Regulation of mitochondrial trafficking, function and quality control by the mitochondrial GTPases Miro1 and Miro2

Regulated trafficking of mitochondria in neurons is essential for providing ATP at the correct spatial location to power neural function and computation, and for providing calcium buffering at sites of calcium entry or release. Indeed the regulation of mitochondrial distribution, morphology and function are proposed to play an important role in neuronal development and survival but the regulatory mechanisms remain unclear. Miro family proteins (Miro1 and Miro2 in mammals) contain a transmembrane domain locating them to the outer mitochondrial membrane, along with two GTPase domains and two calcium-sensing EF-hand domains that face into the cytosol, and play a key role in regulating mitochondrial transport. Miro proteins mediate mitochondrial trafficking in neurons by linking mitochondria to kinesin and dynein motor proteins for their transport in axons and dendrites. Miro proteins are also targets for the Parkinson’s Disease associated PINK1/Parkin mitophagy pathway and are therefore implicated in altered mitochondrial dynamics during mitophagy. Here I will present our recent results on the role played by Miro proteins in regulating mitochondrial trafficking and quality control. The role that Miro-mediated control of mitochondrial trafficking and turnover plays in regulating neuronal development, function and pathology will also be explored.

The nucleus accumbens has long been a major target for studies on the rewarding eff ects of drugs of abuse or physiological reinforcers, whereas the brain regions medial of the medial accumbens shell have received less attention. We investigated if counterconditioning with dyadic (i.e., one-to-one) social interaction, a strong inhibitor of the subsequent reacquisition of cocaine conditioned place preference (CPP), diff erentially modulates the activity of the diverse brain regions oriented along a mediolateral corridor reaching from the interhemispheric sulcus to the anterior commissure, i.e., the nucleus of the vertical limb of the diagonal band, the medial septal nucleus, the major island of Calleja, the intermediate part of the lateral septal nucleus, and the medial accumbens shell and core. EGR1 activation was predominantly found in dynorphin-labeled cells, i.e., presumably D1 receptor-expressing medium spiny neurons (D1-MSNs), with D2-MSNs (immunolabeled with an anti-DRD2 antibody) being less aff ected. Cholinergic interneurons or GABAergic interneurons positive for parvalbumin, neuropeptide Y or calretinin were not involved in these CPP-related EGR1 changes. Glial cells did not show any EGR1 expression either. Cocaine conditioning increased the spike frequency of neurons in the septal nuclei, whereas social interaction conditioning increased the spike frequency in the nucleus accumbens compared to saline control animals. In addition, social interaction conditioning decreased the amount of active neuron clusters in the nucleus accumbens.The present findings could be of relevance for the therapy of impaired social interaction in substance use disorders, depression, psychosis, and autism spectrum disorders.
G protein-coupled receptors (GPCRs) are intensely studied as drug targets and for their role in signaling. High-resolution crystal structures of GPCRs capturing diff erent receptor conformations are now available, which have provided insights into the mechanism of activation and ligand selectivity for this important class of drug targets. I will fi rst present a series of structure-based screens for novel ligands of the A2A adenosine receptor (A2AAR), which is a drug target for Parkinson's disease (antagonists) and ischemia (agonists). As crystal structures for both inactive-and active-like receptor conformations of the A2AAR have now been determined, molecular docking screens for novel ligands can be performed. Virtual screens against diff erent conformations of the A2AAR were carried out to investigate if structure-based methods can be used to identify agonists and antagonists. Our results shed light on the importance of access to crystal structures and the role of the chemical library in screens for ligands with specifi c pharmacological properties. For most GPCRs, no experimental coordinates are available and structure-based screens are forced to rely on homology models. However, it is still unclear if models of GPCRs are suffi ciently accurate to be used in ligand discovery. The determination of crystal structures for dopamine and serotonin receptors, and the challenges to the community to predict these in the GPCR Dock competitions, have enabled us to carry out comparisons of ligand discovery from models versus crystal structures. Our results from these challenges reveal opportunities and limitations of the use of homology models in ligand discovery and design of selective lead candidates.
Brain tumors represent a recognized cause of epilepsy in both children and adults. In principle, any tumor (extra-axial, intra-axial, benign or malignant, common or uncommon) can cause seizures. However, patients with supratentorial low-grade glial tumors are more likely to develop epilepsy. Several clinical studies emphasize that pharmacologically intractable epilepsy critically aff ects the daily life of patients with brain tumors, even if the tumor is under control. Recently, the term of long-term epilepsy associated tumour (LEAT) has been introduced. LEATs are low grade, slowly growing, cortically-based tumours which predominantly occur in young patients with long histories (often 2 years or more) of drug-resistant epilepsy. Glioneuronal tumors (GNT), including gangliogliomas (GG) and dysembryoplastic neuroepithelial tumors (DNTs), represent the most common tumor within the spectrum of LEAT. The advent of the neurosurgical treatment of epilepsy-associated brain lesions confi rmed the strong epileptogenicity of these tumor entities. Understanding the mechanisms that underlie epileptogenesis in LEATs is essential to identify new treatment targets and to develop an eff ective therapy. Mechanisms such as alterations of the balance between excitation and inhibition and alterations in neuron-glia interactions might be involved. Astroglial cells express functional receptors for a variety of neurotransmitters and may critically modulate synaptic transmission. In addition, an increasing number of observations indicate that proepileptogenic infl ammatory pathways are activated in GNT and may contribute to the onset and progression of seizures. The recent advances and likely candidate mechanisms and molecules involved in tumor-associated epileptogenesis will be discussed. progressively more aggressive tumors (respectively, astrocytomas and anaplastic glioblastoma, and glioblastoma multiforme, containing proliferating cells resembling Oligodendrocytes Precursor Cells, OPCs) confers growth advantage and chemoresistance. Characterization of the specifi c P1 and P2 receptors on these tumors may unveil new strategies to reduce cancer growth and/or promote diff erentiation to non-cancerous glial phenotypes. The adenosine A3 receptor (A3AR) has emerged as a potential target. Under hypoxia, a condition typical of gliomas' core, A3AR mediates chemoresistance via the PKB/Akt pathway (leading to inactivation of the pro-apopototic Bad protein) and by upregulating matrix metalloproteinase-9, that degrades extracellular matrix and promotes migration of glioma cells towards healthy brain regions (Ceruti and Abbracchio, and ref therein). Thus, inhibition of A3AR with selective antagonists could represent an appealing therapeutic approach. More recently, the P2X7 receptor has been recently found to be over-expressed in grade IV human gliomas Cannabinoids, originally derived from Cannabis sativa, as well as their endogenous and synthetic counterparts, were shown to induce apoptosis of glioma cells in vitro and tumour regression in vivo via their specifi c receptors, cannabinoid receptors CB1 and/or CB2. CB2 are abnormally expressed in human gliomas and glioma cell lines. Most of the analysed gliomas expressed signifi cant levels of CB2 receptor and the extent of CB2 expression in the tumour specimens was related to tumour malignancy. A synthetic cannabinoid, WIN 55,212-2, down-regulated the Akt and Erk signalling pathways in C6 glioma cells that resulted in reduction of phosphorylated Bad levels, mitochondrial depolarization and activation of caspase cascade leading to apoptosis. We examined whether synthetic cannabinoids with diff erent receptor specifi city: WIN55,212-2 (a non-selective CB1/CB2 agonist) and JWH133 (a CB2selective agonist) aff ect survival of four human glioma cell lines and three primary human glioma cell lines. WIN-55,212-2 decreased cell viability in all examined cell lines and induced cell death. Susceptibility of the cells to JWH133 treatment correlated with the CB2 expression. Cannabinoids triggered a decrease of mitochondrial membrane potential, cleavage of caspase-9 and eff ector caspases. Induction of cell death by cannabinoid treatment led to the generation of a pro-apoptotic sphingolipid ceramide and disruption of signalling pathways crucial for regulation of proliferation and survival. Increased ceramide levels induced ER-stress and autophagy in drug-treated glioblastoma cells. We conclude that cannabinoids are effi cient inhibitors of human glioma cells growth, once