Are Genetics the Predicting Factor for the Success of Migraine Surgery? A Report on Identical Twins.

Migraine affects more than 1 billion people globally, with distinct genetic variations influencing susceptibility. Thereby, genetic variations play a key role in the probability of developing migraine. However, personalized genetic analysis-based treatment options in migraine treatments are limited. Notably, surgical deactivation of extracranial trigger has shown efficacy in the treatment of migraine patients with identifiable trigger points in specific anatomical locations in the head and neck region. We present the first case of monozygotic twin sisters, both experiencing occipital and temporal-triggered migraine headaches with identical history and characteristics and without response to conservative migraine treatments. After surgical intervention, targeting the greater and lesser occipital nerves as well as auriculotemporal nerves, both twin sisters exhibited an over 99% reduction in symptoms without postoperative complications. This case suggests a potential correlation between genetic background, irrespective of environmental factors, and the effectiveness of surgical deactivation of trigger points in migraine management.

Ola1p trafficking indicates an interaction network between mitochondria, lipid droplets, and stress granules in times of stress.

Protein aggregates arise naturally under normal physiological conditions, but their formation is accelerated by age or stress-induced protein misfolding. When the stressful event dissolves, these aggregates are removed by mechanisms, such as aggrephagy, chaperone-mediated autophagy, refolding attempts, or the proteasome. It was recently shown that mitochondria in yeast cells may support these primarily cytosolic processes. Protein aggregates attach to mitochondria, and misfolded proteins are transported into the matrix and degraded by mitochondria-specific proteases. Using a proximity labeling method and colocalization with an established stress granule (SG) marker, we were able to show that these mitochondria-localized aggregates that harbor the "super aggregator" Ola1p are, in fact, SGs. Our in vivo and in vitro studies have revealed that Ola1p can be transferred from mitochondria to lipid droplets (LDs). This "mitochondria to LD" aggregate transfer dampens proteotoxic effects. The LD-based protein aggregate removal system gains importance when other proteolytic systems fail. Furthermore, we were able to show that the distribution of SGs is drastically altered in LD-deficient yeast cells, demonstrating that LDs play a role in the SG life cycle.

The Janus-Faced Role of Lipid Droplets in Aging: Insights from the Cellular Perspective.

It is widely accepted that nine hallmarks-including mitochondrial dysfunction, epigenetic alterations, and loss of proteostasis-exist that describe the cellular aging process. Adding to this, a well-described cell organelle in the metabolic context, namely, lipid droplets, also accumulates with increasing age, which can be regarded as a further aging-associated process. Independently of their essential role as fat stores, lipid droplets are also able to control cell integrity by mitigating lipotoxic and proteotoxic insults. As we will show in this review, numerous longevity interventions (such as mTOR inhibition) also lead to strong accumulation of lipid droplets in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammalian cells, just to name a few examples. In mammals, due to the variety of different cell types and tissues, the role of lipid droplets during the aging process is much more complex. Using selected diseases associated with aging, such as Alzheimer's disease, Parkinson's disease, type II diabetes, and cardiovascular disease, we show that lipid droplets are "Janus"-faced. In an early phase of the disease, lipid droplets mitigate the toxicity of lipid peroxidation and protein aggregates, but in a later phase of the disease, a strong accumulation of lipid droplets can cause problems for cells and tissues.

Lipid Droplets Protect Aging Mitochondria and Thus Promote Lifespan in Yeast Cells.

Besides their role as a storage for neutral lipids and sterols, there is increasing evidence that lipid droplets (LDs) are involved in cellular detoxification. LDs are in close contact to a broad variety of organelles where protein- and lipid exchange is mediated. Mitochondria as a main driver of the aging process produce reactive oxygen species (ROS), which damage several cellular components. LDs as highly dynamic organelles mediate a potent detoxification mechanism by taking up toxic lipids and proteins. A stimulation of LDs induced by the simultaneously overexpression of Lro1p and Dga1p (both encoding acyltransferases) prolongs the chronological as well as the replicative lifespan of yeast cells. The increased number of LDs reduces mitochondrial fragmentation as well as mitochondrial ROS production, both phenotypes that are signs of aging. Strains with an altered LD content or morphology as in the sei1∆ or lro1∆ mutant lead to a reduced replicative lifespan. In a yeast strain defective for the LON protease Pim1p, which showed an enhanced ROS production, increased doubling time and an altered mitochondrial morphology, a LRO1 overexpression resulted in a partially reversion of this "premature aging" phenotype.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

The life in a gradient: calcium, the lncRNA SPRR2C and mir542/mir196a meet in the epidermis to regulate the aging process.

