The (15) N isotope effect as a means for correlating phenotypic alterations and affected pathways in a trait anxiety mouse model

Stable isotope labeling techniques hold great potential for accurate quantitative proteomics comparisons by MS. To investigate the effect of stable isotopes in vivo, we metabolically labeled high anxiety-related behavior (HAB) mice with the heavy nitrogen isotope (15) N. (15) N-labeled HAB mice exhibited behavioral alterations compared to unlabeled ((14) N) HAB mice in their depression-like phenotype. To correlate behavioral alterations with changes on the molecular level, we explored the (15) N isotope effect on the brain proteome by comparing protein expression levels between (15) N-labeled and (14) N HAB mouse brains using quantitative MS. By implementing two complementary in silico pathway analysis approaches, we were able to identify altered networks in (15) N-labeled HAB mice, including major metabolic pathways such as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Here, we discuss the affected pathways with regard to their relevance for the behavioral phenotype and critically assess the utility of exploiting the (15) N isotope effect for correlating phenotypic and molecular alterations.     

Proteomic profiling of lymphedema development in mouse model

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham‐operated and lymphedema‐operated mice for 3 days, using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced‐right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A‐1 preprotein, alpha‐actinin‐3, mCG21744, parkinson disease, serum amyloid P‐component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham‐operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up‐regulation of serum amyloid P‐component precursor, parkinson disease, and apolipoprotein A‐1 preprotein in lymphedema model over sham‐operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.     

Sleep phenotyping in a mouse model of extreme trait anxiety

Background

There is accumulating evidence that anxiety impairs sleep. However, due to high sleep variability in anxiety disorders, it has been difficult to state particular changes in sleep parameters caused by anxiety. Sleep profiling in an animal model with extremely high vs. low levels of trait anxiety might serve to further define sleep patterns associated with this psychopathology.

Methodology/Principal Findings

Sleep-wake behavior in mouse lines with high (HAB), low (LAB) and normal (NAB) anxiety-related behaviors was monitored for 24 h during baseline and recovery after 6 h sleep deprivation (SD). The amounts of each vigilance state, sleep architecture, and EEG spectral variations were compared between the mouse lines. In comparison to NAB mice, HAB mice slept more and exhibited consistently increased delta power during non-rapid eye movement (NREM) sleep. Their sleep patterns were characterized by heavy fragmentation, reduced maintenance of wakefulness, and frequent intrusions of rapid eye movement (REM) sleep. In contrast, LAB mice showed a robust sleep-wake rhythm with remarkably prolonged sleep latency and a long, persistent period of wakefulness. In addition, the accumulation of delta power after SD was impaired in the LAB line, as compared to HAB mice.

Conclusions/Significance

Sleep-wake patterns were significantly different between HAB and LAB mice, indicating that the genetic predisposition to extremes in trait anxiety leaves a biological scar on sleep quality. The enhanced sleep demand observed in HAB mice, with a strong drive toward REM sleep, may resemble a unique phenotype reflecting not only elevated anxiety but also a depression-like attribute.     

Protein biomarkers in a mouse model of extremes in trait anxiety

Brain proteome analysis of mice selectively bred for either high or low anxiety-related behavior revealed quantitative and qualitative protein expression differences. The enzyme glyoxalase-I was consistently expressed to a higher extent in low anxiety as compared with high anxiety mice in several brain areas. The same phenotype-dependent difference was also found in red blood cells with normal and cross-mated animals showing intermediate expression profiles of glyoxalase-I. Another protein that showed a different mobility during two-dimensional gel electrophoresis was identified as enolase phosphatase. The presence of both protein markers in red or white blood cells, respectively, creates the opportunity to screen for their expression in clinical blood specimens from patients suffering from anxiety.     