the cells express specifi c type of cannabinoid receptor. Integrins are a group of molecules expressed by various cells including glioma cells and endothelial cells within the tumor. There are 18 known alpha and beta integrin subunits which form a heterodimer. Integrins regulate diff erent cellular processes such as proliferation, adhesion, motility and survival as shown in numerous preclinical models. Furthermore, integrins control the activity of the transforming growth factor (TGF)-beta pathway and are involved in the process of angiogenesis which is indispensable for continued tumor growth. Because of the high expression levels of some integrins on glioma cells and their numerous functions, inhibition of integrin signaling has been considered a promising strategy for the treatment of glioma patients. Besides blocking antibodies which are currently under clinical investigation in other cancer entities, the integrin inhibitor cilengitide has been tested within several trials in glioblastoma patients over the last years. Cilengitide is a cyclic RDG peptide which targets integrins alphvbeta3 and alphavbeta5. Based on the results of smaller, initial trials suggesting an activity of cilengitide against glioblastoma, 2 larger trials were subsequently performed. However, both trials, which combined temozolomide-based chemoradiation with cilengitide failed to demonstrate an improved outcome with the addition of cilengitide. Ongoing translational analyses suggest that integrin levels in the tumor tissue are neither prognostic nor predict response to cilengitide. While the clinical development of cilengitide has been stopped, integrin inhibition with more eff ective agents may still be a promising approach in clinical neurooncology. Keywords Glioma, integrin, TGF-beta Alzheimer's disease (AD) is a devastating neurodegenerative disease causing irreversible cognitive decline in the elderly. There is no disease-modifying therapy for this condition and the mechanisms underpinning neuronal dysfunction and neurodegeneration are unclear. Compromised cytoskeletal integrity within neurons is reported in AD. This is believed to result from loss-of-function of the microtubule-associated protein tau, which becomes hyperphosphorylated and deposits into neurofi brillary tangles in AD. We have developed a Drosophila model of tauopathy in which abnormal human tau mediates neuronal dysfunction characterised by microtubule destabilisation, axonal transport disruption, synaptic defects and behavioural impairments. Here we show that a microtubule-stabilising drug, NAPVSIPQ (NAP), prevents as well as reverses these phenotypes even after they have become established. Moreover, it does not alter abnormal tau levels indicating that it by-passes toxic tau altogether. Thus, microtubule stabilisation is a disease-modifying therapeutic strategy protecting against tau-mediated neuronal dysfunction, which holds great promise for tauopathies like AD. Keywords Microtubule stabilisation, tau, NAP Current therapies for Alzheimer's disease (AD) and related disorders have demonstrated very modest, symptomatic effi cacy, leaving an unmet medical need for new, more eff ective therapies. While drug development eff orts in the last two decades have primarily focused on the amyloid cascade hypothesis, with disappointing results so far, tau-based strategies have received little attention until recently despite that the presence of extensive tau pathology is central to the disease. The discovery at the turn of the century of mutations within the tau gene that cause fronto-temporal dementia demonstrated that tau dysfunction was per se suffi cient to cause neuronal loss and clinical dementia. Development of tau pathology is associated with progressive neuronal loss and cognitive decline and is the common underlying cause of a group of neurodegenerative disorders collectively known as "tauopathies". Tauopathies are clinically, morphologically and biochemically heterogeneous neurodegenerative diseases characterized by the deposition of abnormal tau protein in the brain. The neuropathological phenotypes are distinguished based on the involvement of diff erent anatomical areas, cell types and presence of distinct isoforms of tau in the pathological deposits. Thus, the spectrum of tauopathy entities expands beyond the traditionally discussed disease forms. Emerging evidence strongly suggests that accumulation of abnormal tau is mediated through spreading of seeds of the protein from cell to cell. This prion-like mechanism would support the concept that in AD brains, tau pathology iinitiates in a very small part of the brain many years before becoming symptomatic, spreading slowly and progressively to the whole brain following an anatomically defi ned pattern. Emerging therapeutic strategies aimed at treating the underlying causes of the tau pathology will be discussed, including some novel therapeutic approaches on the verge of providing new treatment paradigms in upcoming years. Keywords Tau, Alzheimer, dementia Oligodendrocytes, the myelin forming cells of the CNS, enwrap neuronal axons and form multilamellar myelin sheets. They are derived from oligodendrocyte precursor cells which migrate from the subventricular zone into the diff erent regions of the brain. Diff erentiation from the early progenitor to the mature multiprocessed oligodendrocyte is characterized by diff erent morphological stages. To support cell morphology and establish and maintain the myelin membrane, an intact, spatially organized cytoskeleton with dynamic properties is essential. In particular microtubules and their associated proteins play an important role. A variety of microtubule binding proteins, including tau, are present in oligodendrocytes. Oligodendrocytes in culture express all six isoforms of tau which are developmentally regulated. Tau proteins are present in immature and mature oligodendrocytes and specifi cally prominent in the branching points of the cellular processes. Downregulation of tau impairs cell diff erentiation and the process of early myelination. In neurodegenerative diseases collectively termed tauopathies, fi brillary tau accumulations occur not only in neurons but also in glia. Tau positive coiled bodies originating in oligodendrocytes are characteristic for the brains of patients with frontotemporal dementias, such as corticobasal degeneration and progressive supranuclear palsy. These aggregates are further characterized by the presence of heat shock proteins and ubiquitin, indicating that stress situations are causally related to the pathogenesis. In this respect, proteasomal dysfunctions have been discussed to be involved in neurodegenerative disorders and the aging process. Data on the consequences of proteolytic stress in oligodendrocytes and the protein aggregation process will be presented. Our data demonstrate that an intact cytoskeleton is essential for cellular defense mechanisms. Keywords Oligodendrocytes, microtubules, neurodegenerative diseases Tau is a classical microtubule-associated protein known to regulate microtubule stability in neurons. In our study, we have addressed the putative crosstalk between tau and End binding proteins 1 and 3 (EB1/3), the core microtubule plus-end tracking proteins (+TIPs), in diff erentiating neuronal cells. We show that tau and EB proteins interact directly and that the cellular distribution and mobility of EB proteins depends on tau localization and expression levels. Moreover, our data reveal that tau is essential for the proper localization of EB1 at the medial-distal region of the axon shaft in developing neurons. In summary, we provide evidence for a new function of tau protein as a direct regulator of EB1/3 proteins. This further indicates that the interplay between classical MAPs and core +TIPs may be important for the fi ne-tuned regulation of microtubule dynamics and stability during neuronal diff erentiation. Keywords Tau, end binding proteins, microtubule dynamics Orexin/hypocretin peptides are central for the regulation of the sleep/arousal states, and have, in addition, a role in the regulation of e.g. metabolism, addiction, stress response and pain gating. Orexin responses are mediated by G-protein-coupled OX1 and OX2 orexin receptors. Orexin signaling (in diff erent cell types) is very versatile, ranging from excitation to induction of cell death. Orexin receptor coupling is promiscuous, engaging members of at least three diff erent families of G-proteins, namely Gi, Gs and Gq, and some non-G-protein mediators as well. Preferred G-protein-coupling of the receptors appears diff erent in diff erent tissues, but the mechanism determining this are unknown. The primary signal transducers of orexin receptors very eff ectively activate phosholipase cascades, including PLA2, PLC and PLD, and also PLC-diacylglycerol lipasemediated endocannabinoid generation. These cascades may play a signifi cant role in the regulation of K+ and non-selective cation channels, which are the eff ectors for the orexin-mediated neuronal excitation. In some cell types, orexin receptor stimulation induces programmed cell death. Some diff erent mechanisms for his have been proposed, but the picture is still incomplete. Orexin receptors have been a target for multiple drug discovery projects. Main focus has been on the antagonists, with insomnia as the indication. For agonist drugs there are some ongoing smaller academic and semi-academic