Regulatory Roles of Antimicrobial Peptides in the Nervous System: Implications for Neuronal Aging.

Antimicrobial peptides (AMPs) are classically known as important effector molecules in innate immunity across all multicellular organisms. However, emerging evidence begins to suggest multifunctional properties of AMPs beyond their antimicrobial activity, surprisingly including their roles in regulating neuronal function, such as sleep and memory formation. Aging, which is fundamental to neurodegeneration in both physiological and disease conditions, interestingly affects the expression pattern of many AMPs in an infection-independent manner. While it remains unclear whether these are coincidental events, or a mechanistic relationship exists, previous studies have suggested a close link between AMPs and a few key proteins involved in neurodegenerative diseases. This review discusses recent literature and advances in understanding the crosstalk between AMPs and the nervous system at both molecular and functional levels, with the aim to explore how AMPs may relate to neuronal vulnerability in aging.

An Antimicrobial Peptide and Its Neuronal Receptor Regulate Dendrite Degeneration in Aging and Infection.

Infections have been identified as possible risk factors for aging-related neurodegenerative diseases, but it remains unclear whether infection-related immune molecules have a causative role in neurodegeneration during aging. Here, we reveal an unexpected role of an epidermally expressed antimicrobial peptide, NLP-29 (neuropeptide-like protein 29), in triggering aging-associated dendrite degeneration in C. elegans. The age-dependent increase of nlp-29 expression is regulated by the epidermal tir-1/SARM-pmk-1/p38 MAPK innate immunity pathway. We further identify an orphan G protein-coupled receptor NPR-12 (neuropeptide receptor 12) acting in neurons as a receptor for NLP-29 and demonstrate that the autophagic machinery is involved cell autonomously downstream of NPR-12 to transduce degeneration signals. Finally, we show that fungal infections cause dendrite degeneration using a similar mechanism as in aging, through NLP-29, NPR-12, and autophagy. Our findings reveal an important causative role of antimicrobial peptides, their neuronal receptors, and the autophagy pathway in aging- and infection-associated dendrite degeneration.

Lactate's effect on human neuroblastoma cell bioenergetic fluxes.

Lactate, once considered a metabolic dead-end, has been recently proposed to support neuron bioenergetics. To better understand how lactate specifically influences cell energy metabolism, we studied the effects of lactate supplementation on SH-SY5Y human neuroblastoma cell bioenergetic fluxes. Lactate supplementation increased cell respiration, there was no change in respiratory coupling efficiency, and lactate itself appeared to directly support the respiratory flux increase. Conversely, lactate supplementation reduced the glycolysis flux. This apparent pro-aerobic shift in the respiration:glycolysis ratio was accompanied by post-translational modifications and compartmental redistributions of proteins that respond to and modify bioenergetic fluxes, including cAMP-response element binding protein (CREB), p38 mitogen-activated protein kinases (p38 MAPK), AMP-activated protein kinase (AMPK), peroxisome-proliferator activated receptor gamma coactivator 1 ß (PGC-1ß), Akt, mammalian target of rapamycin (mTOR), and forkhead box protein O1 (FOXO1). mRNA levels for PGC-1ß, nuclear respiratory factor 1 (NRF1), and cytochrome c oxidase subunit 1 (COX1) increased. Some effects depended on the direct presence of lactate, while others were durable and evident several hours after lactate was removed. We conclude lactate can be used to manipulate cell bioenergetics.

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.

Effect of high-intensity exercise on aged mouse brain mitochondria, neurogenesis, and inflammation.

In aged mice, we assessed how intensive exercise affects brain bioenergetics, inflammation, and neurogenesis-relevant parameters. After 8 weeks of a supra-lactate threshold treadmill exercise intervention, 21-month-old C57BL/6 mice showed increased brain peroxisome proliferator-activated receptor gamma coactivator-1α protein, mammalian target of rapamycin and phospho-mammalian target of rapamycin protein, citrate synthase messenger RNA, and mitochondrial DNA copy number. Hippocampal vascular endothelial growth factor A (VEGF-A) gene expression trended higher, and a positive correlation between VEGF-A and PRC messenger RNA levels was observed. Brain doublecortin, brain-derived neurotrophic factor, tumor necrosis factor-α, and CCL11 gene expression, as well as plasma CCL11 protein levels, were unchanged. Despite these apparent negative findings, a negative correlation between plasma CCL11 protein levels and hippocampal doublecortin gene expression was observed; further analysis indicated exercise may mitigate this relationship. Overall, our data suggest supra-lactate threshold exercise activates a partial mitochondrial biogenesis in aged mice, and a gene (VEGF-A) known to support neurogenesis. Our data are consistent with another study that found systemic inflammation in general, and CCL11 protein specifically, suppresses hippocampal neurogenesis. Our study supports the view that intense exercise above the lactate threshold may benefit the aging brain; future studies to address the extent to which exercise-generated lactate mediates the observed effects are warranted.

