Temporal Quantitative Proteomics of mGluR-induced Protein Translation and Phosphorylation in Neurons.

At neuronal synapses, activation of group I metabotropic glutamate receptors (mGluR1/5) triggers a form of long-term depression (mGluR-LTD) that relies on new protein synthesis and the internalization of AMPA-type glutamate receptors. Dysregulation of these processes has been implicated in the development of mental disorders such as autism spectrum disorders and therefore merit a better understanding on a molecular level. Here, to study mGluR-induced signaling pathways, we integrated quantitative phosphoproteomics with the analyses of newly synthesized proteins via bio-orthogonal amino acids (azidohomoalanine) in a pulsed labeling strategy in cultured hippocampal neurons stimulated with DHPG, a specific agonist for group I mGluRs. We identified several kinases with important roles in DHPG-induced mGluR activation, which we confirmed using small molecule kinase inhibitors. Furthermore, changes in the AMPA receptor endocytosis pathway in both protein synthesis and protein phosphorylation were identified, whereby Intersectin-1 was validated as a novel player in this pathway. This study revealed several new insights into the molecular pathways downstream of group I mGluR activation in hippocampal neurons, and provides a rich resource for further analyses.

In vivo phosphoproteomics reveals kinase activity profiles that predict treatment outcome in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, we use high-throughput phosphoproteomics to build a functional TNBC taxonomy. A cluster of 159 phosphosites is upregulated in relapsed cases of a training set (n = 34 patients), with 11 hyperactive kinases accounting for this phosphoprofile. A mass-spectrometry-to-immunohistochemistry translation step, assessing 2 independent validation sets, reveals 6 kinases with preserved independent prognostic value. The kinases split the validation set into two patterns: one without hyperactive kinases being associated with a >90% relapse-free rate, and the other one showing ≥1 hyperactive kinase and being associated with an up to 9.5-fold higher relapse risk. Each kinase pattern encompasses different mutational patterns, simplifying mutation-based taxonomy. Drug regimens designed based on these 6 kinases show promising antitumour activity in TNBC cell lines and patient-derived xenografts. In summary, the present study elucidates phosphosites and kinases implicated in TNBC and suggests a target-based clinical classification system for TNBC.

Proteasome Activation by Small Molecules.

Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Proteomic and Metabolomic Analyses of Vanishing White Matter Mouse Astrocytes Reveal Deregulation of ER Functions.

Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho ) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation. We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.

Monitoring light/dark association dynamics of multi-protein complexes in cyanobacteria using size exclusion chromatography-based proteomics.

UNLABELLED: Diurnal rhythms are recurring 24h patterns such as light/dark cycles that affect many natural environmental and biological processes. The cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) produces its energy through photosynthesis and therefore its internal molecular machinery is strongly influenced by these diurnal rhythms. Moreover, it has one of the simplest, self-sustained, circadian rhythms, extensively studied functionally and structurally. These characteristics together with the relatively small genome of S. elongatus, make it an ideal model system for the study of diurnal and circadian rhythms. Although expression of many gene transcripts has been shown to fluctuate in phase with the circadian rhythm, fluctuations at the protein level were less pronounced. This led us to hypothesize that the diurnal adaptation occurs at the level of higher organization of protein complexes. Therefore, we probed the abundance and constituency of S. elongatus protein complexes during the day and night. Following several well-known complexes such as the RNA polymerase, the ribosome and photosynthetic protein complexes, we observe for the first time that these complexes change not only in abundance but also in constituency. Therefore, we conclude that the dynamic assembly of protein complexes is indeed also a key-player in the processes governing the diurnal rhythm. SIGNIFICANCE: The succession of day and night periods imposes drastic changes in all living organisms. Cyanobacteria produce their energy through photosynthesis and are therefore strongly influenced by diurnal rhythms. The cyanobacteria, Synechococcus elongatus PCC 7942 (S. elongatus), also exhibit a self-sustained biological clock. The connection between the central circadian oscillator and its output to the rest of the cell is not completely known. It has been shown that the expression of many gene transcripts heavily fluctuates in phase with the circadian rhythm; however, our recent global proteomics investigation revealed that the diurnal fluctuations seemed to be less pronounced at the protein level. As many known regulatory functions depend on protein-protein interactions (PPIs) and/or protein assemblies and the fact that so few fluctuations in protein abundances were observed earlier, here we investigated the diurnal adaptation at the level of dynamic changes in protein assembly. The paper demonstrates that the combination of native protein complex fractionation and high-resolution proteomics provides insight in the regulation of megadalton protein assemblies in cyanobacteria, including the ribosomal and photosynthetic complexes. The differences observed between the light and dark conditions in these complexes indicate a cyclic regulation of essential cellular processes.

The effect of stress on core and peripheral body temperature in humans.