projects. Use of orexin receptor agonists is suggested to be benefi cial in narcolepsy (as peptide replacement therapy) and putatively in other sleep/wakefulness disturbances, metabolic disorders and cancer. Keywords Orexin, hypocretin, phospholipase through the cell membrane in response to various extracellular stimuli. GPCR have become important targets of many drugs for treatments of very diff erent diseases. During the last decade several fl uorescencebased methods have been implemented for the characterization of signal transduction via GPCRs, starting from ligand binding and including several steps leading up to a response on the level of gene regulation. We have proposed the fl uorescence anisotropy (FA) and fl uorescence intensity (FI) assay to investigate fl uorescent ligand binding properties to diff erent GPCRs . The implementations of budded baculoviruses that display G proteincoupled receptors on their surfaces have signifi cantly increased sensitivity and applicability of these assays . The developed novel assay systems opened new possibilities for real-time monitoring of ligand binding to their receptors for understanding their particular kinetic properties. These assays are also compatible for homogenous HTS suitable fo ligand screening. There has been implemented assay systems for receptors of peptides like melanocortin (MC4R) and neuropeptide Y (NPY1R) as well as for receptors of monoamines like dopamine (D1DAR) and serotonin (5-HT1AR). Opioid receptors (OR) are widely known as mediators of the analgesic eff ects of opioids and also contribute to the development of tolerance and dependence. Moreover, opioids are implicated in cell proliferation and survival. How opioids modulate these downstream signaling pathways is a research receiving a lot of attention. Our group aims to defi ne the signaling pathways through which opioid receptors participate in these physiological processes. Emphasis is given to unconventional interacting partners of the μ and δ-opioid receptors such as the Regulators of G protein signalling (RGS) proteins and STAT5B (Georgoussi et al. 2012). Evidence will be presented with which RGS proteins opioid receptors interact, how RGS members confer selectivity to receptors to choose a specifi c subset of G proteins, how activation of opioid receptors result in recruitment of RGS proteins to the plasma membrane and exert a diff erential modulatory eff ect in ERK1,2 phosphorylation, agonist-driven adenylyl cyclase inhibition and internalization of the opioid receptors (Papakonstantinou et al., 2015). Moreover evidence will be presented on how STAT5B associates with the δ-opioid receptor and forms selective pairs with selective Gα, Gβγ subunits and RGS proteins, and how activation of the δ-opioid receptor with selective agonists promotes a multi-component signaling complex involving the STAT5B transcription factor and other signaling intermediates to mediate neuronal survival and neurite outgrowth (Georganta et al., 2013). Understanding the mechanism that control OR signaling is important to address problems related to phenomena such as pain perception, tolerance and dependence that occur upon chronic opiate administration and defi ne whether disruption of such interactions may contribute to the development of novel therapeutic strategies. Acknowledgements Supported by the EU "Normolife"-LSHC-CT2006-037733 and the GSRT, Excellence II -3722, "NO-ALGOS". Z.G participates in the EU COST Action CM1207 (GLISTEN Autism spectrum disorders (ASD) are heterogeneous, heritable neurodevelopmental conditions, aff ecting ~0.5% of the population across cultures, with a ~4:1 male/female ratio. ASD are characterized by social interaction and communication defi cits, restricted interests, repetitive behaviors, and reduced cognitive fl exibility. Causes likely converge at the synapse, as shown by mutations of synaptic genes or mutations causing quantitative alterations in synaptic protein expression. Neuroligin4 (NLGN4X) mutations are among the most frequent causes of heritable ASD. But monogenetic forms altogether account for 1200 schizophrenic subjects and validated it in Asperger autists. We hypothesized that a coincidence of unfortunate normal variation in synaptic or synapse-regulating genes rather than mutations underlies most autistic phenotypes. We identifi ed 'proautistic' variants in synaptic genes, which in aggregate are associated with high autism severity. A transcranial magnetic stimulation study on respective individuals revealed enhanced glutamatergic and GABAergic activity. IPS-derived cortical neurons from these subjects are now functionally characterized. Keywords Autism, synapse, ambra1 Motor neuron disease (MND) is a neurodegenerative disease of mid-life, with average survival of 3-5 years after diagnosis. The only approved treatment, riluzole, is only moderately eff ective and so there is a great need for novel therapeutics. TAR DNA binding protein 43 (TDP-43) has held a particular centrality in MND research since its discovery as the principal protein component of the characteristic protein aggregates found in the disease. Subsequent work has investigated its role as an RNA-binding protein and regulator of approximately 3000 RNA transcripts. In the majority of disease cases, both familial and sporadic, normally nuclear TDP-43 is sequestered in the cytoplasm in protein aggregates. Using diff erentiated motor neuron-like cells (NSC-34) and primary motor neurons, we have treated cells with the disease-relevant ER stressor tunicamycin. Overnight treatment with a low concentration of tunicamycin resulted in the formation of aggregates immunoreactive for endogenous TDP-43, as visualised by immunocytochemistry and Western blotting of the RIPA-insoluble fraction. We have found that these aggregates do not co-stain for TIAR, a well-known marker of stress granules. However, using the oxidative stressor sodium arsenite, we are able to induce formation of stress granules, though they do not co-stain for TDP-43. The tunicamycin treatment also led to a moderate decrease in cell viability, as shown by MTT and Trypan Blue Exclusion assays. In this study, we have recapitulated a known pathological process (ER stress) of MND in vitro, which resulted in the formation of TDP-43 aggregates. The signalling pathways driving TDP-43 aggregation are unknown, hence work detailing these mechanisms is likely to present wide scope for therapeutic intervention. Spinal and bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are motoneuron diseases. A mutation in the androgen receptor (ARpolyQ) gene is responsible for SBMA. Mutations in the SOD1, in the TDP-43, in the FUS-TLS or in the C9ORF72 genes are responsible for familiar form of ALS. The mutated coded proteins misfold and aggregates. Effi cient protein quality control (PQC) is required for the maintenance of physiological and soluble protein pool in aff ected motoneuron. The balance between autophagy and ubiquitinproteasome system (UPS) prevents protein aggregation and increases degradation of SBMA and ALS misfolded proteins. Dynein binds the cochaperone BAG3 and transports the mutant proteins at microtubule organization center where misfolded proteins interact, aggregate and can be degraded by autophagy. However, here misfolded proteins may blocks autophagy fl ux. In NSC34 cells, dynein is sequestered into ARpolyQ aggregates suggesting the role of dynein into aggregates formation process. Unexpectedly, the silencing of dynein heavy chain resulted in a drastic reduction of ARpolyQ retained in fi lter retardation assay (FRA). Moreover, dynein silencing drastically altered autophagic markers localization (LC3 and p62) by immunofl uorescence. Notably, treatment with a dynein inhibitor (EHNA) drastically reduced the retention of ARpolyQ, mutSOD1 and mutTDP43 aggregates in FRA, even when autophagy was inhibit with 3-MA. Conversely UPS blockage with MG132 counteracted the reduction induced by altered dynein transports. RTq-PCR on NSC34 cells treated with EHNA showed an increased BAG1:BAG3 ratio that can targeting the misfolded proteins to UPS. Moreover, in NSC34 cells, EHNA increased the degradation of proteasome reporter GFPu, while BAG1 overexpression reduced the level of aggregates retained in FTA. These data suggest that, when autophagy is overload, by misfolded proteins, dynein inhibition restores the physiological and soluble protein pool via UPS. The main objective of this study was to investigate and compare the neuroprotective and anti-infl ammatory eff ects of DHEA and its spiroepoxy derivatives BNN27 and BNN20 (Calogeropoulou et al., J Med Chem, 2009) (not metabolized to estrogens and androgens), in the rat streptozotocin model of DR. BNN27, via TrkA activation, protected in a dose-dependent manner (2, 10, 50 mg/kg, ip) bNOS (brain nitric oxide synthetase) and TH (tyrosine hydroxylase) expressing amacrine cells and ganglion axons (NFL immunoreactivity) similar to DHEA's actions, while BNN20 was less eff ective. BNN27 activated the TrkA prosurvival signaling pathway ERK1/2 kinase. It reduced the activation of SAPK/ JNK kinase and the expression of p75NTR. BNN27 also increased the expression of anti-infl ammatory cytokines (IL10). These results suggest that NGF TrkA receptor is involved in the neuroprotective and antiinfl ammatory eff ects of BNN27 and is a valuable target via which BNN27 could aff ord effi cacious therapeutics for the treatment of DR.