The turnover of the epidermis beginning with the progenitor cells in the basal layer to the fully differentiated corneocytes is tightly regulated by calcium. Calcium more than anything else promotes the differentiation of keratinocytes which implies the need for a calcium gradient with low concentrations in the stratum basale and high concentrations in the stratum granulosum. One of the hallmarks of skin aging is a collapse of this gradient that has a direct impact on the epidermal fitness. The rise of calcium in the stratum basale reduces cell proliferation, whereas the drop of calcium in the stratum granulosum leads to a changed composition of the cornified envelope. We showed that keratinocytes respond to the calcium induced block of cell division by a large increase of the expression of several miRNAs (hsa-mir542-5p, hsa-mir125a, hsa-mir135a-5p, hsa-mir196a-5p, hsa-mir491-5p and hsa-mir552-5p). The pitfall of this rescue mechanism is a dramatic change in gene expression which causes a further impairment of the epidermal barrier. This effect is attenuated by a pseudogene (SPRR2C) that gives rise to a lncRNA. SPRR2C specifically resides in the stratum granulosum/corneum thus acting as a sponge for miRNAs.

Actin Cytoskeleton Regulation by the Yeast NADPH Oxidase Yno1p Impacts Processes Controlled by MAPK Pathways.

Reactive oxygen species (ROS) that exceed the antioxidative capacity of the cell can be harmful and are termed oxidative stress. Increasing evidence suggests that ROS are not exclusively detrimental, but can fulfill important signaling functions. Recently, we have been able to demonstrate that a NADPH oxidase-like enzyme (termed Yno1p) exists in the single-celled organism Saccharomyces cerevisiae. This enzyme resides in the peripheral and perinuclear endoplasmic reticulum and functions in close proximity to the plasma membrane. Its product, hydrogen peroxide, which is also produced by the action of the superoxide dismutase, Sod1p, influences signaling of key regulatory proteins Ras2p and Yck1p/2p. In the present work, we demonstrate that Yno1p-derived H2O2 regulates outputs controlled by three MAP kinase pathways that can share components: the filamentous growth (filamentous growth MAPK (fMAPK)), pheromone response, and osmotic stress response (hyperosmolarity glycerol response, HOG) pathways. A key structural component and regulator in this process is the actin cytoskeleton. The nucleation and stabilization of actin are regulated by Yno1p. Cells lacking YNO1 showed reduced invasive growth, which could be reversed by stimulation of actin nucleation. Additionally, under osmotic stress, the vacuoles of a ∆yno1 strain show an enhanced fragmentation. During pheromone response induced by the addition of alpha-factor, Yno1p is responsible for a burst of ROS. Collectively, these results broaden the roles of ROS to encompass microbial differentiation responses and stress responses controlled by MAPK pathways.

Friend or Foe: Lipid Droplets as Organelles for Protein and Lipid Storage in Cellular Stress Response, Aging and Disease.

Lipid droplets (LDs) were considered as a mere lipid storage organelle for a long time. Recent evidence suggests that LDs are in fact distinct and dynamic organelles with a specialized proteome and functions in many cellular roles. As such, LDs contribute to cellular signaling, protein and lipid homeostasis, metabolic diseases and inflammation. In line with the multitude of functions, LDs interact with many cellular organelles including mitochondria, peroxisomes, lysosomes, the endoplasmic reticulum and the nucleus. LDs are highly mobile and dynamic organelles and impaired motility disrupts the interaction with other organelles. The reduction of interorganelle contacts results in a multitude of pathophysiologies and frequently in neurodegenerative diseases. Contacts not only supply lipids for ß-oxidation in mitochondria and peroxisomes, but also may include the transfer of toxic lipids as well as misfolded and harmful proteins to LDs. Furthermore, LDs assist in the removal of protein aggregates when severe proteotoxic stress overwhelms the proteasomal system. During imbalance of cellular lipid homeostasis, LDs also support cellular detoxification. Fine-tuning of LD function is of crucial importance and many diseases are associated with dysfunctional LDs. We summarize the current understanding of LDs and their interactions with organelles, providing a storage site for harmful proteins and lipids during cellular stress, aging inflammation and various disease states.