Proteomic and metabolomic profiling of Valencia orange fruit after natural frost exposure

The aim of this study was to evaluate the response of orange fruit (Citrus sinensis var. Valencia Late) to freezing stress in planta, both immediately after the natural event and after a week, in order to understand the biochemical and molecular basis of the changes that later derive in internal and external damage symptoms. Using two‐dimensional differential gel electrophoresis to analyze exposed and non‐exposed fruit, 27 differential protein spots were detected in juice sacs and flavedo, among all comparisons made. Also, primary and secondary metabolites relative contents were analyzed in both tissues by gas chromatography‐mass spectrometry and liquid chromatography‐mass spectrometry, respectively. Proteins and compounds involved in regulatory functions, iron metabolism, oxidative damage and carbohydrate metabolism were the most affected. Interestingly, three glycolytic enzymes were induced by cold, and there was an increase in fermentation products (volatiles); all of that suggests that more energy generation might be required from glycolysis to counter the cold stress. Moreover, a notable increase in sugar levels was observed after frost, but it was not at the expense of organic acids utilization. Consequently, these results suggest a probable redistribution of photoassimilates in the frost‐exposed plants, tending to restore the homeostasis altered by that severe type of stress. Isosinensetin was the most cold‐sensitive secondary metabolite because it could not be detected at all after the frost, constituting a possible tool to early diagnose freezing damage.     

Enhanced fear expression in a psychopathological mouse model of trait anxiety: pharmacological interventions

The propensity to develop an anxiety disorder is thought to be determined by genetic and environmental factors. Here we investigated the relationship between a genetic predisposition to trait anxiety and experience-based learned fear in a psychopathological mouse model. Male CD-1 mice selectively bred for either high (HAB), or normal (NAB) anxiety-related behaviour on the elevated plus maze were subjected to classical fear conditioning. During conditioning both mouse lines showed increased fear responses as assessed by freezing behaviour. However, 24 h later, HAB mice displayed more pronounced conditioned responses to both a contextual or cued stimulus when compared with NAB mice. Interestingly, 6 h and already 1 h after fear conditioning, freezing levels were high in HAB mice but not in NAB mice. These results suggest that trait anxiety determines stronger fear memory and/or a weaker ability to inhibit fear responses in the HAB line. The enhanced fear response of HAB mice was attenuated by treatment with either the α(2,3,5)-subunit selective benzodiazepine partial agonist L-838,417, corticosterone or the selective neurokinin-1 receptor antagonist L-822,429. Overall, the HAB mouse line may represent an interesting model (i) for identifying biological factors underlying misguided conditioned fear responses and (ii) for studying novel anxiolytic pharmacotherapies for patients with fear-associated disorders, including post-traumatic stress disorder and phobias.     

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.     

Proteomic profiling of accessory structures from the mouse sperm flagellum

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.     

Serum metabolomic markers for traumatic brain injury: a mouse model

Introduction

Traumatic brain injury (TBI) is physical injury to brain tissue that temporarily or permanently impairs brain function.

Objectives

Evaluate the use of metabolomics for the development of biomarkers of TBI for the diagnosis and timing of injury onset.

Methods

A validated model of closed injury TBI was employed using 10 TBI mice and 8 sham operated controls. Quantitative LC–MS/MS metabolomic analysis was performed on the serum.

Results

Thirty-six (24.0 %) of 150 metabolites were altered with TBI. Principal component analysis (PCA) and Partial least squares discriminant analysis (PLS-DA) analyses revealed clear segregation between TBI versus control sera. The combination of methionine sulfoxide and the lipid PC aa C34:4 accurately diagnosed TBI, AUC (95 % CI) 0.85 (0.644–1.0). A combination of metabolite markers were highly accurate in distinguishing early (4 h post TBI) from late (24 h) TBI: AUC (95 % CI) 1.0 (1.0–1.0). Spermidine, which is known to have an antioxidant effect and which is known to be metabolically disrupted in TBI, was the most discriminating biomarker based on the variable importance ranking in projection (VIP) plot. Several important metabolic pathways were found to be disrupted including: pathways for arginine, proline, glutathione, cysteine, and sphingolipid metabolism.

