Chronic Cerebral Hypoperfusion-Induced Disturbed Proteostasis of Mitochondria and MAM Is Reflected in the CSF of Rats by Proteomic Analysis.

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. We carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. We found 19, 35, and 12 significantly altered proteins in the mitochondria, MAM, and CSF, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. We confirmed decreased levels of proteins involved in protein folding and amino acid catabolism, such as P4hb and Hibadh in the mitochondria by western blot. We detected reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

Neuronal-specific septin-3 binds Atg8/LC3B, accumulates and localizes to autophagosomes during induced autophagy.

In synapses that show signs of local apoptosis and mitochondrial stress and undergo neuro-immunological synapse pruning, an increase in the levels of the presynaptic protein, neuronal-specific septin-3 can be observed. Septin-3 is a member of the septin GTPase family with the ability to form multimers and contribute to the cytoskeleton. However, the function of septin-3 remains elusive. Here, we provide evidence that septin-3 is capable of binding the most-studied autophagy protein Atg8 homolog microtubule-associated protein 1 light chain 3B (LC3B), besides another homolog, GABA receptor-associated protein-like 2 (GABARAPL2). Moreover, we demonstrate that colocalization of septin-3 and LC3B increases upon chemical autophagy induction in primary neuronal cells. Septin-3 is accumulated in primary neurons upon autophagy enhancement or blockade, similar to autophagy proteins. Using electron microscopy, we also show that septin-3 localizes to LC3B positive membranes and can be found at mitochondria. However, colocalization results of septin-3 and the early mitophagy marker PTEN-induced kinase 1 (PINK1) do not support that binding of septin-3 to mitochondria is mitophagy related. We conclude that septin-3 correlates with synaptic/neuronal autophagy, binds Atg8 and localizes to autophagic membranes that can be enhanced with chemical autophagy induction. Based on our results, elevated septin-3 levels might indicate enhanced or impeded autophagy in neurons.

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins.

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.

Proteomic identification of Placental Protein 1 (PP1), PP8, and PP22 and characterization of their placental expression in healthy pregnancies and in preeclampsia.

INTRODUCTION: Placental Protein 1 (PP1), PP8, and PP22 were isolated from the placenta. Herein, we aimed to identify PP1, PP8, and PP22 proteins and their placental and trophoblastic expression patterns to reveal potential involvement in pregnancy complications. METHODS: We analyzed PP1, PP8, and PP22 proteins with LC-MS. We compared the placental behaviors of PP1, PP8, and PP22 to the predominantly placenta-expressed PP5/TFPI-2. Placenta-specificity scores were generated from microarray data. Trophoblasts were isolated from healthy placentas and differentiated; total RNA was isolated and subjected to microarray analysis. We assigned the placentas to the following groups: preterm controls, early-onset preeclampsia, early-onset preeclampsia with HELLP syndrome, term controls, and late-onset preeclampsia. After histopathologic examination, placentas were used for tissue microarray construction, immunostaining with anti-PP1, anti-PP5, anti-PP8, or anti-PP22 antibodies, and immunoscoring. RESULTS: PP1, PP8, and PP22 were identified as 'nicotinate-nucleotide pyrophosphorylase', 'serpin B6', and 'protein disulfide-isomerase', respectively. Genes encoding PP1, PP8, and PP22 are not predominantly placenta-expressed, in contrast with PP5. PP1, PP8, and PP22 mRNA expression levels did not increase during trophoblast differentiation, in contrast with PP5. PP1, PP8, and PP22 immunostaining were detected primarily in trophoblasts, while PP5 expression was restricted to the syncytiotrophoblast. The PP1 immunoscore was higher in late-onset preeclampsia, while the PP5 immunoscore was higher in early-onset preeclampsia. DISCUSSION: PP1, PP8, and PP22 are expressed primarily in trophoblasts but do not have trophoblast-specific regulation or functions. The distinct dysregulation of PP1 and PP5 expression in either late-onset or early-onset preeclampsia reflects different pathophysiological pathways in these preeclampsia subsets.

Synaptic mitochondrial dysfunction and septin accumulation are linked to complement-mediated synapse loss in an Alzheimer's disease animal model.