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3.

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.

The transfer of specific mitochondrial lipids and proteins to lipid droplets contributes to proteostasis upon stress and aging in the eukaryotic model system Saccharomyces cerevisiae.

Originally Lipid droplets (LDs) were considered as being droplets for lipid storage only. Increasing evidence, however, demonstrates that LDs fulfill a pleiotropy of additional functions. Among them is the modulation of protein as well as lipid homeostasis. Under unfavorable pro-oxidative conditions, proteins can form aggregates which may exceed the overall proteolytic capacity of the proteasome. After stress termination LDs can adjust and support the removal of these aggregates. Additionally, LDs interact with mitochondria, specifically take over certain proteins and thus prevent apoptosis. LDs, which are loaded with these harmful proteins, are subsequently eliminated via lipophagy. Recently it was demonstrated that this autophagic process is a modulator of longevity. LDs do not only eliminate potentially dangerous proteins, but they are also able to prevent lipotoxicity by storing specific lipids. In the present study we used the model organism Saccharomyces cerevisiae to compare the proteome as well as lipidome of mitochondria and LDs under different conditions: replicative aging, stress and apoptosis. In this context we found an accumulation of proteins at LDs, supporting the role of LDs in proteostasis. Additionally, the composition of main lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols, triacylglycerols, ceramides, phosphatidic acids and ergosterol of LDs and mitochondria changed during stress conditions and aging.

The defense and signaling role of NADPH oxidases in eukaryotic cells : Review.

This short review article summarizes what is known clinically and biochemically about the seven human NADPH oxidases. Emphasis is put on the connection between mutations in the catalytic and regulatory subunits of Nox2, the phagocyte defense enzyme, with syndromes like chronic granulomatous disease, as well as a number of chronic inflammatory diseases. These arise paradoxically from a lack of reactive oxygen species production needed as second messengers for immune regulation. Both Nox2 and the six other human NADPH oxidases display signaling functions in addition to the functions of these enzymes in specialized biochemical reactions, for instance, synthesis of the hormone thyroxine. NADPH oxidases are also needed by Saccharomyces cerevisiae cells for the regulation of the actin cytoskeleton in times of stress or developmental changes, such as pseudohyphae formation. The article shows that in certain cancer cells Nox4 is also involved in the re-structuring of the actin cytoskeleton, which is required for cell mobility and therefore for metastasis.

The Influence of Calcium on the Skin pH and Epidermal Barrier During Aging.

During aging, the pH of the epidermis goes up and the calcium gradient goes down. Both have negative effects on the protective function of the epidermis and both are connected to each other as is discussed here. In the aging process, the pH rises from ∼5 to ∼5.5-6. The establishment of the skin pH is the joint effort of several independent factors including the activity of sodium-hydrogen antiporters and the presence of lactate, urocanic acid, free fatty acids and melanophores in the outermost layers of the skin. All these elements are under the control of a small ion: calcium. Calcium is organized in the form of a gradient in the epidermis with low concentrations in the stratum basale and peak concentrations in the stratum granulosum. During the aging process, this epidermal gradient collapses. In this chapter, we describe how a drop of calcium in the stratum granulosum affects the expression as well as the activity of proteins and enzymes that are involved in the establishment of the skin pH. This rise of the pH combined with a rearrangement of the cornified envelope is a main driver for a reduced epidermal barrier in old age leading to an increased prevalence for infections, reduced resistance against mechanical stress and reduced wound healing.

Behead and live long or the tale of cathepsin L.