Conclusion

Using serum metabolomic analysis we were able to identify novel putative serum biomarkers of TBI. They were accurate for detecting and determining the timing of TBI. In addition, pathway analysis provided important insights into the biochemical mechanisms of brain injury. Potential clinical implications for diagnosis, timing, and monitoring brain injury are discussed.

     

Proteomic profiling in an animal model of acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disease of the pancreas, which evolves in approximately 20% of the patients to a severe illness associated with a high mortality rate. In this study, we performed a comparative proteomic analysis of pancreatic tissue extracts from rats with AP and healthy rodent controls in order to identify changes in protein expression related to the pathobiological processes of this disease. Pancreatic extracts from diseased and controls rats were analyzed by 2-DE and MS/MS. A total of 125 proteins were identified from both samples. Comparative analysis allowed the detection of 42 proteins or protein fragments differentially expressed between diseased and control pancreas, some of them being newly described in AP. Interestingly, these changes were representative of the main pathobiological pathways involved in this disease. We observed activation of digestive proteases and increased expression of various inflammatory markers, including several members of the alpha-macroglobulin family. We also detected changes related to oxidative and cell stress responses. Finally, we highlighted modifications of 14-3-3 proteins that could be related to apoptosis regulation. These results showed the interest of proteomic analysis to identify changes characterizing pancreatic tissue damage and, therefore, to highlight new potential biomarkers of AP.     

Proteomic profiling of the hypothalamus in two mouse models of narcolepsy

Narcolepsy is a disabling neurological disorder of sleepiness linked to the loss of neurons producing orexin neuropeptides in the hypothalamus. Two well‐characterized phenotypic mouse models of narcolepsy, loss‐of‐function (orexin‐knockout), and progressive loss of orexin (orexin/ataxin‐3) exist. The open question is whether the proteomics signatures of the hypothalamus would be different between the two models. To address this gap, we utilized a label‐free proteomics approach and conducted a hypothalamic proteome analysis by comparing each disease model to that of wild type. Following data processing and statistical analysis, 14 484 peptides mapping to 2282 nonredundant proteins were identified, of which 39 proteins showed significant differences in protein expression across groups. Altered proteins in both models showed commonalties in pathways for mitochondrial dysfunction and neuronal degeneration, as well as altered proteins related to inflammatory demyelination, insulin resistance, metabolic responses, and the dopaminergic and monoaminergic systems. Model‐specific alterations in insulin degraded enzyme (IDE) and synaptosomal‐associated protein‐25 were unique to orexin‐KO and orexin/ataxin‐3, respectively. For both models, proteomics not only identified clinically suspected consequences of orexin loss on energy homeostasis and neurotransmitter systems, but also identified commonalities in inflammation and degeneration despite the entirely different genetic basis of the two mouse models.     

Proteomic analysis of muscle affected by motor neuron degeneration: the wobbler mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is the most common form of motor neuron disease in adult patients and characterized by progressive paralysis. The wobbler mouse (phenotype WR, genotype wr/wr) is an established animal model of human motor neuron disease and is characterized by a large variety of cellular abnormalities including muscular atrophy. In analogy to recent proteomic studies of cerebrospinal fluid and spinal cord, we have used here fluorescence difference in-gel electrophoresis to analyze global changes in the skeletal muscle proteome from WR versus normal mice. Relative concentrations of 21 proteins were found to be increased and 3 proteins were decreased. Mass spectrometric analysis identified these proteins to be associated with key metabolic pathways, the contractile apparatus, intermediate filaments and the cellular stress response. Drastically increased levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase were confirmed by immunoblotting and this finding agrees with the idea of an oxidative-to-glycolytic shift in disease-related muscular atrophy. The establishment of novel disease-specific biomarkers of motor neuron disease might be helpful in the design of improved diagnostic tools and the identification of novel therapeutic targets.     

