentially enhance the sensitivity of exosome-based biomarker assays intended to inform the status of an individual's mitochondria.

Mitochondrial Membrane Potential Influences Amyloid-ß Protein Precursor Localization and Amyloid-ß Secretion.

BACKGROUND: Amyloid-ß (Aß), which derives from the amyloid-ß protein precursor (AßPP), forms plaques and serves as a fluid biomarker in Alzheimer's disease (AD). How Aß forms from AßPP is known, but questions relating to AßPP and Aß biology remain unanswered. AD patients show mitochondrial dysfunction, and an Aß/AßPP mitochondria relationship exists. OBJECTIVE: We considered how mitochondrial biology may impact AßPP and Aß biology. METHODS: SH-SY5Y cells were transfected with AßPP constructs. After treatment with FCCP (uncoupler), Oligomycin (ATP synthase inhibitor), or starvation Aß levels were measured. ß-secretase (BACE1) expression was measured. Mitochondrial localized full-length AßPP was also measured. All parameters listed were measured in ρ0 cells on an SH-SY5Y background. iPSC derived neurons were also used to verify key results. RESULTS: We showed that mitochondrial depolarization routes AßPP to, while hyperpolarization routes AßPP away from, the organelle. Mitochondrial AßPP and cell Aß secretion inversely correlate, as cells with more mitochondrial AßPP secrete less Aß, and cells with less mitochondrial AßPP secrete more Aß. An inverse relationship between secreted/extracellular Aß and intracellular Aß was observed. CONCLUSION: Our findings indicate mitochondrial function alters AßPP localization and suggest enhanced mitochondrial activity promotes Aß secretion while depressed mitochondrial activity minimizes Aß secretion. Our data complement other studies that indicate a mitochondrial, AßPP, and Aß nexus, and could help explain why cerebrospinal fluid Aß is lower in those with AD. Our data further suggest Aß secretion could serve as a biomarker of cell or tissue mitochondrial function.

The impact of foot shock-induced stress on pain-related behavior associated with burn injury.

Acute pain is prevalent following burn injury and can often transition to chronic pain. Prolonged acute pain is an important risk factor for chronic pain and there is little preclinical research to address this problem. Using a mouse model of second-degree burn, we investigated whether pre-existing stress influences pain(sensitivity) after a burn injury. We introduced a contribution of stress in two different ways: (1) the use of foot-shock as a pre-injury stressor or (2) the use of A/J mice to represent higher pre-existing stress compared to C57Bl/6 mice. C57Bl/6 and A/J mice were exposed to repeated mild foot shock to induce stress for 10 continuous days and mice underwent either burn injury or sham burn injury of the plantar surface of the right hind paw. Assessments of mechanical and thermal sensitivities of the injured and uninjured paw were conducted during the shock protocol and at intervals up to 82-day post-burn injury. In both strains of mice that underwent burn injury, thermal hypersensitivity and mechanical allodynia appeared rapidly in the ipsilateral paw. Mice that were stressed took much longer to recover their hind paw mechanical thresholds to baseline compared to non-stressed mice in both burn and non-burn groups. Analysis of the two mouse strains revealed that the recovery of mechanical thresholds in A/J mice which display higher levels of baseline anxiety was shorter than C57Bl/6 mice. No differences were observed regarding thermal sensitivities between strains. Our results support the view that stress exposure prior to burn injury affects mechanical and thermal thresholds and may be relevant to as a risk factor for the transition from acute to chronic pain. Finally, genetic differences may play a key role in modality-specific recovery following burn injury.

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.