Synaptic functional disturbances with concomitant synapse loss represent central pathological hallmarks of Alzheimer's disease. Excessive accumulation of cytotoxic amyloid oligomers is widely recognized as a key event that underlies neurodegeneration. Certain complement components are crucial instruments of widespread synapse loss because they can tag synapses with functional impairments leading to their engulfment by microglia. However, an exact understanding of the affected synaptic functions that predispose to complement-mediated synapse elimination is lacking. Therefore, we conducted systematic proteomic examinations on synaptosomes prepared from an amyloidogenic mouse model of Alzheimer's disease (APP/PS1). Synaptic fractions were separated according to the presence of the C1q-tag using fluorescence-activated synaptosome sorting and subjected to proteomic comparisons. The results raised the decline of mitochondrial functions in the C1q-tagged synapses of APP/PS1 mice based on enrichment analyses, which was verified using flow cytometry. Additionally, proteomics results revealed extensive alterations in the level of septin protein family members, which are known to dynamically form highly organized pre- and postsynaptic supramolecular structures, thereby affecting synaptic transmission. High-resolution microscopy investigations demonstrated that synapses with considerable amounts of septin-3 and septin-5 show increased accumulation of C1q in APP/PS1 mice compared to the wild-type ones. Moreover, a strong positive correlation was apparent between synaptic septin-3 levels and C1q deposition as revealed via flow cytometry and confocal microscopy examinations. In sum, our results imply that deterioration of synaptic mitochondrial functions and alterations in the organization of synaptic septins are associated with complement-dependent synapse loss in Alzheimer's disease.

Identification of Neuronal Pentraxins as Synaptic Binding Partners of C1q and the Involvement of NP1 in Synaptic Pruning in Adult Mice.

Elements of the immune system particularly that of innate immunity, play important roles beyond their traditional tasks in host defense, including manifold roles in the nervous system. Complement-mediated synaptic pruning is essential in the developing and healthy functioning brain and becomes aberrant in neurodegenerative disorders. C1q, component of the classical complement pathway, plays a central role in tagging synapses for elimination; however, the underlying molecular mechanisms and interaction partners are mostly unknown. Neuronal pentraxins (NPs) are involved in synapse formation and plasticity, moreover, NP1 contributes to cell death and neurodegeneration under adverse conditions. Here, we investigated the potential interaction between C1q and NPs, and its role in microglial phagocytosis of synapses in adult mice. We verified in vitro that NPs interact with C1q, as well as activate the complement system. Flow cytometry, immunostaining and co-immunoprecipitation showed that synapse-bound C1q colocalizes and interacts with NPs. High-resolution confocal microscopy revealed that microglia-surrounded C1q-tagged synapses are NP1 positive. We have also observed the synaptic occurrence of C4 suggesting that activation of the classical pathway cannot be ruled out in synaptic plasticity in healthy adult animals. In summary, our results indicate that NPs play a regulatory role in the synaptic function of C1q. Whether this role can be intensified upon pathological conditions, such as in Alzheimer's disease, is to be disclosed.

Local apoptotic-like mechanisms underlie complement-mediated synaptic pruning.

C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. However, the molecular mechanisms underlying C1q-controlled synaptic pruning are mostly unknown. This study addresses distortions in the synaptic proteome leading to C1q-tagged synapses. Our data demonstrated the preferential localization of C1q to the presynapse. Proteomic investigation and pathway analysis of C1q-tagged synaptosomes revealed the presence of apoptotic-like processes in C1q-tagged synapses, which was confirmed experimentally with apoptosis markers. Moreover, the induction of synaptic apoptotic-like mechanisms in a model of sensory deprivation-induced synaptic depression led to elevated C1q levels. Our results unveiled that C1q label-based synaptic pruning is triggered by and directly linked to apoptotic-like processes in the synaptic compartment.

Retino-cortical stimulus frequency-dependent gamma coupling: evidence and functional implications of oscillatory potentials.

