Comparative analysis of hippocampal extracellular space uncovers widely altered peptidome upon epileptic seizure in urethane-anaesthetized rats.

BACKGROUND: The brain extracellular fluid (ECF), composed of secreted neurotransmitters, metabolites, peptides, and proteins, may reflect brain processes. Analysis of brain ECF may provide new potential markers for synaptic activity or brain damage and reveal additional information on pathological alterations. Epileptic seizure induction is an acute and harsh intervention in brain functions, and it can activate extra- and intracellular proteases, which implies an altered brain secretome. Thus, we applied a 4-aminopyridine (4-AP) epilepsy model to study the hippocampal ECF peptidome alterations upon treatment in rats. METHODS: We performed in vivo microdialysis in the hippocampus for 3-3 h of control and 4-AP treatment phase in parallel with electrophysiology measurement. Then, we analyzed the microdialysate peptidome of control and treated samples from the same subject by liquid chromatography-coupled tandem mass spectrometry. We analyzed electrophysiological and peptidomic alterations upon epileptic seizure induction by two-tailed, paired t-test. RESULTS: We detected 2540 peptides in microdialysate samples by mass spectrometry analysis; and 866 peptides-derived from 229 proteins-were found in more than half of the samples. In addition, the abundance of 322 peptides significantly altered upon epileptic seizure induction. Several proteins of significantly altered peptides are neuropeptides (Chgb) or have synapse- or brain-related functions such as the regulation of synaptic vesicle cycle (Atp6v1a, Napa), astrocyte morphology (Vim), and glutamate homeostasis (Slc3a2). CONCLUSIONS: We have detected several consequences of epileptic seizures at the peptidomic level, as altered peptide abundances of proteins that regulate epilepsy-related cellular processes. Thus, our results indicate that analyzing brain ECF by in vivo microdialysis and omics techniques is useful for monitoring brain processes, and it can be an alternative method in the discovery and analysis of CNS disease markers besides peripheral fluid analysis.

LPS-induced acute neuroinflammation, involving interleukin-1 beta signaling, leads to proteomic, cellular, and network-level changes in the prefrontal cortex of mice.

Neuroinflammation induced by peripheral infections leads to various neuropsychiatric symptoms both in humans and laboratory animals, e.g., to the manifestation of sickness behavior that resembles some features of clinical depression. However, in addition to depression-like behavior, there are other symptoms of acute systemic inflammation that can be associated with the impairment of prefrontal cortex (PFC)-regulated cognitive functions. Thus, we investigated the electrophysiological and proteomic alterations of the PFC using brain slices and the lipopolysaccharide (LPS) model of acute peripheral infection in male mice. Based on the gene expression differences of the coreceptor (Il1rap) of interleukin-1 beta (IL-1ß) between neuron types in our previous single-cell sequencing dataset, we first compared the electrophysiological effects of IL-1ß on PFC pyramidal cells and interneurons. We found that pyramidal cells are more responsive to IL-1ß, as could be presumed from our transcriptomic data. To examine the possible circuit-level correlates of the cellular changes, frontal electroencephalographic (EEG) activity and fronto-occipital functional connectivity were analyzed in LPS-treated mice and significant changes were found in the fronto-occipital EEG correlation and coherence in the delta and high-gamma frequency bands. The upregulation of the prefrontal IL-1 system (IL-1ß and its receptor) after LPS treatment was revealed by immunoassays simultaneously with the observed EEG changes. Furthermore, we investigated the LPS-induced alterations of the synaptic proteome in the PFC using 2-D differential gel electrophoresis and mass spectrometry and found 48 altered proteins mainly related to cellular signaling, cytoskeletal organization, and carbohydrate/energy metabolism. Thus, our results indicate remarkable electrophysiological and molecular changes in the PFC related to acute systemic inflammation that may explain some of the concomitant behavioral and physiological symptoms.

The Effect of Sleep Deprivation and Subsequent Recovery Period on the Synaptic Proteome of Rat Cerebral Cortex.