Even though there are indications that stress influences body temperature in humans, no study has systematically investigated the effects of stress on core and peripheral body temperature. The present study therefore aimed to investigate the effects of acute psychosocial stress on body temperature using different readout measurements. In two independent studies, male and female participants were exposed to a standardized laboratory stress task (the Trier Social Stress Test, TSST) or a non-stressful control task. Core temperature (intestinal and temporal artery) and peripheral temperature (facial and body skin temperature) were measured. Compared to the control condition, stress exposure decreased intestinal temperature but did not affect temporal artery temperature. Stress exposure resulted in changes in skin temperature that followed a gradient-like pattern, with decreases at distal skin locations such as the fingertip and finger base and unchanged skin temperature at proximal regions such as the infra-clavicular area. Stress-induced effects on facial temperature displayed a sex-specific pattern, with decreased nasal skin temperature in females and increased cheek temperature in males. In conclusion, the amplitude and direction of stress-induced temperature changes depend on the site of temperature measurement in humans. This precludes a direct translation of the preclinical stress-induced hyperthermia paradigm, in which core temperature uniformly rises in response to stress to the human situation. Nevertheless, the effects of stress result in consistent temperature changes. Therefore, the present study supports the inclusion of body temperature as a physiological readout parameter of stress in future studies.

Measurement of alcohol hangover severity: development of the Alcohol Hangover Severity Scale (AHSS).

OBJECTIVE: This study aims to develop a new alcohol hangover symptom severity scale and compare its effectiveness with the Hangover Symptoms Scale (HSS), the Acute Hangover Scale (AHS), and a one-item hangover score. METHODS: Data from 1,410 Dutch students (Penning et al., Alcohol Alcohol 47:248-252, 2012) on the severity of 47 hangover symptoms were re-analyzed to develop the Alcohol Hangover Severity Scale (AHSS). The psychometric properties of the AHSS were compared with those of the HSS and the AHS. A survey among 1,000 students compared the AHSS and HSS with a one-item hangover severity score. The AHSS was further tested in a naturalistic hangover experiment. RESULTS: The 12 items of the AHSS were fatigue, clumsiness, dizziness, apathy, sweating, shivering, nausea, heart pounding, confusion, stomach pain, concentration problems, and thirst. The Penning et al. (Alcohol Alcohol 47:248-252, 2012) data revealed that the predictive validity of the AHSS (92.4 %) for the overall hangover score was significantly higher than that of the HSS (81.5 %) and the AHS (71.0 %). The survey data (N = 966) showed that scores on the AHSS (39.7 %) and the HSS (47.6 %) only moderately predicted the one-item hangover score. A total of 119 subjects completed the naturalistic study. On average, they consumed 9.7 alcoholic consumptions, yielding a mean estimated blood alcohol concentration (BAC) of 0.16 %. During hangover, the AHSS score correlated significantly with the number of alcoholic consumptions (r = 0.38, p < 0.0001) and estimated BAC (r = 0.40, p < 0.0001). CONCLUSIONS: The AHS, HSS, and AHSS all seem appropriate for application in hangover research. The use of a one-item hangover scale is not recommended.

Alcohol hangover symptoms and their contribution to the overall hangover severity.

AIMS: Scientific literature suggests a large number of symptoms that may be present the day after excessive alcohol consumption. The purpose of this study was to explore the presence and severity of hangover symptoms, and determine their interrelationship. METHODS: A survey was conducted among n = 1410 Dutch students examining their drinking behavior and latest alcohol hangover. The severity of 47 presumed hangover symptoms were scored on a 10-point scale ranging from 0 (absent) to 10 (maximal). Factor analysis was conducted to summarize the data into groups of associated symptoms that contribute significantly to the alcohol hangover and symptoms that do not. RESULTS: About half of the participants (56.1%, n = 791) reported having had a hangover during the past month. Most commonly reported and most severe hangover symptoms were fatigue (95.5%) and thirst (89.1%). Factor analysis revealed 11 factors that together account for 62% of variance. The most prominent factor 'drowsiness' (explained variance 28.8%) included symptoms such as drowsiness, fatigue, sleepiness and weakness. The second factor 'cognitive problems' (explained variance 5.9%) included symptoms such as reduced alertness, memory and concentration problems. Other factors, including the factor 'disturbed water balance' comprising frequently reported symptoms such as 'dry mouth' and 'thirst', contributed much less to the overall hangover (explained variance <5%). CONCLUSION: Drowsiness and impaired cognitive functioning are the two dominant features of alcohol hangover.

Next day effects of naturalistic alcohol consumption on tasks of attention.