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x-linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of β-dystroglycan and key Ca²⁺-regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca²⁺-handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response.     

Proteomic and cell biological profiling of the renal phenotype of the mdx-4cv mouse model of Duchenne muscular dystrophy

The X-linked inherited muscle wasting disease Duchenne muscular dystrophy, which is caused by primary abnormalities in the membrane cytoskeletal protein dystrophin, is a multi-system disorder. Highly progressive forms of dystrophinopathy are associated with a complex secondary pathophysiology, including renal dysfunction. It was therefore of interest to carry out a systematic survey of potential proteome-wide changes in the kidney of the established mdx-4cv mouse model of dystrophinopathy. Of 5878 mass spectrometrically identified kidney proteins, 82 versus 142 proteins were shown to be decreased or increased, respectively, in association with muscular dystrophy. The most decreased versus increased protein species are the ACSM3 isoform of mitochondrial acyl-coenzyme A synthetase and the FABP1 isoform of fatty acid binding protein, respectively. Both proteomic findings were verified by immunofluorescence microscopy and immunoblot analysis. Interestingly, haematoxylin/eosin staining indicated diffuse whitish deposits in the mdx-4cv kidney, and an increased intensity of Sudan Black labelling of kidney cells revealed ectopic fat deposition. Although the proteomic results and cell biological findings do not demonstrate a direct functional link between increased FABP1 and fat accumulation, the results suggest that the up-regulation of FABP1 may be related to abnormal fat metabolism. This makes FABP1 potentially a novel pathobiochemical indicator for studying kidney abnormalities in the mdx-4cv model of dystrophinopathy.     

Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse

Lipid droplets in adipocytes serve as the principal long-term energy storage depot of animals. There is increasing recognition that lipid droplets are not merely a static neutral lipid storage site, but in fact dynamic and multi-functional organelles. Structurally, lipid droplet consists of a neutral lipid core surrounded by a phospholipid monolayer and proteins embedded in or bound to the phospholipid layer. Proteins on the surface of lipid droplets are crucial to droplet structure and dynamics. To understand the lipid droplet-associated proteome of primary adipocyte with a large central lipid droplet, lipid droplets of white adipose tissue from C57BL/6 mice were isolated. And the proteins were extracted and analyzed by liquid chromatography coupled with tandem mass spectrometry. A total of 193 proteins including 73 previously unreported proteins were identified. Furthermore, the isotope-coded affinity tags (ICAT) was used to compare the difference of lipid droplet-associated proteomes between the normal lean and the high-fat diet-induced obese C57BL/6 mice. Of 23 proteins quantified by ICAT analysis, 3 proteins were up-regulated and 4 proteins were down-regulated in the lipid droplets of adipose tissue from the obese mice. Importantly, two structural proteins of lipid droplets, perilipin A and vimentin, were greatly reduced in the lipid droplets of the adipose tissue from the obese mice, implicating reduced protein machinery for lipid droplet stability.     

Proteomic profiling of skeletal muscle in an animal model of overtraining

Excessive training (i.e. overtraining, OT) may result in underperformance, which can be characterized by the time needed to re-establish performance (i.e. functional overreaching (FOR), nonfunctional overreaching, OT syndrome). The present study is an initial screening for proteins presenting altered abundance in the red (RG) and white (WG) portions of the gastrocnemius muscle from rats submitted to an OT protocol that induced FOR. In the RG, compared to the nontrained control, FOR demonstrated an increased abundance of proteins normally related to adaptation to endurance training (e.g. proteins of oxidative phosphorylation complexes, proteins related to lipid metabolism, antioxidants, and chaperones). In the WG, spots identified as mitochondrial aconitase and a component of the succinate dehydrogenase complex were downregulated in FOR, as were proteins related to myofibril stabilization; these latter were upregulated in the RG. This initial study shows that skeletal muscles with different fiber-type compositions respond differently to an OT period. Also, it is likely that actin-interacting proteins have an important role in muscle adaptation to endurance exercise.