Sleep deprivation (SD) is commonplace in the modern way of life and has a substantial social, medical, and human cost. Sleep deprivation induces cognitive impairment such as loss of executive attention, working memory decline, poor emotion regulation, increased reaction times, and higher cognitive functions are particularly vulnerable to sleep loss. Furthermore, SD is associated with obesity, diabetes, cardiovascular diseases, cancer, and a vast majority of psychiatric and neurodegenerative disorders are accompanied by sleep disturbances. Despite the widespread scientific interest in the effect of sleep loss on synaptic function, there is a lack of investigation focusing on synaptic transmission on the proteome level. In the present study, we report the effects of SD and recovery period (RP) on the cortical synaptic proteome in rats. Synaptosomes were isolated after 8 h of SD performed by gentle handling and after 16 h of RP. The purity of synaptosome fraction was validated with western blot and electron microscopy, and the protein abundance alterations were analyzed by mass spectrometry. We observed that SD and RP have a wide impact on neurotransmitter-related proteins at both the presynaptic and postsynaptic membranes. The abundance of synaptic proteins has changed to a greater extent in consequence of SD than during RP: we identified 78 proteins with altered abundance after SD and 39 proteins after the course of RP. Levels of most of the altered proteins were upregulated during SD, while RP showed the opposite tendency, and three proteins (Gabbr1, Anks1b, and Decr1) showed abundance changes with opposite direction after SD and RP. The functional cluster analysis revealed that a majority of the altered proteins is related to signal transduction and regulation, synaptic transmission and synaptic assembly, protein and ion transport, and lipid and fatty acid metabolism, while the interaction network analysis revealed several connections between the significantly altered proteins and the molecular processes of synaptic plasticity or sleep. Our proteomic data implies suppression of SNARE-mediated synaptic vesicle exocytosis and impaired endocytic processes after sleep deprivation. Both SD and RP altered GABA neurotransmission and affected protein synthesis, several regulatory processes and signaling pathways, energy homeostatic processes, and metabolic pathways.

The Single-Cell Transcriptomic Analysis of Prefrontal Pyramidal Cells and Interneurons Reveals the Neuronal Expression of Genes Encoding Antimicrobial Peptides and Immune Proteins.

The investigation of the molecular background of direct communication of neurons and immune cells in the brain is an important issue for understanding physiological and pathological processes in the nervous system. Direct contacts between brain-infiltrating immune cells and neurons, and the neuromodulatory effect of immune cell-derived regulatory peptides are well established. Several aspects of the role of immune and glial cells in the direct neuro-immune communication are also well known; however, there remain many questions regarding the molecular details of signaling from neurons to immune cells. Thus, we report here on the neuronal expression of genes encoding antimicrobial and immunomodulatory peptides, as well as proteins of immune cell-specific activation and communication mechanisms. In the present study, we analyzed the single-cell sequencing data of our previous transcriptomic work, obtained from electrophysiologically identified pyramidal cells and interneurons of the murine prefrontal cortex. We filtered out the genes that may be associated with the direct communication between immune cells and neurons and examined their expression pattern in the neuronal transcriptome. The expression of some of these genes by cortical neurons has not yet been reported. The vast majority of antimicrobial (~53%) and immune cell protein (~94%) transcripts was identified in the transcriptome of the 84 cells, owing to the high sensitivity of ultra-deep sequencing. Several of the antimicrobial and immune process-related protein transcripts showed cell type-specific or enriched expression. Individual neurons transcribed only a fraction of the investigated genes with low copy numbers probably due to the bursting kinetics of gene expression; however, the comparison of our data with available transcriptomic datasets from immune cells and neurons suggests the functional relevance of the reported findings. Accordingly, we propose further experimental and in silico studies on the neuronal expression of immune system-related genes and the potential role of the encoded proteins in neuroimmunological processes.

Cell Surface Protein mRNAs Show Differential Transcription in Pyramidal and Fast-Spiking Cells as Revealed by Single-Cell Sequencing.