Tau regulates the localization and function of End Binding proteins in neuronal cells
Perinatal complications are a serious clinical problem, in particular hypoxic-ischemic (HI) episodes, caused by birth asphyxia or uterine and fetal blood fl ow interruption. HI corresponds to 23% of neonatal deaths, being one of the top 20 leading causes of disease burden. Preconditioning (PC) is a stimulation below the injury threshold that activates endogenous protective mechanisms to prevent damage. Low doses of carbon monoxide (CO) play a benefi cial role through PC induction. Herein, CO cytoprotection was explored in distinct brain models. The used experimental models range from monoculture of astrocytes, co-cultures of neurons and astrocytes, to the whole organism with the rat model of perinatal ischemia. In primary cultures of astrocytic cells, CO not only impairs mitochondrial membrane permeabilization, by ANT glutathionylation, but also strengths mitochondrial oxidative metabolism, by modulating COX activity, increasing mitochondrial biogenesis and ATP amounts. Also, CO reinforces astrocytes-neurons communication towards neuronal survival. Purinergic molecules are the main mediators for this non-cell autonomous eff ect. Our results seem to indicate that the main pathway involved includes ATP release from astrocytes, its metabolization and A2A receptor binding to initiate protective mechanisms within the neurons. Rat pups were exposed to CO before hypoxia-ischemia induction (Vannucci model). 24 h after HI the brains were collected for cell death and tissue protection assessment (histological and immunohistochemical analysis). It was found limited apoptosis in hippocampus following cerebral ischemia: lower cytochrome c release and caspase-3 activation yielding an increased Bcl-2 expression. Altogether, one can conclude that there is not just a unique pathway for the CO-induced endogenous protection, brain tolerance is the result of a complex cellular change in response to injury. Indeed, CO regulates cell death pathways and modulates cellular metabolism. Keywords Carbon monoxide, preconditioning, ischemia Severe hypobaric hypoxia (180 mm Hg) is a harmful stimulus that induces structural and functional injures of susceptible brain neurons in the neocortex and hippocampus. In contrast, moderate hypobaric hypoxia (360 mm Hg) activates endogenous cellular defensive mechanisms. The rate of neuroprotection aff orded by the mild hypoxia depends on the quantity of hypoxic sessions. In particular, preconditioning (preexposure) by three trials of mild hypoxia protects from deleterious eff ects of subsequent severe hypoxia whereas preconditioning with one mild hypoxic trial does not. The molecular mechanisms induced by mild hypoxic preconditioning are unclear. Pro-survival proteins, such as neurotrophic factor BDNF and anti-apoptotic factor Bcl-2 are supposed to be involved in this process. In the present study, the eff ects of threetrial and one-trial hypoxic preconditioning on the expression of prosurvival proteins BDNF and Bcl-2 as well as their up-stream activator pCREB, have been studied in the neocortex and hippocampus of rats. As revealed by quantitative immunocytochemistry, the severe hypobaric hypoxia didn't aff ect or down-regulated the neuronal levels of pCREB, BDNF and Bcl-2 at 3-24 h after the exposure. The onetrial preconditioning did not change this eff ect of severe hypoxia. In contrast, preconditioning by three trials of mild hypoxia (360 Torr, 2 h, 24 h intervals, 3 times) signifi cantly enhanced the pCREB, BDNF and Bcl-2 neuronal expression in response to severe hypoxic challenge. Threetrial mild hypoxia alone also up-regulated the expression of molecular factors examined in the neocortex and hippocampus at 24 h whereas one trial of the mild hypoxia did not. The results of the present study indicate that development of the neuronal hypoxic tolerance induced by the three-trial, in contrast to one-trial, mild hypoxic preconditioning is apparently largely associated with the activation of CREB, as well as BDNF and Bcl-2 overexpression. Keywords Hypoxic preconditioning, brain hypoxic tolerance, pCREB Ankrd11 is a potential transcriptional regulator that is implicated in cognitive dysfunction and ASD, but has no known function in the brain. We show that Ankrd11 is expressed in the embryonic cortex, and that when it was knocked-down in murine cortical precursors this caused decreased proliferation, reduced neurogenesis, and aberrant neuronal positioning in developing cortex. Knockdown of Ankrd11 in human forebrain neural precursors phenocopied Ankrd11 knockdown in murine neural precursors. Decreased proliferation of embryonic and adult neural precursors and neuronal mispositioning were also observed in Yoda mice carrying a point mutation in the histone deacetylase (HDAC)-binding domain of Ankrd11. Yoda mice also displayed ASD-like behavioural abnormalities. Consistent with a role for Ankrd11 in histone acetylation, Ankrd11 was associated with chromatin and co-localized with HDAC3. Also, expression and histone acetylation of Ankrd11 target genes were altered in Yoda neural precursors. Finally, the Ankrd11 knockdown-mediated decrease in precursor proliferation was rescued by inhibiting histone acetyltransferase activity or expressing HDAC3. Thus, Ankrd11 is a crucial epigenetic regulator of neural development that regulates histone acetylation and gene expression, thereby providing a likely explanation for its association with cognitive dysfunction and ASD. Keywords Chromatin, stem cells, brain development Endocannabinoids (eCBs) are bioactive lipids which primarily infl uence synaptic communication in the nervous system. They are synthesized by neurons but also by microglia, especially under infl ammation. To exert their function, eCBs travel across the intercellular space. However, how eCBs move extracellularly remains obscure. Our recent evidence indicates that reactive microglia release extracellular vesicles (EVs), which may represent an ideal vehicle for the transport of hydrophobic eCBs. Hence, in this study we investigated whether microglial EVs carry eCBs and may infl uence neurotransmission. First we analyzed the eCB content of EVs and found a clear enrichment of anandamide (AEA) in EVs relative to parental microglia. This analysis revealed higher AEA levels in EVs shed from the plasma membrane (microvesicles), compared to those which originate from the endocytic compartment (exosomes). To bioassay the activity of vesicular AEA, we used patch clamp analysis of miniature inhibitory post-synaptic currents (mIPSC) on hippocampal neurons in vitro. Exposure of neurons to microvesicles (MVs) induced a signifi cant decrease in mIPSC frequency, mimicking the action of CB1R agonists. The involvement of vesicular AEA in this phenomenon was inferred from the ability of the CB1R antagonist SR141716A to block the reduction of mIPSC frequency evoked by MVs. Western blot analysis showed that MVs induces an increase in ERK phosphorylation, which was completely inhibited by SR141716A. This indicate that CB1R activation by AEA-storing MVs translates into downstream signaling. Finally, consistent with a surface localization of AEA, MVs membranes maintained their capability to decrease mIPSC frequency. Overall, this study shows that microglial MVs carry AEA on their surface to stimulate CB1R on target GABAergic neurons thus playing a crucial role in the modulation of inhibitory transmission. Keywords Endocannabinoids, extracellular vesicles, microglia-neuron signaling Reference Peripheral nerve injuries are an important part of everyday medical practice since there are reported over 600,000 cases in Europe and in the United States annually [1]. Many of such nerve injures cause gaps between nerve stumps. Without intervention, they can lead to the formation of a stump neuroma, what can result in functional impairment of the nerve fi ber. The current approaches to regeneration of damaged peripheral nerves include: autografting, allografting, and, last but not least, the implantation of polymeric tubes and conduits between nerve stumps. Nerve autografting is considered as the "gold standard" technique for the repair of peripheral nerve discontinuities, but it has a number of limitations, such as the requirement for the second surgical procedure to harvest the graft tissue, the donor site morbidity, additional injuries and scarring as well as the increased recovery time. Allografts (e.g., cadaver nerve grafts) and xenografts (e.g. animal nerve grafts) can be an alternative to autografts, but their main drawback lies in the high possibility of an undesirable immune response. The most promising materials for peripheral nerve conduits preparation are natural biopolymers. They are obtained from natural sources, exhibit similar properties to the tissues they are replacing and reveal good cell adhesion. Furthermore, they