Altering O-linked ß-N-acetylglucosamine cycling disrupts mitochondrial function.

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.

Lactate administration reproduces specific brain and liver exercise-related changes.

The effects of exercise are not limited to muscle, and its ability to mitigate some chronic diseases is under study. A more complete understanding of how exercise impacts non-muscle tissues might facilitate design of clinical trials and exercise mimetics. Here, we focused on lactate's ability to mediate changes in liver and brain bioenergetic-associated parameters. In one group of experiments, C57BL/6 mice underwent 7 weeks of treadmill exercise sessions at intensities intended to exceed the lactate threshold. Over time, the mice dramatically increased their lactate threshold. To ensure that plasma lactate accumulated during the final week, the mice were run to exhaustion. In the liver, mRNA levels of gluconeogenesis-promoting genes increased. While peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α) expression increased, there was a decrease in PGC-1ß expression, and overall gene expression changes favored respiratory chain down-regulation. In the brain, PGC-1α and PGC-1ß were unchanged, but PGC-1-related co-activator expression and mitochondrial DNA copy number increased. Brain tumor necrosis factor alpha expression fell, whereas vascular endothelial growth factor A expression rose. In another group of experiments, exogenously administered lactate was found to reproduce some but not all of these observed liver and brain changes. Our data suggest that lactate, an exercise byproduct, could mediate some of the effects exercise has on the liver and the brain, and that lactate itself can act as a partial exercise mimetic.

Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines.

Bioenergetic dysfunction occurs in Alzheimer's disease (AD) and mild cognitive impairment (MCI), a clinical syndrome that frequently precedes symptomatic AD. In this study, we modeled AD and MCI bioenergetic dysfunction by transferring mitochondria from MCI, AD and control subject platelets to mtDNA-depleted SH-SY5Y cells. Bioenergetic fluxes and bioenergetics-related infrastructures were characterized in the resulting cytoplasmic hybrid (cybrid) cell lines. Relative to control cybrids, AD and MCI cybrids showed changes in oxygen consumption, respiratory coupling and glucose utilization. AD and MCI cybrids had higher ADP/ATP and lower NAD+/NADH ratios. AD and MCI cybrids exhibited differences in proteins that monitor, respond to or regulate cell bioenergetic fluxes including HIF1α, PGC1α, SIRT1, AMPK, p38 MAPK and mTOR. Several endpoints suggested mitochondrial mass increased in the AD cybrid group and probably to a lesser extent in the MCI cybrid group, and that the mitochondrial fission-fusion balance shifted towards increased fission in the AD and MCI cybrids. As many of the changes we observed in AD and MCI cybrid models are also seen in AD subject brains, we conclude reduced bioenergetic function is present during very early AD, is not brain-limited and induces protean retrograde responses that likely have both adaptive and mal-adaptive consequences.

Glycolysis-respiration relationships in a neuroblastoma cell line.

BACKGROUND: Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. METHODS: We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. RESULTS: Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. CONCLUSIONS: These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. GENERAL SIGNIFICANCE: Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches.

Role of mitochondrial homeostasis and dynamics in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects a staggering percentage of the aging population and causes memory loss and cognitive decline. Mitochondrial abnormalities can be observed systemically and in brains of patients suffering from AD, and may account for part of the disease phenotype. In this review, we summarize some of the key findings that indicate mitochondrial dysfunction is present in AD-affected subjects, including cytochrome oxidase deficiency, endophenotype data, and altered mitochondrial morphology. Special attention is given to recently described perturbations in mitochondrial autophagy, fission-fusion dynamics, and biogenesis. We also briefly discuss how mitochondrial dysfunction may influence amyloidosis in Alzheimer's disease, why mitochondria are a valid therapeutic target, and strategies for addressing AD-specific mitochondrial dysfunction.