Long-range gamma band EEG oscillations mediate information transmission between distant brain regions. Gamma band-based coupling may not be restricted to cortex-to-cortex communication but may include extracortical parts of the visual system. The retinogram and visual event-related evoked potentials exhibit time-locked, forward propagating oscillations that are candidates of gamma oscillatory coupling between the retina and the visual cortex. In this study, we tested if this gamma coupling is present as indicated by the coherence of gamma-range (70-200 Hz) oscillatory potentials (OPs) recorded simultaneously from the retina and the primary visual cortex in freely moving, adult rats. We found significant retino-cortical OP coherence in a wide range of stimulus duration (0.01-1000 msec), stimulus intensity (800-5000 mcd/mm2), interstimulus interval (10-400 msec), and stimulus frequency (0.25-25 Hz). However, at low stimulus frequencies, the OPs were time-locked, flickering light at 25 Hz entrained continuous OP coherence (steady-state response, SSR). Our results suggest that the retina and the visual cortex exhibit oscillatory coupling at high-gamma frequency with precise time locking and synchronization of information transfer from the retina to the visual cortex, similar to cortico-cortical gamma coupling. The temporal fusion of retino-cortical gamma coherence at stimulus rates of theater movies may explain the mechanism of the visual illusion of continuity. How visual perception depends on early transformations of ascending sensory information is incompletely understood. By simultaneous measurement of flash-evoked potentials in the retina and the visual cortex in awake, freely moving rats, we demonstrate for the first time that time-locked gamma oscillatory potentials exhibit stable retino-cortical synchrony across a wide range of stimulus parameters and that the temporal continuity of coherence changes with stimulus frequency according to the expected change in the visual illusion of continuity.

Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system.

Widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats.

An increasing number of studies have revealed associations between pre- and perinatal immune activation and the development of schizophrenia and autism spectrum disorders (ASDs). Accordingly, neuroimmune crosstalk has a considerably large impact on brain development during early ontogenesis. While a plethora of heterogeneous abnormalities have already been described in established maternal immune activation (MIA) rodent and primate animal models, which highly correlate to those found in human diseases, the underlying molecular background remains obscure. In the current study, we describe the long-term effects of MIA on the neocortical pre- and postsynaptic proteome of adolescent rat offspring in detail. Molecular differences were revealed in sub-synaptic fractions, which were first thoroughly characterized using independent methods. The widespread proteomic examination of cortical samples from offspring exposed to maternal lipopolysaccharide administration at embryonic day 13.5 was conducted via combinations of different gel-based proteomic techniques and tandem mass spectrometry. Our experimentally validated proteomic data revealed more pre- than postsynaptic protein level changes in the offspring. The results propose the relevance of altered synaptic vesicle recycling, cytoskeletal structure and energy metabolism in the presynaptic region in addition to alterations in vesicle trafficking, the cytoskeleton and signal transduction in the postsynaptic compartment in MIA offspring. Differing levels of the prominent signaling regulator molecule calcium/calmodulin-dependent protein kinase II in the postsynapse was validated and identified specifically in the prefrontal cortex. Finally, several potential common molecular regulators of these altered proteins, which are already known to be implicated in schizophrenia and ASD, were identified and assessed. In summary, unexpectedly widespread changes in the synaptic molecular machinery in MIA rats were demonstrated which might underlie the pathological cortical functions that are characteristic of schizophrenia and ASD.

Modulatory effects of inosine, guanosine and uridine on lipopolysaccharide-evoked increase in spike-wave discharge activity in Wistar Albino Glaxo/Rijswijk rats.

We showed previously that the number of spike-wave discharges (SWDs) was increased after intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), inosine (Ino) and muscimol alone whereas i.p. guanosine (Guo), uridine (Urd), bicuculline, theophylline and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) alone decreased the SWD number in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. These drugs may exert their effects on absence epileptic activity mainly via proinflammatory cytokines-evoked increase in cortical excitability (such as LPS), GABAergic system (LPS, Ino, Urd, muscimol and bicuculline), glutamatergic system (LPS, Guo and MK-801) and adenosinergic system (LPS, Ino, Guo, Urd and theophylline). Both GABAergic system and glutamatergic system are involved in the pathomechanism of absence epilepsy, the LPS-evoked increase in absence epileptic activity and the pro- or antiepileptic effects of non-adenosine (non-Ado) nucleosides Ino, Guo and Urd. Moreover, Ino, Guo and Urd have modulatory effects on inflammatory processes. Thus, we investigated whether Ino, Guo and Urd have also modulatory influence on LPS-evoked increase in SWD number using two different concentrations of each nucleoside in WAG/Rij rats. We demonstrated that Ino dose-dependently aggravated whereas Guo and Urd attenuated the LPS-evoked increase in SWD number. Our results suggest that different nucleosides have diverse effects on LPS-induced changes in absence epileptic activity.