The prefrontal cortex (PFC) plays a key role in higher order cognitive functions and psychiatric disorders such as autism, schizophrenia, and depression. In the PFC, the two major classes of neurons are the glutamatergic pyramidal (Pyr) cells and the GABAergic interneurons such as fast-spiking (FS) cells. Despite extensive electrophysiological, morphological, and pharmacological studies of the PFC, the therapeutically utilized drug targets are restricted to dopaminergic, glutamatergic, and GABAergic receptors. To expand the pharmacological possibilities as well as to better understand the cellular and network effects of clinically used drugs, it is important to identify cell-type-selective, druggable cell surface proteins and to link developed drug candidates to Pyr or FS cell targets. To identify the mRNAs of such cell-specific/enriched proteins, we performed ultra-deep single-cell mRNA sequencing (19 685 transcripts in total) on electrophysiologically characterized intact PFC neurons harvested from acute brain slices of mice. Several selectively expressed transcripts were identified with some of the genes that have already been associated with cellular mechanisms of psychiatric diseases, which we can now assign to Pyr (e.g., Kcnn2, Gria3) or FS (e.g., Kcnk2, Kcnmb1) cells. The earlier classification of PFC neurons was also confirmed at mRNA level, and additional markers have been provided.

Proteomic comparison of different synaptosome preparation procedures.

Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect.

Chronic stepwise cerebral hypoperfusion differentially induces synaptic proteome changes in the frontal cortex, occipital cortex, and hippocampus in rats.

During chronic cerebral hypoperfusion (CCH), the cerebral blood flow gradually decreases, leading to cognitive impairments and neurodegenerative disorders, such as vascular dementia. The reduced oxygenation, energy supply induced metabolic changes, and insufficient neuroplasticity could be reflected in the synaptic proteome. We performed stepwise bilateral common carotid occlusions on rats and studied the synaptic proteome changes of the hippocampus, occipital and frontal cortices. Samples were prepared and separated by 2-D DIGE and significantly altered protein spots were identified by HPLC-MS/MS. We revealed an outstanding amount of protein changes in the occipital cortex compared to the frontal cortex and the hippocampus with 94, 33, and 17 proteins, respectively. The high alterations in the occipital cortex are probably due to the hypoxia-induced retrograde degeneration of the primary visual cortex, which was demonstrated by electrophysiological experiments. Altered proteins have functions related to cytoskeletal organization and energy metabolism. As CCH could also be an important risk factor for Alzheimer's disease (AD), we investigated whether our altered proteins overlap with AD protein databases. We revealed a significant amount of altered proteins associated with AD in the two neocortical areas, suggesting a prominent overlap with the AD pathomechanism.

Placenta-Specific Genes, Their Regulation During Villous Trophoblast Differentiation and Dysregulation in Preterm Preeclampsia.

The human placenta maintains pregnancy and supports the developing fetus by providing nutrition, gas-waste exchange, hormonal regulation, and an immunological barrier from the maternal immune system. The villous syncytiotrophoblast carries most of these functions and provides the interface between the maternal and fetal circulatory systems. The syncytiotrophoblast is generated by the biochemical and morphological differentiation of underlying cytotrophoblast progenitor cells. The dysfunction of the villous trophoblast development is implicated in placenta-mediated pregnancy complications. Herein, we describe gene modules and clusters involved in the dynamic differentiation of villous cytotrophoblasts into the syncytiotrophoblast. During this process, the immune defense functions are first established, followed by structural and metabolic changes, and then by peptide hormone synthesis. We describe key transcription regulatory molecules that regulate gene modules involved in placental functions. Based on transcriptomic evidence, we infer how villous trophoblast differentiation and functions are dysregulated in preterm preeclampsia, a life-threatening placenta-mediated obstetrical syndrome for the mother and fetus. In the conclusion, we uncover the blueprint for villous trophoblast development and its impairment in preterm preeclampsia, which may aid in the future development of non-invasive biomarkers for placental functions and early identification of women at risk for preterm preeclampsia as well as other placenta-mediated pregnancy complications.

Proteomic Analysis of the Maternal Preoptic Area in Rats.