Effect of cholinergic signaling on neuronal cell bioenergetics.

Alzheimer's disease (AD) patients have reduced brain acetylcholine and reversing this deficit yields clinical benefits. In this study we explored how increased cholinergic tone impacts cell bioenergetics, which are also perturbed in AD. We treated SH-SY5Y neuroblastoma cells with carbachol, a cholinergic agonist, and tested for bioenergetic flux and bioenergetic infrastructure changes. Carbachol rapidly increased both oxidative phosphorylation and glycolysis fluxes. ATP levels rose slightly, as did cell energy demand, and AMPK phosphorylation occurred. At least some of these effects depended on muscarinic receptor activation, ER calcium release, and ER calcium re-uptake. Our data show that increasing cholinergic signaling enhances cell bioenergetics, and reveal mechanisms that mediate this effect. Phenomena we observed could potentially explain why cholinesterase inhibitor therapy increases AD brain glucose utilization and N-acetyl aspartate levels. The question of whether cholinesterase inhibitors have a disease modifying effect in AD has long been debated; our data suggest a theoretical mechanism through which such an effect could potentially arise.

Effect of exercise on mouse liver and brain bioenergetic infrastructures.

To assess the effects of exercise on liver and brain bioenergetic infrastructures, we exposed C57BL/6 mice to 6 weeks of moderate-intensity treadmill exercise. During the training period, fasting blood glucose was lower in exercised mice than in sedentary mice, but serum insulin levels were not reduced. At week 6, trained mice showed a paradoxical decrease in plasma lactate during exercise, which was accompanied by an increase in the liver monocarboxylate transporter 2 protein level (∼30%, P < 0.05). Exercise increased liver peroxisomal proliferator-activated receptor-γ coactivator 1α expression (approximately twofold, P < 0.001), NAD-dependent deacetylase sirtuin-1 protein (∼30%, P < 0.05), p38 protein (∼15%, P < 0.05), cytochrome c oxidase subunit 4 isoform 1 protein (∼50%, P < 0.05) and AMP-activated protein kinase phosphorylation (∼40%, P < 0.05). Despite this, liver mitochondrial DNA copy number (∼30%, P = 0.05), mitochondrial transcription factor A expression (∼15%, P < 0.05), cytochrome c oxidase subunit 2 expression (∼10%, P < 0.05), cAMP-response element binding protein phosphorylation (∼60%, P < 0.05) and brain-derived neurotrophic factor expression (∼40%, P < 0.05) were all reduced, while cytochrome oxidase and citrate synthase activities were unchanged. The only altered brain parameter observed was a reduction in tumour necrosis factor α expression (∼35%, P < 0.05); tumour necrosis factor α expression was unchanged in liver. Our data suggest that lactate produced by exercising muscle modifies the liver bioenergetic infrastructure, and enhanced liver uptake may in turn limit the ability of exercise-generated lactate to modify brain bioenergetics. Also, it appears that, at least in the liver, a dissociated mitochondrial biogenesis, in which some components are strategically enhanced while others are minimized, can occur.

Mitochondrial abnormalities in Alzheimer's disease: possible targets for therapeutic intervention.

Mitochondria from persons with Alzheimer's disease (AD) differ from those of age-matched control subjects. Differences in mitochondrial morphology and function are well documented, and are not brain-limited. Some of these differences are present during all stages of AD, and are even seen in individuals who are without AD symptoms and signs but who have an increased risk of developing AD. This chapter considers the status of mitochondria in AD subjects, the potential basis for AD subject mitochondrial perturbations, and the implications of these perturbations. Data from multiple lines of investigation, including epidemiologic, biochemical, molecular, and cytoplasmic hybrid studies, are reviewed. The possibility that mitochondria could potentially constitute a reasonable AD therapeutic target is discussed, as are several potential mitochondrial medicine treatment strategies.

Potentiation of dietary restriction-induced lifespan extension by polyphenols.

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF+PAO). Both IF and IF+PAO groups outlived the control group and the IF+PAO group outlived the IF group (all p<0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.

Reduced mitochondria cytochrome oxidase activity in adult children of mothers with Alzheimer's disease.