OBJECTIVES: The aim of this study was to examine the next day effects of alcohol consumption on a range of attention tasks. METHODS: The study followed a counterbalanced repeated measure design, with participants tested the morning following normal/usual alcohol consumption and again the morning after no alcohol consumption. Participants were 48 social drinkers (15 men and 33 women), who performed attention tests at 9 am, 11 am, or 1 pm. Performance was assessed by tasks measuring sustained attention, divided attention, selective attention, and spatial attention and by the Stroop test. RESULTS: The morning after alcohol consumption, a significantly higher proportion of missed targets was observed (F(1, 40) = 6.43, p < 0.05) in the sustained attention task. In the Stroop test, participants responded significantly slower (F(1, 42) = 8.72, p < 0.005) in the interference condition (when naming the color of the ink of the words) the morning after alcohol consumption. In the selective attention task, the consumption of alcohol the night before eliminated the robust distance by compatibility interaction, which was observed the morning after no alcohol consumption (F(1, 43) = 10.41, p < 0.01). No influence of alcohol was observed in the divided attention test nor in the spatial attention task. CONCLUSION: Alcohol consumption has a negative impact on some but not all facets of attentional processing the morning after a normal nights drinking.

Treatment and prevention of alcohol hangover.

The search for alcohol hangover cures is as old as alcohol itself. Many cures and prophylactic agents are available, but scientific evidence for their effectiveness is generally lacking. This review summarizes and discusses the limited number of studies that examined the effectiveness of alcohol hangover treatments. From these studies it must be concluded that most remedies do not significantly reduce overall hangover severity. Some compounds reduce specific symptoms such as vomiting and headache, but are not effective in reducing other common hangover symptoms such as drowsiness and fatigue. Hangover cures that showed positive effects were those inhibiting prostaglandin synthesis or accelerating alcohol metabolism. Future studies should elucidate the pathology of alcohol hangover. Until then, it is unlikely that an effective hangover cure will be developed.

The alcohol hangover research group consensus statement on best practice in alcohol hangover research.

Alcohol-induced hangover, defined by a series of symptoms, is the most commonly reported consequence of excessive alcohol consumption. Alcohol hangovers contribute to workplace absenteeism, impaired job performance, reduced productivity, poor academic achievement, and may compromise potentially dangerous daily activities such as driving a car or operating heavy machinery. These socioeconomic consequences and health risks of alcohol hangover are much higher when compared to various common diseases and other health risk factors. Nevertheless, unlike alcohol intoxication the hangover has received very little scientific attention and studies have often yielded inconclusive results. Systematic research is important to increase our knowledge on alcohol hangover and its consequences. This consensus paper of the Alcohol Hangover Research Group discusses methodological issues that should be taken into account when performing future alcohol hangover research. Future research should aim to (1) further determine the pathology of alcohol hangover, (2) examine the role of genetics, (3) determine the economic costs of alcohol hangover, (4) examine sex and age differences, (5) develop common research tools and methodologies to study hangover effects, (6) focus on factor that aggravate hangover severity (e.g., congeners), and (7) develop effective hangover remedies.

The pathology of alcohol hangover.

Research on human subjects analyzing blood and urine samples determined biological correlates that may explain the pathology of alcohol hangover. These analyses showed that concentrations of various hormones, electrolytes, free fatty acids, triglycerides, lactate, ketone bodies, cortisol, and glucose were not significantly correlated with reported alcohol hangover severity. Also, markers of dehydration (e.g., vasopressin) were not significantly related to hangover severity. Some studies report a significant correlation between blood acetaldehyde concentration and hangover severity, but most convincing is the significant relationship between immune factors and hangover severity. The latter is supported by studies showing that hangover severity may be reduced by inhibitors of prostaglandin synthesis. Several factors do not cause alcohol hangover but can aggravate its severity. These include sleep deprivation, smoking, congeners, health status, genetics and individual differences. Future studies should more rigorously study these factors as well as biological correlates to further elucidate the pathology of alcohol hangover.

Drugs of abuse, driving and traffic safety.

Roadside studies indicate that 1-15% of drivers drive under the influence of one or more drugs of abuse. After drug use, drivers are more often culpable for an accident than non-users. Information on drugs and traffic safety comes from roadside studies, epidemiological research, experimental studies on driving-related skills, and on-the-road driving tests. Road-side studies show that drivers most frequently test positive for the use of alcohol and/or cannabis. These two drugs affect driving ability in a dose-dependent matter and result in poor vehicle control, especially when used in combination. Drivers on cocaine, ecstasy and amphetamine show no impairment on basic driving skills, but often overestimate their driving skills. In combination with impaired decision making, this increases risk taking during driving. Only few studies looked at the effects on driving of other drugs of abuse, such as ketamine, inhalants and anabolic steroids, but suggest a negative effect on driving performance. In conclusion, most drugs of abuse negatively affect driving ability, especially when used in combination with alcohol or another drug. It is of concern that a substantial number of drug users are not aware that their driving is impaired.