The behavior of female rats changes profoundly as they become mothers. The brain region that plays a central role in this regulation is the preoptic area, and lesions in this area eliminates maternal behaviors in rodents. The molecular background of the behavioral changes has not been established yet; therefore, in the present study, we applied proteomics to compare protein level changes associated with maternal care in the rat preoptic area. Using 2-dimensional fluorescence gel electrophoresis followed by identification of altered spots with mass spectrometry, 12 proteins were found to be significantly increased, and 6 proteins showed a significantly reduced level in mothers. These results show some similarities with a previous proteomics study of the maternal medial prefrontal cortex and genomics approaches applied to the preoptic area. Gene ontological analysis suggested that most altered proteins are involved in glucose metabolism and neuroplasticity. These proteins may support the maintenance of increased neuronal activity in the preoptic area, and morphological changes in preoptic neuronal circuits are known to take place in mothers. An increase in the level of alpha-crystallin B chain (Cryab) was confirmed by Western blotting. This small heat shock protein may also contribute to maintaining the increased activity of preoptic neurons by stabilizing protein structures. Common regulator and target analysis of the altered proteins suggested a role of prolactin in the molecular changes in the preoptic area. These results first identified the protein level changes in the maternal preoptic area. The altered proteins contribute to the maintenance of maternal behaviors and may also be relevant to postpartum depression, which can occur as a molecular level maladaptation to motherhood.

Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.

Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different "omics," clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental "virtual" liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal-fetal-placental immune interactions in preeclampsia. The description of these novel pathways in the "molecular phase" of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.

Early Presymptomatic Changes in the Proteome of Mitochondria-Associated Membrane in the APP/PS1 Mouse Model of Alzheimer's Disease.

Intracellular ß-amyloid (Aß) accumulation is an early event in Alzheimer's disease (AD) progression. Recently, it has been uncovered that presenilins (PSs), the key components of the amyloid precursor protein (APP) processing and the ß-amyloid producing γ-secretase complex, are highly enriched in a special sub-compartment of the endoplasmic reticulum (ER) functionally connected to mitochondria, called mitochondria-associated ER membrane (MAM). A current hypothesis of pathogenesis of Alzheimer's diseases (AD) suggests that MAM is involved in the initial phase of AD. Since MAM supplies mitochondria with essential proteins, the increasing level of PSs and ß-amyloid could lead to metabolic dysfunction because of the impairment of ER-mitochondrion crosstalk. To reveal the early molecular changes of this subcellular compartment in AD development MAM fraction was isolated from the cerebral cortex of 3 months old APP/PS1 mouse model of AD and age-matched C57BL/6 control mice, then mass spectrometry-based quantitative proteome analysis was performed. The enrichment and purity of MAM preparations were validated with EM, LC-MS/MS and protein enrichment analysis. Label-free LC-MS/MS was used to reveal the differences between the proteome of the transgenic and control mice. We obtained 77 increased and 49 decreased protein level changes in the range of - 6.365 to + 2.988, which have mitochondrial, ER or ribosomal localization according to Gene Ontology database. The highest degree of difference between the two groups was shown by the ATP-binding cassette G1 (Abcg1) which plays a crucial role in cholesterol metabolism and suppresses Aß accumulation. Most of the other protein changes were associated with increased protein synthesis, endoplasmic-reticulum-associated protein degradation (ERAD), oxidative stress response, decreased mitochondrial protein transport and ATP production. The interaction network analysis revealed a strong relationship between the detected MAM protein changes and AD. Moreover, it explored several MAM proteins with hub position suggesting their importance in Aß induced early MAM dysregulation. Our identified MAM protein changes precede the onset of dementia-like symptoms in the APP/PS1 model, suggesting their importance in the development of AD.

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex.

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer's disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia.

Synaptic proteome changes in the hypothalamus of mother rats.