Effects of nucleosides on glia - neuron interactions open up new vistas in the development of more effective antiepileptic drugs.

One-third of epileptic patients are drug refractory due to the limited efficacy of antiepileptic therapy. Thus, there is an immense need to find more effective, safer and well-tolerated antiepileptic drugs. A great deal of results suggests that adenosine (Ado), guanosine (Guo), inosine (Ino) or uridine (Urd) are endogenous antiepileptogenic modulators. Furthermore, nucleosides and their derivatives may be safe and effective potential drugs in the treatment of epilepsy. Conversely, nucleosidergic modulatory system implying nucleoside levels, metabolism, receptors and transporters may also be involved in seizure pathomechanisms. Application of Ado receptor agonists as well as antagonists, elevation of nucleoside levels (e.g., by nucleoside metabolism inhibitors, and Adoreleasing implants) or utilization of non-Ado nucleosides may also turn to be useful approaches to decrease epileptic activity. However, all drugs exerting their effects on the nucleosidergic modulatory system may affect the fine regulation of glia-neuron interactions that are intimately governed by various nucleosidergic processes. Perturbation of the complex, bidirectional communication between neurons and astrocytes through these nucleosidergic modulatory mechanisms may lead to pathological changes in the central nervous system (CNS) and therefore may cause significant side effects. Thus, a deeper understanding of the nucleosidergic modulatory control over glia-neuron interactions is essential in order to develop more effective and safe nucleoside-based antiepileptic drugs. In this review article we focus on the role of Ado and Urd in glia-neuron interactions, placing emphasis on their implications for the treatment of epilepsy.

Non-adenosine nucleoside inosine, guanosine and uridine as promising antiepileptic drugs: a summary of current literature.

Adenosine (Ado) and some non-adenosine (non-Ado) nucleosides including inosine (Ino), guanosine (Guo) and uridine (Urd) are modulatory molecules in the central nervous system (CNS), regulating different physiological and pathophysiological processes in the brain such as sleep and epilepsy. Indeed, different drugs effective on adenosinergic system (e.g., Ado metabolism inhibitors, agonists and antagonists of Ado receptors) are being used in drug development for the treatment of epileptic disorders. Although (i) endogenous Ino, Guo and Urd showed anticonvulsant/antiepileptic effects (e.g., in quinolinic acid - induced seizures and in different epilepsy models such as hippocampal kindling models), and (ii) there is a need to generate new and more effective antiepileptic drugs for the treatment of drug-resistant epilepsies, our knowledge about antiepileptic influence of non-Ado nucleosides is far from complete. Thus, in this review article, we give a short summary of anticonvulsant/antiepileptic effects and mechanisms evoked by Ino, Guo, and Urd. Finally, we discuss some non-Ado nucleoside derivatives and their structures, which may be candidates as potential antiepileptic agents.

Lipopolysaccharide induced increase in seizure activity in two animal models of absence epilepsy WAG/Rij and GAERS rats and Long Evans rats.