tend to accelerate regeneration processes due to specifi c chemical interactions within the human body, e.g. with extracellular matrix (ECM) molecules. The purpose of our study is to create a conduit with properties that will mimic the ones of the extracellular matrix of the peripheral nervous system. Our focus is put on natural polymers, especially chitosan. In addition, we use chemotactic factors which exhibit properties benefi cial in regeneration of the peripheral nervous tissue. Keywords Biomaterials, hydrogel, nerve regeneration Reference Ginkgo biloba extract is a neuroactive agent that is widely used for correction of age-associated impairment of memory, attention defi cit and other cognitive functions. It has been shown that ginkgolides and bilobalides, Ginkgo biloba extract components, are potent blockers of glycine receptors [1,2]. However, the eff ect of ginkgolic acid, the other Ginkgo biloba extract constituent, on ligand-gated ion channels was not investigated. In the present study, using patch-clamp technique and transient transfection of diff erent subunits in CHO cells we have shown that glycine receptors (GlyRs) are modulated by ginkgolic acid in a subunit-specifi c manner. After pre-application of ginkgolic acid (0.5-2 min), glycine-induced currents mediated by α1 GlyRs were strongly potentiated, while currents mediated by α2 GlyRs exhibited weak inhibition. There was no signifi cant eff ect of ginkgolic acid on amplitudes of currents mediated by α3 GlyRs or on GABAARs composed of 1α/1β/2γ subunits. In order to further investigate subunit-specifi c eff ect of ginkgolic acid we have focused on possible interaction sites for this compound inside diff erent GlyR domains. We found that mutation of three residues (T59A/A261G/A303S) in α2 subunit can convert the inhibitory action of ginkgolic acid into potentiation. Indeed, application of ginkgolic acid to cells expressing α2 T59A/A261G/A303S subunits resulted in an increase of responses to low concentrations of glycine and abolishment of the inhibitory eff ect, typical for wild type α2 GlyR. Our results suggest that (i) ginkgolic acid selectively enhances the function of α1 GlyRs and attenuates the function of α2 GlyRs, (ii) mutation of α2 subunit converts eff ect of ginkgolic acid from inhibition to potentiation. Keywords Glycine receptor, ginkgolic acid, patch-clamp References Iron oxide nanoparticles (IONPs) are frequently used for biomedical applications including magnetic resonance imaging, drug delivery or tumor treatment by hyperthermia. Since IONPs can reach the brain from periphery or are directly applied into the brain, the investigation of potential adverse eff ects of IONPs on properties and functions of the diff erent types of brain cells is an important task. In order to directly compare diff erent types of brain cells concerning the uptake and toxicity of IONPs, we synthesized fl uorescent IONPs and characterized these BODIPY-labeled particles regarding their physicochemical properties. In the medium used for the cell studies, these particles had a hydrodynamic diameter of around 158 ± 28 nm and a negative surface charge with a zeta-potential of -10 ± 2 mV. Exposure of primary cultures of rat astrocytes, neurons and microglial cells revealed that among these cell types, microglial cells accumulated IONPs most rapidly. However, microglial cells were also most vulnerable towards acute IONP-induced stress while astrocytes and neurons were not acutely damaged by IONPs. In microglial cells, but not in astrocytes or neurons, IONPs were found to be localized in lysosomes and large amounts of reactive oxygen species (ROS) were observed after IONPexposure. The IONP-induced toxicity in microglial cells was prevented by neutralizing lysosomes or by chelation of intracellular ferrous iron ions, suggesting that the toxic potential of IONPs in microglia involves rapid particle uptake, liberation of ferrous iron from the internalized IONPs in the acidic lysosomal compartment and iron-catalyzed ROS formation. These data suggest that also in brain IONPs may harm microglial cells and compromise microglial functions. Keywords Toxicity, protection, nanoparticles Regulated traffi cking of mitochondria in neurons is essential for providing ATP at the correct spatial location to power neural function and computation, and for providing calcium buff ering at sites of calcium entry or release. Indeed the regulation of mitochondrial distribution, morphology and function are proposed to play an important role in neuronal development and survival but the regulatory mechanisms remain unclear. Miro family proteins (Miro1 and Miro2 in mammals) contain a transmembrane domain locating them to the outer mitochondrial membrane, along with two GTPase domains and two calcium-sensing EF-hand domains that face into the cytosol, and play a key role in regulating mitochondrial transport. Miro proteins mediate mitochondrial traffi cking in neurons by linking mitochondria to kinesin and dynein motor proteins for their transport in axons and dendrites. Miro proteins are also targets for the Parkinson's Disease associated PINK1/Parkin mitophagy pathway and are therefore implicated in altered mitochondrial dynamics during mitophagy. Here I will present our recent results on the role played by Miro proteins in regulating mitochondrial traffi cking and quality control. The role that Miro-mediated control of mitochondrial traffi cking and turnover plays Mitochondrial fusion-fi ssion dynamics play a crucial role in many important cell processes. These dynamics control mitochondrial morphology, which in turn infl uences several important mitochondrial properties including mitochondrial bioenergetics and quality control, and they appear to be aff ected in several neurodegenerative diseases. The molecular machineries behind mitochondrial fusion and fi ssion events are relatively well known. The regulation of fusion and fi ssion events beyond the molecular machinery involved is less clear, fusion and fi ssion are not random occurrences but form a cycle whereby fi ssion typically follows fusion. Mitochondrial fi ssion machinery may somehow sense mitochondrial length and become active when the mitochondrion is oversized and cease when mitochondria are smaller. In contrast, mitochondrial fusion events depend heavily on mitochondrial traffi cking. Fusion only takes place when two mitochondria meet and motile mitochondria will be more likely to encounter one another. In cultured cortical neurons, for example, only one in every 14th contact between mitochondria results in fusion. The purpose of this presentation is to provide insight into the complex crosstalk between diff erent processes involved in mitochondrial fusion-fi ssion dynamics and to discuss the potential physiological purpose of mitochondrial fusion and fi ssion. Keywords Mitochondrial dynamics, neurons, mitophagy The genes encoding the E3 ubiquitin protein ligase Parkin (PARK2) and the mitochondrial serine/threonine kinase PINK1 (PARK6) are mutated in clinically similar, autosomal recessive early onset Parkinson's disease (PD) forms. Over the past ten years, a number of studies in diff erent model systems have demonstrated that PINK1 and Parkin regulate jointly several processes relevant to maintenance of mitochondrial quality, including mitochondrial traffi cking and dynamics, mitophagy and biogenesis. By using a combination of approaches of cell biology, confocal imaging and biochemistry in diff erent cell models, we recently showed that loss of protein import effi ciency triggers recruitment of Parkin by PINK1 in proximity of the translocase of outer mitochondrial membrane (TOM). We provided evidence that the degradation of specifi c TOM subunits plays a key role in initiating the autophagic degradation of damaged mitochondria. We also showed that PINK1 and Parkin interact with the TOM machinery on polarized mitochondria. Our results suggests that this interaction modulates the import of the multifunctional mitochondrion-protective matrix enzyme 17beta-hydroxysteroid dehydrogenase 10, which is depleted in Parkin-defi cient mice and Parkinson's disease patients. Electron and confocal microscopy, and calcium imaging approaches used to characterize the endoplasmic reticulum (ER)-mitochondria interface, a compartment previously linked to neurodegenerative processes, revealed enhanced juxtaposition between these organelles, associated with increased ER-to-mitochondria calcium transfer in cells from Parkindefi cient mice and patients with PARK2 mutations. Our current work aims at investigating the relevance of mitochondrial quality control mechanisms regulated by PINK1 and Parkin in diff erent cell types of the We have originally discovered activity-dependent neuroprotective protein (ADNP) as a major regulatory gene, a component of the SWI/ SNF chromatin remodeling complex, essential for brain formation.