To establish synaptic proteome changes associated with motherhood, we isolated synaptosome fractions from the hypothalamus of mother rats and non-maternal control females at the 11th postpartum day. Proteomic analysis by two-dimensional differential gel electrophoresis combined with mass spectrometric protein identification established 26 significant proteins, 7 increasing and 19 decreasing protein levels in the dams. The altered proteins are mainly involved in energy homeostasis, protein folding, and metabolic processes suggesting the involvement of these cellular processes in maternal adaptations. The decrease in a significantly altered protein, complement component 1q subcomponent-binding protein (C1qbp) was validated with Western blotting. Furthermore, immunohistochemistry showed its presence in hypothalamic fibers and terminals in agreement with its presence in synaptosomes. We also found the expression of C1qbp in different hypothalamic nuclei including the preoptic area and the paraventricular hypothalamic nucleus at the protein and at the mRNA level using immunohistochemistry and in situ hybridization histochemistry, respectively. Bioinformatical network analysis revealed that cytokines, growth factors, and protein kinases are common regulators, which indicates a complex regulation of the proteome change in mothers. The results suggest that maternal responsiveness is associated with synaptic proteins level changes in the hypothalamus, and that growth factors and cytokines may govern these alterations. BIOLOGICAL SIGNIFICANCE: The period of motherhood is accompanied with several behavioral, neuroendocrine, emotional and metabolic adaptations in the brain. Although it is established that various hypothalamic networks participate in the maternal adaptations of the rodent brain, our knowledge on the molecular background of these alterations remains seriously limited. In the present study, we first determined that the functional alterations of the maternal brain can be detected at the level of the synaptic proteome in the hypothalamus. Independent confirmation of synaptic localization, and also the established decrease in the level of C1qbp protein suggest the validity of the data. Common regulators of altered proteins belonging to the growth factor and cytokine family suggest that the synaptic adaptation is governed by these extracellular signals and future studies should focus on their specific roles. Our study was also the first to describe the expression pattern of C1qbp in the hypothalamus, a protein potentially involved in mitochondrial and neuroimmunological regulations of synaptic plasticity. Its presence in the preoptic area responsible for maternal behaviors and also in the paraventricular hypothalamic and arcuate nuclei regulating hormonal levels suggests that the same proteins may be involved in different aspects of maternal adaptations. The conclusions of the present work contribute to establishing the molecular alterations that determine different maternal adaptations in the brain. Since maternal changes are models of neuronal plasticity in all social interactions, the reported results can affect a wide field of molecular and behavioral neuroscience.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Maternal alterations in the proteome of the medial prefrontal cortex in rat.

Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC). A 2-D DIGE minimal dye technique combined with LC-MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR. The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression. BIOLOGICAL SIGNIFICANCE: The behavior and emotional state of females change robustly when they become mothers. The brain, which governs these changes, may also undergo molecular alterations in mothers. As no proteomics approaches have been applied regarding maternal changes in the brain, we addressed this issue in the mPFC as this brain area is the uppermost cortical center of maternal control and the associated mood changes. The high number of protein-level alterations found between mothers taking care of their litter and those without pups indicates that pup nursing is associated with cortical protein-level changes. Alterations in proteins participating in synaptic transport, plasticity and neuron development suggest neuroplastic changes in the maternal brain. In turn, the relatively high number of altered proteins in the mPFC associated with depression suggests that the physiological effects of the protein-level alterations in the maternal mPFC could promote the incidence of postpartum depression. Cryab, a protein confirmed to be increased during maternal behaviors, was selectively found in parvalbumin cells, which, as fast-spiking interneurons, are associated with depression. The function of Cryab should be further investigated to establish whether it can be used to identify drug targets for future drug development.

Mitochondrial Proteome Changes Correlating with ß-Amyloid Accumulation.