We showed previously that the number and time of spike-wave discharges (SWDs) were increased after intraperitoneal (i.p.) injection of lipopolysaccharide (LPS), an effect, which was completely abolished by cyclooxygenase-2 (COX-2) inhibitor indomethacin (IND) pretreatment in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. These and other results suggest that injection of LPS to genetically absence epileptic animals, such as WAG/Rij rats, may allow us to investigate relationships between absence epilepsy and LPS evoked neuroinflammation processes. However, LPS may evoke different effects on absence epileptic activity in various animal strains. Thus, to extend our previous results, we injected two doses of LPS (50 µg/kg and 350 µg/kg i.p.) alone and in combination with IND (10mg/kg IND i.p. +50 µg/kg LPS) into rats of two model animal strains (WAG/Rij rats; GAERS rats: Genetic Absence Epileptic Rats from Strasbourg) and into Long Evans rats. The effects of treatments on SWD number and SWD duration were examined. Both doses of LPS increased the SWD number and the total time of SWDs dose-dependently during the whole 4-h recording period, which was abolished by IND pretreatment in all three investigated strains. These results extend our previous results suggesting that our methods using LPS injection into freely moving absence epileptic rats is applicable not only in well-established animal models of absence epilepsy such as WAG/Rij rats and GAERS rats but also in Long Evans rats to investigate links between inflammation and absence epilepsy.

Brain protein expression changes in WAG/Rij rats, a genetic rat model of absence epilepsy after peripheral lipopolysaccharide treatment.

Peripheral injection of bacterial lipopolysaccharide (LPS) facilitates 8-10Hz spike-wave discharges (SWD) characterizing absence epilepsy in WAG/Rij rats. It is unknown however, whether peripherally administered LPS is able to alter the generator areas of epileptic activity at the molecular level. We injected 1mg/kg dose of LPS intraperitoneally into WAG/Rij rats, recorded the body temperature and EEG, and examined the protein expression changes of the proteome 12h after injection in the fronto-parietal cortex and thalamus. We used fluorescent two-dimensional differential gel electrophoresis to investigate the expression profile. We found 16 differentially expressed proteins in the fronto-parietal cortex and 35 proteins in the thalamus. It is known that SWD genesis correlates with the transitional state of sleep-wake cycle thus we performed meta-analysis of the altered proteins in relation to inflammation, epilepsy as well as sleep. The analysis revealed that all categories are highly represented by the altered proteins and these protein-sets have considerable overlap. Protein network modeling suggested that the alterations in the proteome were largely induced by the immune response, which invokes the NFkB signaling pathway. The proteomics and computational analysis verified the known functional interplay between inflammation, epilepsy and sleep and highlighted proteins that are involved in their common synaptic mechanisms. Our physiological findings support the phenomenon that high dose of peripheral LPS injection increases SWD-number, modifies its duration as well as the sleep-wake stages and decreases body temperature.

5'-nucleotidases, nucleosides and their distribution in the brain: pathological and therapeutic implications.

Elements of the nucleoside system (nucleoside levels, 5'-nucleotidases (5'NTs) and other nucleoside metabolic enzymes, nucleoside transporters and nucleoside receptors) are unevenly distributed in the brain, suggesting that nucleosides have region-specific functions in the human brain. Indeed, adenosine (Ado) and non-Ado nucleosides, such as guanosine (Guo), inosine (Ino) and uridine (Urd), modulate both physiological and pathophysiological processes in the brain, such as sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington's disease, Alzheimer's disease and Parkinson's disease. Interactions have been demonstrated in the nucleoside system between nucleoside levels and the activities of nucleoside metabolic enzymes, nucleoside transporters and Ado receptors in the human brain. Alterations in the nucleoside system may induce pathological changes, resulting in central nervous system (CNS) diseases. Moreover, several CNS diseases such as epilepsy may be treated by modulation of the nucleoside system, which is best achieved by modulating 5'NTs, as 'NTs exhibit numerous functions in the CNS, including intracellular and extracellular formation of nucleosides, termination of nucleoside triphosphate signaling, cell adhesion, synaptogenesis and cell proliferation. Thus, modulation of 5'NT activity may be a promising new therapeutic tool for treating several CNS diseases. The present article describes the regionally different activities of the nucleoside system, demonstrates the associations between these activities and 5'NT activity and discusses the therapeutic implications of these associations.

Neonatal tricyclic antidepressant clomipramine treatment reduces the spike-wave discharge activity of the adult WAG/Rij rat.