Others found ADNP as a most frequent autism spectrum disorder (ASD)associated gene. Furthermore, ADNP is the only protein signifi cantly decreasing in the serum of Alzheimer's disease (AD) patients. Our most recent results revealed sex-related learning/memory diff erences in mice, refl ecting hippocampal expression changes in ADNP and ADNPcontrolled AD/ASD risk genes1. Hippocampal ADNP transcript content was doubled in male vs. female mice, with females showing equal expression to ADNP+/-males and no signifi cant genotype-associated reduction. Increased male ADNP expression was replicated in human postmortem hippocampal samples. The hippocampal transcript for ApoE (the major risk gene for AD) was doubled in female mice compared with males, and further doubled in the ADNP+/-females, contrasting a decrease in young ADNP+/-males. ADNP regulates >400 genes during embryonic development, with ApoE being a major target. Other AD related proteins regulated by ADNP include tau (with pathological tau constituting the neurofi brillary tangles and with AD being the major tauopathy). Furthermore, ADNP associates with microtubule end binding proteins, controlling dendritic spine density, which is compromised in AD and ASD. Previously, overexpression of the eukaryotic translation initiation factor 4E (eIF4E) led to ASDlike phenotype in mice and we have shown that hippocampal eIF4E expression was specifi cally increased in young ADNP+/-male mice.
Understanding ADNP expression, positioned as a master regulator of key ASD and AD risk genes, introduces a novel concept of hippocampal gene-regulated sexual dimorphism toward gender-based biology and therapeutics. Neurotrophin brain-derived neurotrophic factor (BDNF) is a growth factor that has important roles in the development and functioning of nervous system by promoting the survival, diff erentiation and synaptic plasticity of specifi c neuronal populations. Modifi ability of neuronal connectivity by formation of new synapses, and alteration of the strength and stability of existing synapses, is regarded as the main cellular basis for memory and long-term behavioral adaptations. The gene encoding BDNF is considered to be one of the master genes of synaptic plasticity. BDNF has also received particular interest for its deregulation in nervous system disorders. Decreases of BDNF and its receptor TrkB levels and activity are accompanied by and are believed to lead to several pathologies, particularly nervous system diseases like neurodegenerative, psychiatric and cognitive diseases. Therefore knowledge about the regulatory mechanisms of BDNF gene is important both for understanding of nervous system function and for fi nding new drug targets. Results of our studies on the molecular mechanisms of neuronal activity-regulated expression of BDNF gene in the nervous system will be presented and discussed. Hypoxic preconditioning is a pre-exposure to the repetitive mild hypoxia which results in development of brain hypoxic/ischemic tolerance and cross-tolerance to injurious factors of another nature. The endogenous defense processes mobilized by hypoxic preconditioning and resulting in formation of brain tolerance are based on evolutionary acquired gene-determined mechanisms of neuroprotection and adaptation. Key event is an activation of pro-adaptive transcriptional factors HIF-1, CREB, NF-kB, c-Fos, NGFI-A and down-stream expression of their target genes in vulnerable brain neurons. An important role can thus be suggested for the epigenetic regulation of gene expression, in particular, acetylation of core nucleosome histones leading to changes in chromatin structure which ensure access of the transcriptional factors activated by the preconditioning to the promoters of target genes. It has been shown that hypoxic preconditioning considerably increases an acetylation status of all histones and, particularly, H3 in the neurons of rat hippocampus and neocortex, whereas injurious severe hypoxia causes global repression of histone acetylation. Diverse eff ects of the severe hypoxia and mild hypoxic preconditioning on the methylation of DNA and histones have also been observed. The complex of the epigenetic modifi cations induced by the hypoxic preconditioning is attributed to the relaxed DNA which becomes available for activation of gene expression by pro-adaptive transcriptional factors up-regulated by the preconditioning. Acknowledgements The work was supported by Russian Foundation for Basic Research (grant No. 14-04-00516). Keywords Hypoxic preconditioning, brain hypoxic/ischemic tolerance, epigenetic mechanisms Understanding the mechanisms regulating gene expression in the course of development and adaptation of the organism to the permanently changing environment and in the case of pathology is fundamentally important. Special attention in recent years has been paid to the processes of epigenetic regulation of neuronal genes involving chromatin modifi cations at the level of DNA methylation and histone acetylation. In these processes an important role belongs to the histone deacetylases (HDAC) which control gene silencing.

Acknowledgements
Recently it was shown that the amyloid precursor protein (APP) and its intracellular domain (AICD) participate in regulation of expression of a number of neuronal genes, including those involved in amyloid metabolism and clearance. The list of APP-regulated genes includes the major amyloid-degrading enzyme neprilysin (NEP), a transport protein transthyretin (TTR), aquaporin and others. Our studies strongly indicate that AICD regulation of NEP and TTR is APP isoform-dependent and cell-type specifi c. In this process AICD competes for gene regulation with HDACs and treatment of cells or animals with HDAC inhibitors such as valproic acid results in up-regulation of NEP and TTR mRNA and protein levels and increased NEP activity leading to a reduction in total cellular amyloid (Aβ) peptide levels. Regulation of other proteins, e.g. acetylcholinesterase, does not involve AICD but requires full length APP molecules. APP overexpression in neuronal cells was also shown to aff ect the levels of HDAC gene products which might explain its role in gene regulation. Further studies of the APP interactome are important for better understanding of its role in brain development and functioning and for designing the drugs protecting the brain against neurodegeneration, in particular Alzheimer's disease. Acknowledgements Supported by RFBR (13-04-00388), Program of RAS "Fundamental Sciences for Medicine", ARUK. Keywords Amyloid precursor protein, neprilysin, histone decetylases

Anti-infl ammatory activities of carbon monoxide-releasing molecules (CO-RMs) in the brain
The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are ubiquitous protective systems against oxidative stress, infl ammation and tissue injury. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), possess interesting anti-oxidant and anti-infl ammatory properties suggesting that exploitation of this pathway as a target for drug discovery may off er therapeutic avenues in a variety of disorders [1]. In this context we have developed carbon monoxide-releasing molecules (CO-RMs), a class of compounds that deliver CO in a controlled fashion and exert a variety of pharmacological eff ects [2]. Data will be presented showing that CO-RMs modulate neuroinfl ammation and neuroprotection in vitro and in vivo. In BV2 microglia cells challenged with thrombin and interferon gamma, CO-RMs reduced the production of infl ammatory mediators both in normoxic and hypoxic conditions. Similarly, in a rat model of collagenase-induced intracerebral hemorrhage, CO-RMs SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 modulated microglia activation and the production of TNF-α while partially protecting against brain damage [3]. The mechanism(s) by which CO liberated from CO-RMs exert protection remains elusive. However, our laboratory has recently produced fi ndings in BV2 microglia pointing to mitochondria as a plausible target for the antiinfl ammatory action of CO. Studies are also currently ongoing on the synthesis and characterization of novel hybrid molecules that potently activate Nrf2 and simultaneously release CO in order to maximize the anti-infl ammatory eff ects of the HO-1 pathway [4]. The endogenously produced gasotransmitter carbon monoxide (CO) has been studied as a factor involved in cytoprotection, homeostasis and anti-infl ammation. Small amounts of reactive oxygen species (ROS) are described as signaling factors in CO's biological mode of action. Mitochondria are the main source of ROS and are also key organelles in orchestrating cell function: metabolism, cell death control and redox signaling. Astrocytes are most abundant glial cells and essential for neuronal function, namely metabolic and physical support, expression of neurotransmitters and promotion of neuroprotection. In this work it is shown that CO prevents astrocytic cell death and improves cell metabolism by targeting mitochondria, and some of the underlying molecular mechanism are disclosed. CO directly targets non-synaptic mitochondria and inhibits their mitochondrial membrane permeabilization, by preventing mitochondrial swelling, depolarization and inner membrane permeabilization. Thus, CO limits the release of cytochrome c into the cytosol and the activation of apoptotic cascade in astrocytes. All these events are ROS-dependent and involve glutathionylation of adenine nucleotide translocator (ANT), whose activity is ATP/ADP transport through mitochondrial inner membrane. In addition, low amounts of exogenous CO increase ATP production by improving oxidative metabolism. Mitochondrial population and specifi c cytochrome c oxidase activity are higher upon CO treatment. The COinduced metabolic improvement is dependent on Bcl-2 expression. Dysfunctional mitochondrial can be eliminated by mitophagy, which is a crucial process for maintaining their function and quality control. In astrocytes, CO promotes mitophagy at 1 h of treatment, while following 24 h mitochondrial population is back to basal levels, indicating that CO contributes to mitochondrial turnover. Furthermore, CO limits astrocytic cell death in an autophagic dependent manner. Stopping the succession of events that lead to development of Parkinson's disease (PD) is a principal challenge that requires a brain protective approach in early diagnosed patients. Although PD ethiopathology may not have a single causative factor, information from sporadic and familial cases together with chemical and genetic animal models strongly suggests that low-grade chronic infl ammation and oxidative stress play a critical role. Our team is studying the relevance of the transcription factor NRF2 (Nuclear factor (erythroidderived 2)-like 2), a master regulator of oxidant and infl ammatory defense, as a new therapeutic target in PD. The pro-infl ammatory activation of microglia and astroglia in response to LPS, MPTP and human α-synuclein over-expression is exacerbated in Nrf2-defi cient mice, thus demonstrating an immunomodulatory role of this protein.