Alzheimer's disease (AD) is a multifactorial disease of wide clinical heterogenity. Overproduction of amyloid precursor protein (APP) and accumulation of ß-amyloid (Aß) and tau proteins are important hallmarks of AD. The identification of early pathomechanisms of AD is critically important for discovery of early diagnosis markers. Decreased brain metabolism is one of the earliest clinical symptoms of AD that indicate mitochondrial dysfunction in the brain. We performed the first comprehensive study integrating synaptic and non-synaptic mitochondrial proteome analysis (two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry) in correlation with Aß progression in APP/PS1 mice (3, 6, and 9 months of age). We identified changes of 60 mitochondrial proteins that reflect the progressive effect of APP overproduction and Aß accumulation on mitochondrial processes. Most of the significantly affected proteins play role in the mitochondrial electron transport chain, citric acid cycle, oxidative stress, or apoptosis. Altered expression levels of Htra2 and Ethe1, which showed parallel changes in different age groups, were confirmed also by Western blot. The common regulator bioinformatical analysis suggests the regulatory role of tumor necrosis factor (TNF) in Aß-mediated mitochondrial protein changes. Our results are in accordance with the previous postmortem human brain proteomic studies in AD in the case of many proteins. Our results could open a new path of research aiming early mitochondrial molecular mechanisms of Aß accumulation as a prodromal stage of human AD.

Proteomic investigation of the prefrontal cortex in the rat clomipramine model of depression.

Neonatal rodents chronically treated with the tricyclic antidepressant clomipramine show depression-like behavior, which persists throughout adulthood. Therefore, this animal model is suitable to investigate the pathomechanism of depression, which is still largely unknown at the molecular level beyond monoaminergic dysfunctions. Here, we describe protein level changes in the prefrontal cortex of neonatally clomipramine-treated adult rats correlating with behavioral abnormalities. Clomipramine was administered to rat pups twice daily between postnatal days 8-21, while controls received saline injections. Behavioral tests were performed on 3months old rats. The proteomic study was conducted using two-dimensional differential gel electrophoresis. We have identified 32 proteins by mass spectrometry analysis of the significantly altered protein spots. The changed proteins are related to several biological functions, such as inflammation, transcription, cell metabolism and cytoskeleton organization. Among the altered proteins, the level of macrophage migration inhibitory factor showed the largest alteration, which was confirmed with Western blot. Macrophage migration inhibitory factor showed widespread distribution and was predominantly expressed in astrocytes in the forebrain of rats which were described using immunohistochemistry. We conclude that neonatal clomipramine exposure induces sustained modification in the proteome, which may form the molecular basis of the observed depression-like behavior in adult rats. BIOLOGICAL SIGNIFICANCE: It is known that some of the psychiatric disorders, such as autism, depression or schizophrenia may be at least in part, developmental disorders. We hypothesized that clomipramine treatment in early stage of brain development, which is known to induce depression-like behavior in adult rats, results in pathological distortion in neuronal and glial network development, which can be reflected by the cellular proteome in adulthood. Thus, we performed an unbiased proteomics experiment in adult rats, which were neonatally administered with clomipramine to reveal protein level changes three months after treatment. Many of the identified changed proteins are previously associated with depressive symptoms, e.g., the macrophage migration inhibitory factor (MIF), the level of which showed the largest alteration among the identified proteins. Based on our data, we suggest that neonatal clomipramine treatment is a reliable model to study the developmental effect of psychoactive drugs applied in the sensitive early phase of brain development. Furthermore, our findings support the idea that the alteration of early development of the brain induced by antidepressant treatment could result in sustained pathological changes in the cellular phenotype in the prefrontal cortex leading to depression-like behavioral symptoms.

Synaptic mitochondria: a brain mitochondria cluster with a specific proteome.