Recently it was revealed that the absence-like epileptic activity of the WAG/Rij (Wistar Albino Glaxo/Rijswijk) rat is associated with depression-like behavioural symptoms. Whether these depressive-like symptoms are accompanying epileptic activity (manifested in spike-wave discharges, SWDs, in the EEG) or whether they are causative for each other are open questions. Neonatally administered tricyclic antidepressant clomipramine is a well characterized animal model of major depression. It evokes behavioural symptoms of depression and changes sleep pattern in normal adult rats. We investigated whether in the WAG/Rij rat the neonatally administered clomipramine would aggravate the depression-like behavioural symptoms and the SWD activity. Male WAG/Rij pups from postnatal day 8 (PD8) to PD21 were treated with clomipramine (20mg/kg) or saline (control animals) twice daily intraperitoneally (i.p.). In the 8 months old rats, sleep parameters and sucrose solution intake (as hedonic index) as well as the SWD activity were measured. While the neonatal clomipramine treatment significantly increased the rapid eye movement sleep (REM) amount and decreased the sucrose preference score, it surprisingly attenuated the adult (8 months old) SWD activity. We concluded that neonatal clomipramine treatment produced aggravation of depression-like symptoms while decreased the SWD activity in the adult (8 months old) WAG/Rij rat.

Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2.

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.

Crystal structure of the S100A4-nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism.

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. It binds to the nonmuscle myosin IIA (NMIIA) tail near the assembly competence domain (ACD) promoting filament disassembly, which could be associated with increasing metastatic potential of tumor cells. Here, we investigate the mechanism of S100A4-NMIIA interaction based on binding studies and the crystal structure of S100A4 in complex with a 45-residue-long myosin heavy chain fragment. Interestingly, we also find that S100A4 binds as strongly to a homologous heavy chain fragment of nonmuscle myosin IIC as to NMIIA. The structure of the S100A4-NMIIA complex reveals a unique mode of interaction in the S100 family: A single, predominantly α-helical myosin chain is wrapped around the Ca(2+)-bound S100A4 dimer occupying both hydrophobic binding pockets. Thermal denaturation experiments of coiled-coil forming NMIIA fragments indicate that the coiled-coil partially unwinds upon S100A4 binding. Based on these results, we propose a model for NMIIA filament disassembly: Part of the random coil tailpiece and the C-terminal residues of the coiled-coil are wrapped around an S100A4 dimer disrupting the ACD and resulting in filament dissociation. The description of the complex will facilitate the design of specific drugs that interfere with the S100A4-NMIIA interaction.

Thalamic Gap Junctions Control Local Neuronal Synchrony and Influence Macroscopic Oscillation Amplitude during EEG Alpha Rhythms.

Although EEG alpha (α; 8-13 Hz) rhythms are often considered to reflect an "idling" brain state, numerous studies indicate that they are also related to many aspects of perception. Recently, we outlined a potential cellular substrate by which such aspects of perception might be linked to basic α rhythm mechanisms. This scheme relies on a specialized subset of rhythmically bursting thalamocortical (TC) neurons (high-threshold bursting cells) in the lateral geniculate nucleus (LGN) which are interconnected by gap junctions (GJs). By engaging GABAergic interneurons, that in turn inhibit conventional relay-mode TC neurons, these cells can lead to an effective temporal framing of thalamic relay-mode output. Although the role of GJs is pivotal in this scheme, evidence for their involvement in thalamic α rhythms has thus far mainly derived from experiments in in vitro slice preparations. In addition, direct anatomical evidence of neuronal GJs in the LGN is currently lacking. To address the first of these issues we tested the effects of the GJ inhibitors, carbenoxolone (CBX), and 18ß-glycyrrhetinic acid (18ß-GA), given directly to the LGN via reverse microdialysis, on spontaneous LGN and EEG α rhythms in behaving cats. We also examined the effect of CBX on α rhythm-related LGN unit activity. Indicative of a role for thalamic GJs in these activities, 18ß-GA and CBX reversibly suppressed both LGN and EEG α rhythms, with CBX also decreasing neuronal synchrony. To address the second point, we used electron microscopy to obtain definitive ultrastructural evidence for the presence of GJs between neurons in the cat LGN. As interneurons show no phenotypic evidence of GJ coupling (i.e., dye-coupling and spikelets) we conclude that these GJs must belong to TC neurons. The potential significance of these findings for relating macroscopic changes in α rhythms to basic cellular processes is discussed.