In PD patients, some evidence gathered from epidemiological, genetic and anatomopathologic studies also support a protective role of NRF2. Several compounds activate NRF2 and provide an immunomodulator and cytoprotective response in preclinical animal models of PD. At this time a crucial point to translate these promising results to the clinic is the discovery of a drug with good pharmacokinetics and pharmacodynamics that fulfi ll criteria of safety, tolerability and effi cacy. Carbon monoxide (CO) is a gaseous second messenger produced when heme oxygenase (HO) enzymes catabolize heme. We have demonstrated that CO can be therapeutic in ischemia-reperfusion brain injury; however, it is unclear whether CO can also off er protection in permanent ischemic stroke or what mechanism underlies the eff ect. HO1 neuroprotection is shown to be regulated by Nrf2; therefore, we investigated whether CO might partially exert neuroprotection by modulating the Nrf2 pathway. To evaluate potential protective eff ects of CO, we exposed wildtype and Nrf2-/-mice to 250 ppm CO or control air for 18 h immediately after permanent middle cerebral artery occlusion. Infarct volume and neurological defi cits were assessed on day 7. Molecular mechanisms of Nrf2 pathway activation by CO were also investigated. Mice exposed to CO after permanent ischemia had 29.6 ± 12.6% less brain damage than did controls at 7 days. Additionally, 18 h CO treatment led to Nrf2 dissociation from Keap1, nuclear translocation, increased binding activity of Nrf2 to HO1 antioxidant response elements, and elevated HO1 expression 6-48 h after CO exposure. The CO neuroprotection was essentially completely abolished in Nrf2-/-mice. Low-concentration of exogenous CO represents a neuroprotective agent for stroke combination treatment and its benefi cial eff ect would be at least partially mediated by activation of the endogenous Nrf2 pathway. have been associated with peripheral neuropathy [1], but the specifi c role of peripheral nerve FA synthesis in myelin formation and function is poorly understood. We explored the extent to which lack of the key regulator of FA synthesis as Sterol Regulatory Element Binding Factor-1c (Srebf-1c) could result in the development of peripheral neuropathy. We found that Srebf-1c null mice display a neuropathic phenotype consisting in hypermyelinated small caliber fi bers, the result of changes in myelin periodicity. Unexpectedly, transcriptomics and metabolomics revealed activation of peroxisome proliferator activated receptor α (Pparα) signaling in Srebf-1c null peripheral nerve as a result of increased levels of two distinct phosphatidylcholine-based Pparα ligands, PC-C16:0/C18:1 and PC-C18:0/C18:1 [2,3]. Pparα is a nuclear receptor that directs uptake, utilization and catabolism of FAs [4]. As a consequence of abnormal local Pparα activation, Srebf-1c null peripheral nerve exhibit increased fatty acid utilization, a detrimental condition leading to peripheral neuropathy. Treatment with a Pparα antagonist rescues the neuropathy of Srebf-1c null mice. These fi ndings reveal the importance of FA synthesis to sustain peripheral nerve structure and function.

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The molecular control of GnRH neuron development Anna Cariboni 1 , Andre´ Valentina 1 , Kathryn Davidson 2 , John Parnavelas 2 Fertility critically depends by a small number of hypothalamic neurons secreting the neurohormone GnRH. During development GnRH neurons migrate from the nasal placode to the hypothalamus by following the migratory path formed by the vomeronasal axons. Developmental defects of this process can cause congenital GnRH defi ciency (GD), characterized by absent/delayed puberty and consequent infertility. The underlying mutated loci are for the majority of GD cases unknown, partially because of a poor understanding of the molecular mechanisms that control the development of these crucial neuroendocrine cells.
Here we provide evidence of the importance of class 3 semaphorins and their receptors in this process. Specifi cally, I will explain how two diff erent semaphorins play distinct roles during the migration of GnRH neuron.s Thus, semaphorin3A aff ects the migration of GnRH neurons in the nasal compartment, via the co-receptors Neuropilin-1 and 2, whereas semaphorin3E via its receptor plexind1 controls the survival of GnRH neurons once positioned in the hypothalamus. Accordingly, disrupted semaphorin signalling may be involved in the aethiopathogenesis of genetic diseases characterized by GnRH defi ciency. Keywords GnRH neuron, semaphorin, migration We analyzed the pathological consequences of abnormal Wnt/βcatenin signaling in endothelial cells of brain vessels using a murine model of Cerebral Cavernous Malformation (CCM) disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report increased transcription activity of β-catenin in CCM3-knockout endothelial cells in in-vitro and in-vivo models. Such activation is cellautonomous, independent of Wnt-receptor stimulation, does not induce canonical Wnt/β-catenin signaling and represents an early response to CCM3 ablation that initiates the expression of EndMT makers before the onset of Tgf-β/BMP signaling which is required for the progression of the pathology, as we have previously described.
We also show that the NSAIDs sulindac sulfi de and sulindac sulfone, which attenuate β-catenin transcription activity, signifi cantly reduce the number and dimension of vascular lesions in the central nervous system of mice with endothelial cell CCM3 knockout. Wnt signalling represents an ancient and highly versatile signalling system, which plays diverse critical roles in embryonic development. Sensory neurons of the dorsal root ganglia require Wnt signalling for initial cell fate determination as well as patterning and synapse formation over later development. Recent studies have functionally linked misregulation of Wnt signalling to cancer, bone disorders and abnormal synaptic function in adult life. We will discuss a novel, functional role of Wnt signalling pathways in sensitization of peripheral adult sensory neurons in a pathophysiological context, and the underlying molecular mechanisms. Using an interdisciplinary approach, through diff erent technique spanning molecular, genetic, and behavioural experiment in vitro as well as in vivo pain models in mice, we show that Wnt3a is able to recruit diff erent Wnt-pathways, to alter pain sensitivity in a modality-specifi c manner, acting via intracellular kinases in peripheral nerves. We found evidence for an intriguing dichotomy of noncanonical signalling pathways in mediating mechanical and thermal hypersensitivity. Indeed, while the calcium pathway is mainly involved in thermal hypersensitivity, mechanical hypersensitivity is driven by the planar cell polarity pathway. Interestingly we did not fi nd clear functional evidence for the canonical Wnt signalling pathway in Wnt3ainduced sensitization of nociceptors. Finally, we will provides evidence for a translational potential for targeting peripheral Wnt signalling in tumour-nerve interactions and pathological pain hypersensitivity, paving the way for therapeutic interventions.
withdrawal of medication. Human pluripotent stem cells are currently regarded as the main candidate cell type for CRT because they are readily available, expandable, and can be standardized and diff erentiated into mDA neurons capable of inducing functional recovery in animal models of PD. However, protocols for mDA diff erentiation are still far from optimal and require further improvement. We previously found that members of the Wnt family of morphogens regulate multiple aspects of mDA neuron development [1]. Diff erent branches of the Wnt signaling pathway, such as Wnt/β-catenin, activated by Wnt1, and Wnt/ PCP, activated by Wnt5a, have been thought to regulate separate or opposing functions. However, we found that Wnt5a cooperates with Wnt1 to promote mDA neurogenesis and that Wnt1 cooperates with Wnt5a to promote the diff erentiation of postmitotic mDA neuroblasts [2]. We are currently applying this knowledge to improve protocols for the diff erentiation of human stem cells into mDA neurons suitable for transplantation and functional recovery in animal models for PD [3].