The synapse is a particularly important compartment of neurons. To reveal its molecular characteristics we isolated whole brain synaptic (sMito) and non-synaptic mitochondria (nsMito) from the mouse brain with purity validated by electron microscopy and fluorescence activated cell analysis and sorting. Two-dimensional differential gel electrophoresis and mass spectrometry based proteomics revealed 22 proteins with significantly higher and 34 proteins with significantly lower levels in sMito compared to nsMito. Expression differences in some oxidative stress related proteins, such as superoxide dismutase [Mn] (Sod2) and complement component 1Q subcomponent-binding protein (C1qbp), as well as some tricarboxylic acid cycle proteins, including isocitrate dehydrogenase subunit alpha (Idh3a) and ATP-forming ß subunit of succinyl-CoA ligase (SuclA2), were verified by Western blot, the latter two also by immunohistochemistry. The data suggest altered tricarboxylic acid metabolism in energy supply of synapse while the marked differences in Sod2 and C1qbp support high sensitivity of synapses to oxidative stress. Further functional clustering demonstrated that proteins with higher synaptic levels are involved in synaptic transmission, lactate and glutathione metabolism. In contrast, mitochondrial proteins associated with glucose, lipid, ketone metabolism, signal transduction, morphogenesis, protein synthesis and transcription were enriched in nsMito. Altogether, the results suggest a specifically tuned composition of synaptic mitochondria. BIOLOGICAL SIGNIFICANCE: Neurons communicate with each other through synapse, a compartment metabolically isolated from the cell body. Mitochondria are concentrated in presynaptic terminals by active transport to provide energy supply for information transfer. Mitochondrial composition in the synapse may be different than in the cell body as some examples have demonstrated altered mitochondrial composition with cell type and cellular function in the muscle, heart and liver. Therefore, we posed the question whether protein composition of synaptic mitochondria reflects its specific functions. The determined protein difference pattern was in accordance with known functional specialties of high demand synaptic mitochondria. The data also suggest specifically tuned metabolic fluxes for energy production by means of interaction with glial cells surrounding the synapse. These findings provide possible mechanisms for dynamically adapting synaptic mitochondrial output to actual demand. In turn, an increased vulnerability of synaptic mitochondria to oxidative stress is implied by the data. This is important from theoretical but potentially also from therapeutic aspects. Mitochondria are known to be affected in some neurodegenerative and psychiatric disorders, and proteins with elevated level in synaptic mitochondria, e.g. C1qbp represent targets for future drug development, by which synaptic and non-synaptic mitochondria can be differentially affected.

Altered functional protein networks in the prefrontal cortex and amygdala of victims of suicide.

Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.

Different electrophysiological actions of 24- and 72-hour aggregated amyloid-beta oligomers on hippocampal field population spike in both anesthetized and awake rats.

Diffusible oligomeric assemblies of the amyloid beta-protein (Abeta) could be the primary factor in the pathogenic pathway leading to Alzheimer's disease (AD). Converging lines of evidence support the notion that AD begins with subtle alterations in synaptic efficacy, prior to the occurrence of extensive neuronal degeneration. Recently, however, a shared or overlapping pathogenesis for AD and epileptic seizures occurred as aberrant neuronal hyperexcitability, as well as nonconvulsive seizure activity were found in several different APP transgenic mouse lines. This generated a renewed attention to the well-known comorbidity of AD and epilepsy and interest in how Abeta oligomers influence neuronal excitability. In this study therefore, we investigated the effect of various in vitro-aged Abeta(1-42) oligomer solutions on the perforant pathway-evoked field potentials in the ventral hippocampal dentate gyrus in vivo. Firstly, Abeta oligomer solutions (1 microl, 200 microM) which had been aggregated in vitro for 0, 24 or 72h were injected into the hippocampus of urethane-anesthetized rats, in parallel with in vitro physico-chemical characterization of Abeta oligomerization (atomic force microscopy, thioflavin-T fluorescence). We found a marked increase of hippocampal population spike (pSpike) after injection of the 24-h Abeta oligomer solution and a decrease of the pSpike amplitude after injection of the 72-h Abeta oligomer. Since urethane anesthesia affects the properties of hippocampal evoked potentials, we repeated the injection of these two Abeta oligomer solutions in awake, freely moving animals. Evoked responses to perforant pathway stimulation revealed a 70% increase of pSpike amplitude 50 min after the 24-h Abeta oligomer injection and a 55% decrease after the 72-h Abeta oligomer injection. Field potentials, that reflect synaptic potentials, were not affected by the Abeta injection. These results demonstrate that oligomeric Abeta aggregates elicit opposite electrophysiological effects on neuronal excitability which depend on their degree of oligomerization.