In recent decades Saccharomyces cerevisiae has proven to be one of the most valuable model organisms of aging research. Pathways such as autophagy or the effect of substances like resveratrol and spermidine that prolong the replicative as well as chronological lifespan of cells were described for the first time in S. cerevisiae. In this study we describe the establishment of an aging reporter that allows a reliable and relative quick screening of substances and genes that have an impact on the replicative lifespan. A cDNA library of the flatworm Dugesia tigrina that can be immortalized by beheading was screened using this aging reporter. Of all the flatworm genes, only one could be identified that significantly increased the replicative lifespan of S.cerevisiae. This gene is the cysteine protease cathepsin L that was sequenced for the first time in this study. We were able to show that this protease has the capability to degrade such proteins as the yeast Sup35 protein or the human α-synuclein protein in yeast cells that are both capable of forming cytosolic toxic aggregates. The degradation of these proteins by cathepsin L prevents the formation of these unfolded protein aggregates and this seems to be responsible for the increase in replicative lifespan.

The Human NADPH Oxidase, Nox4, Regulates Cytoskeletal Organization in Two Cancer Cell Lines, HepG2 and SH-SY5Y.

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets.

In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two α-helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response.

Specific circulating phospholipids, acylcarnitines, amino acids and biogenic amines are aerobic exercise markers.

OBJECTIVES: Regular aerobic exercise provides beneficial effects on human health and reduces all-cause mortality. Aerobic exercise has profound metabolic effects, and specific metabolites may reflect physiological changes. We aimed to identify endogenous metabolites that distinguish the trained from the untrained state to increase the spectrum of analytes amenable for hypothesis testing and to expand understanding of putative beneficial pathways. DESIGN: Cross sectional laboratory repeated measures study. METHODS: Exercise testing was performed in 37 healthy male participants and serum samples were obtained before and after completion of a ten-week standardized exercise program. Samples were analyzed for routine clinical parameters and for 188 endogenous metabolites by LC-MS/MS. RESULTS: Indicating the effectiveness of the intervention program, parameters of sport physiology were different after training. After correcting for multiple testing, serum concentrations of several metabolites differed between the trained and untrained state. Serine and glutamate decreased in response to exercise, whereas sarcosine and kynurenine increased. Phosphatidylcholines showed a mixed response in that four species increased and three decreased. However, all seven lysophosphatidylcholines and all four plasmalogens that differed between the trained and untrained state, increased. One short-chain acylcarnitine also decreased. In receiver operator characteristics analyses, sarcosine displayed the highest AUC value (0.839; 95% CI: 0.734-0.926) in discriminating the pre- from the post-trained state. CONCLUSIONS: Our study detected metabolites that clearly differentiate the trained from the untrained state. These metabolites may be targeted in mechanistic studies to understand underlying biochemical pathways and could serve to improve the design, monitoring and individualization of training programs.

Regulation of Ion Channels, Cellular Carriers and Na(+)/K(+)/ATPase by Janus Kinase 3.

Janus kinase-3 (JAK3), a tyrosine kinase, is expressed in a variety of tissues, including the brain and is involved in the signaling of cytokine receptors. JAK3 participates in numerous functions, such as cell survival and proliferation, neuroprotection, apoptosis and the cellular response to hypoxia and ischemia-reperfusion. This kinase further contributes to the signaling of hematopoietic cell cytokine receptors, activation of dendritic cells, maturation, and immune suppression as well as to cell volume regulation. Recently, JAK3 has been demonstrated to be an important regulator of transport processes across the plasma membrane. Either directly or indirectly JAK3 affects the expression of transport proteins, including various ion channels, a number of cellular carriers and the Na+/K+ pump. More specifically, JAK3 is involved in the regulation of various potassium, sodium, and chloride ion channels, a wide variety of Na+-coupled cellular carriers including the high-affinity Na+ coupled glucose transporter SGLT1, the excitatory amino acid transporters EAAT1, EAAT2, EAAT3 and EAAT4, the peptide transporters PepT1 and PepT2, CreaT1 and theNa+/K+-ATPase. Via these transporters this kinase plays a role in various physiological and pathophysiological processes. Additional research is needed to investigate the effects of JAK3 on other cellular transporters and the underlying mechanisms.

The control of translational accuracy is a determinant of healthy ageing in yeast.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.