Biomarker studies demonstrate inheritance of glucose hypometabolism and increased amyloid-ß deposition in adult offspring of mothers, but not fathers, affected by late-onset Alzheimer's disease (LOAD). The underlying genetic mechanisms are unknown. We investigated whether cognitively normal (NL) individuals with a maternal history of LOAD (MH) have reduced platelet mitochondrial cytochrome oxidase activity (COX, electron transport chain complex IV) compared to those with paternal (PH) or negative family history (NH). Thirty-six consecutive NL individuals (age 55 ± 15 y, range 27-71 y, 56% female, CDR = 0, MMSE ≥28, 28% APOE-4 carriers), including 12 NH, 12 PH, and 12 MH, received a blood draw to measure platelet mitochondrial COX activity. Citrate synthase activity (CS) was measured as a reference. Groups were comparable for clinical and neuropsychological measures. We found that after correcting for CS, COX activity was reduced by 29% in MH compared to NH, and by 30% in MH compared to PH (p ≤ 0.006). Results remained significant controlling for age, gender, education, and APOE. No differences were found between PH and NH. COX measures discriminated MH from the other groups with accuracy ≥75%, and relative risk ≥3 (p ≤ 0.005). Among NL with LOAD-parents, only those with MH showed reduced COX activity in platelet mitochondria compared to PH and NH. The association between maternal history of LOAD and systemic COX reductions suggests transmission via mitochondrial DNA, which is exclusively maternally inherited in humans.

Alternate day fasting impacts the brain insulin-signaling pathway of young adult male C57BL/6 mice.

Dietary restriction (DR) has recognized health benefits that may extend to brain. We examined how DR affects bioenergetics-relevant enzymes and signaling pathways in the brains of C57BL/6 mice. Five-month-old male mice were placed in ad libitum or one of two repeated fasting and refeeding (RFR) groups, an alternate day (intermittent fed; IF) or alternate day plus antioxidants (blueberry, pomegranate, and green tea extracts) (IF + AO) fed group. During the 24-h fast blood glucose levels initially fell but stabilized within 6 h of starting the fast, thus avoiding frank hypoglycemia. DR in general appeared to enhance insulin sensitivity. After six weeks brain AKT and glycogen synthase kinase 3 beta phosphorylation were lower in the RFR mice, suggesting RFR reduced brain insulin-signaling pathway activity. Pathways that mediate mitochondrial biogenesis were not activated; AMP kinase phosphorylation, silent information regulator 2 phosphorylation, peroxisomal proliferator-activated receptor-gamma coactivator 1 alpha levels, and cytochrome oxidase subunit 4 levels did not change. ATP levels also did not decline, which suggests the RFR protocols did not directly impact brain bioenergetics. Antioxidant supplementation did not affect the brain parameters we evaluated. Our data indicate in young adult male C57BL/6 mice, RFR primarily affects brain energy metabolism by reducing brain insulin signaling, which potentially results indirectly as a consequence of reduced peripheral insulin production.

Polymorphic variation in cytochrome oxidase subunit genes.

Cytochrome oxidase (COX) activity varies between individuals and low activities associate with Alzheimer's disease. Whether genetic heterogeneity influences function of this multimeric enzyme is unknown. To explore this we sequenced three mitochondrial DNA (mtDNA) and ten nuclear COX subunit genes from at least 50 individuals. 20% had non-synonymous mtDNA COX gene polymorphisms, 12% had a COX4I1 non-synonymous G to A transition, and other genes rarely contained non-synonymous polymorphisms. Frequent untranslated region (UTR) polymorphisms were seen in COX6A1, COX6B1, COX6C, and COX7A1; heterogeneity in a COX7A1 5' UTR Sp1 site was extensive. Synonymous polymorphisms were common and less frequent in the more conserved COX1 than the less conserved COX3, suggesting at least in mtDNA synonymous polymorphisms experience selection pressure and are not functionally silent. Compound gene variations occurred within individuals. To test whether variations could have functional consequences, we studied the COX4I1 G to A transition and an AGCCCC deletion in the COX7A1 5' UTR Sp1 site. Cells expressing the COX4I1 polymorphism had reduced COX Vmax activity. In reporter construct-transduced cells where green fluorescent protein expression depended on the COX7A1 Sp1 site, AGCCCC deletion reduced fluorescence. Our findings indicate COX subunit gene heterogeneity is pervasive and may mediate COX functional variation.