An Integrative Overview of Non-Amyloid and Non-Tau Pathologies in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease that devastates the lives of its victims, and challenges the family members and health care infrastructures that care for them. Clinically, attempts to understand AD have focused on trying to predict the presence of, and more recently demonstrate the presence of, its characteristic amyloid plaque and neurofibrillary tangle pathologies. Fundamental research has also traditionally focused on understanding the generation, content, and pathogenicity of plaques and tangles, but in addition to this there is now an emerging independent interest in other molecular phenomena including apolipoprotein E, lipid metabolism, neuroinflammation, and mitochondrial function. While studies emphasizing the role of these phenomena have provided valuable AD insights, it is interesting that at the molecular level these entities extensively intertwine and interact. In this review, we provide a brief overview of why apolipoprotein E, lipid metabolism, neuroinflammation, and mitochondrial research have become increasingly ascendant in the AD research field, and present the case for studying these phenomena from an integrated perspective.

Intrinsic Activity of C57BL/6 Substrains Associates with High-Fat Diet-Induced Mechanical Sensitivity in Mice.

Pain is significantly impacted by the increasing epidemic of obesity and the metabolic syndrome. Our understanding of how these features impact pain is only beginning to be developed. Herein, we have investigated how small genetic differences among C57BL/6 mice from 2 different commercial vendors lead to important differences in the development of high-fat diet-induced mechanical sensitivity. Two substrains of C57BL/6 mice from Jackson Laboratories (Bar Harbor, ME; C57BL/6J and C57BL/6NIH), as well as C57BL/6 from Charles Rivers Laboratories (Wilmington, MA; C57BL/6CR) were placed on high-fat diets and analyzed for changes in metabolic features influenced by high-fat diet and obesity, as well as measures of pain-related behaviors. All 3 substrains responded to the high-fat diet; however, C57BL/6CR mice had the highest weights, fat mass, and impaired glucose tolerance of the 3 substrains. In addition, the C57BL/6CR mice were the only strain to develop significant mechanical sensitivity over the course of 8 weeks. Importantly, the C57BL/6J mice were protected from mechanical sensitivity, which may be based on increased physical activity compared with the other 2 substrains. These findings suggest that activity may play a powerful role in protecting metabolic changes associated with a high-fat diet and that these may also be protective in pain-associated changes as a result of a high-fat diet. These findings also emphasize the importance of selection and transparency in choosing C57BL/6 substrains in pain-related research. PERSPECTIVE: Obesity and the metabolic syndrome play an important role in pain. This study identifies key differences in the response to a high-fat diet among substrains of C57BL/6 mice and differences in intrinsic physical activity that may influence pain sensitivity. The results emphasize physical activity as a powerful modulator of obesity-related pain sensitivity.

A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice.

Current experiments investigated whether a ketogenic diet impacts neuropathy associated with obesity and prediabetes. Mice challenged with a ketogenic diet were compared to mice fed a high-fat diet or a high-fat diet plus exercise. Additionally, an intervention switching to a ketogenic diet following 8 weeks of high-fat diet was performed to compare how a control diet, exercise, or a ketogenic diet affects metabolic syndrome-induced neural complications. When challenged with a ketogenic diet, mice had reduced bodyweight and fat mass compared to high-fat-fed mice, and were similar to exercised, high-fat-fed mice. High-fat-fed, exercised and ketogenic-fed mice had mildly elevated blood glucose; conversely, ketogenic diet-fed mice were unique in having reduced serum insulin levels. Ketogenic diet-fed mice never developed mechanical allodynia contrary to mice fed a high-fat diet. Ketogenic diet fed mice also had increased epidermal axon density compared all other groups. When a ketogenic diet was used as an intervention, a ketogenic diet was unable to reverse high-fat fed-induced metabolic changes but was able to significantly reverse a high-fat diet-induced mechanical allodynia. As an intervention, a ketogenic diet also increased epidermal axon density. In vitro studies revealed increased neurite outgrowth in sensory neurons from mice fed a ketogenic diet and in neurons from normal diet-fed mice given ketone bodies in the culture medium. These results suggest a ketogenic diet can prevent certain complications of prediabetes and provides significant benefits to peripheral axons and sensory dysfunction.