Keywords WNT, dopaminergic neurons, Parkinson's disease References WNT signaling in microglia and the glioma microenvironment Gunnar Schulte WNT signaling is important during embryonic development and organogenesis having specifi c roles in the development of the CNS such as regulation of neural tube formation, axon guidance and CNS stem cell regulation. Our work has recently established a role of WNT signaling in the regulation of the brain's macrophages, the microglia and thus WNTs emerge as novel regulators of CNS infl ammatory responses. First of all, it appeared that b-catenin levels are elevated in microglia in Alzheimer disease (AD) brains as well as microglia cells in AD mouse models. Employing in vitro studies of primary mouse microglia isolated from newborn mouse pups indicated that both WNT-3A and WNT-5A induce diverse signaling routes in microglia leading to diff erential proinfl ammatory modulation of the cells. Interestingly, the net eff ect of WNT stimulation on the infl ammatory potential of mouse microglia is context dependent. While WNTs increase infl ammatory markers when giving to microglia alone, they are able to act in an antiinfl ammatory manner when microglia are activated by prestimulation with lipopolysaccharides. Our fi ndings thereby indicate that WNTs act on microglia as a homeostatic regulator, further underlined by yet unpublished data that suggest that WNT-5A is elevated in human glioma associated with a distinct infl ammatory signature of the tumor as well as a substantial invasion of microglia.
The major focus of ALS research has recently moved to RNA control of motor neuron functions, as most of the newly identifi ed genes, that alone account for more than half of ALS familial cases, are clearly associated to RNA regulation. These include FUS and TDP43, two DNA/ RNA binding proteins with a role in the regulation of RNA transcription, splicing, transport and translation, and C9orf72, a gene that is marked by the presence, in carriers, of an highly expanded GGGGCC repeat that is believed to provide the mutant gene of an acquired, toxic feature by an RNA-dependent gain of function mechanism. Thus, RNA dysmetabolism is likely to represent a central issue in ALS pathogenesis. Yet, whether a specifi c step of RNA processing is particularly aff ected in ALS motor neurons is unclear. We have recently obtained evidence that a prominent eff ect of FUS and C9orf72 expression is the induction of stress granules-associated translational repression. In this presentation I will discuss our recent work on the molecular mechanisms underlying these eff ects and their potential relevance in ALS pathogenesis. TDP43 is a ubiquitously expressed prevalently nuclear protein involved in RNA splicing, RNA stability and miRNA processing. Amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and inclusion body myopathy (IBM) are characterized by TDP43 being depleted from the nucleus and accumulating in cytoplasmic inclusions, which are the pathological hallmark of the disease -these diseases are also defi ned as "TDP43 proteinopathies". Mutations in TDP43 have been found to be causative of a proportion of ALS cases reinforcing the primary importance of this molecule in disease pathogenesis. The pathogenic mechanism by which TDP43 acts is unclear, and both loss of nuclear function (LOF) and gain of function (GOF) mechanisms have been proposed. We here discuss how we used muscle tissue, which provides high quality RNA of patient disease tissue, to investigate the consequences of TDP43 mislocalization. Further, we characterize two novel mouse TDP43 mutant lines, carrying ENU-induced mutations in order to study the eff ects of TDP43 mutations expressed at physiological levels in the mammalian central nervous system. One mutation (deltaRNA) strongly reduces the RNA-binding capacity of the protein; the second mutation (C-TERM) is in the glycine-rich C-terminal domain where the majority of human pathogenic mutations are found. Our results underline the importance of studying models with physiological expression levels of TDP43 mutations and shed light on the diff erent eff ects on RNA metabolism caused by the TDP43 loss and gain of function. Analysis of the mouse model, which faithfully mimics the SMN insuffi ciency and the motor neuron phenotype, shows that motor neuron degeneration starts in the axons, specifi cally at the neuromuscular junctions. SMN is a house-keeping factor that is necessary for the assembly of the seven-membered ring of Sm proteins around the spliceosomal snRNAs and that is therefore required for pre-mRNA splicing. Such a function cannot easily explain the selective motor neuron phenotype. It has been proposed that insuffi cient supply of the SMN protein aff ects alternative splicing, especially of U11/U12-dependent introns, in mRNAs that are crucially required in motor neurons. Details, however, remain elusive. In addition to the Sm proteins, there are the like-Sm (LSm) proteins that also form heptameric complexes and participate in various steps of mRNA metabolism. We have previously shown that one such LSm complex is involved in the transport of mRNAs to the neurites, especially the axons of motor neurons. mRNA transport and local protein synthesis is an important mechanism to bring about sudden changes in the proteome at distal regions of the cell. Here, we further explore the role of the LSm proteins in neuronal mRNA regulation and how this could be relevant for the SMA pathology. Interestingly, we fi nd that one such LSm protein is signifi cantly depleted in the SMA mouse model before the onset of the disease, possibly indicating a causal involvement. Keywords SMA, mRNA regulation, Sm-like proteins A number of diff erent genes have been found mutated in patients with Amyotrophic Lateral Sclerosis (ALS). Several of these genes encode for proteins involved in multiple steps of RNA processing, suggesting that mRNA dys-metabolism has a role in the degeneration of motor neurons. This is the case also for FUS-linked ALS. FUS (Fused in Sarcoma) is a DNA/RNA binding proteins with an established, yet not completely clear, role in the regulation of RNA transcription, splicing, transport and translation. However, recent evidence indicates that (similarly to mutant ALS-linked SOD1) the toxic function of this protein may lie also in its propensity to aggregate and sequester other proteins, and/ or in its ability to induce mitochondrial damage and oxidative stress. In this presentation I will discuss our recent work on the molecular mechanisms underlying these eff ects and their potential relevance in the pathogenesis of ALS.

Diverse roles of FUS in Amyotrophic Lateral Sclerosis
Spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are two related motorneuron diseases (MNDs), both characterized by the presence of inclusions o aggregates of proteinaceous materials. In SBMA, aggregates contain mutant androgen receptors (AR) with an elongated polyglutamine tract (ARpolyQ), while in ALS aggregates contain TDP43, ubiquilin, optineurin, etc. Exceptions are familial ALS (fALS) forms linked to superoxide dismutase 1 (SOD1) mutations, in which aggregates are composed of mutant SOD1. Protein aggregation occurs when a large excess of proteins with aberrant conformations (misfolding) in produced and poorly cleared from the cells. Neurons contains an effi cient protein quality control (PQC) system, but this may be insuffi cient to correctly remove misfolded proteins, especially during aging. The PQC system requires the activities of effi cient chaperones and of the two major intracellular degradative systems: the ubiquitinproteasome (UPS) and the autophagic systems. When misfolded protein are recognized by chaperones, they can be removed via autophagy by their engulfment into autophagosomes which then fuseto lysosomes. We found that motoneurons may responds to misfolded species by activating the expression of a small HSP, HSPB8, which facilitate the clearance of misfolded species via autophagy, usually acting by restoring the proper autophagic fl ux, found altered in MNDs. HSPB8 requires its co-chaperone BAG3. BAG3 binds the protein 14-3-3 and with this it interacts with dynein in a complex which also includes Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse occurrence and functions. One of the most well-known eff ects of PACAP is its strong neuroprotective eff ect. In this presentation we give an insight into recently described neurochemical changes induced by PACAP or altered by PACAP the lack of it. In an invertebrate model for Parkinson's disease we found that PACAP eff ectively counteracts the dopamine-decreasing eff ect of rotenone, a mitochondrial neurotoxin. Similarly, in a 6-hydroxydopamine-induced rat model of Parkinson's disease, we found that PACAP eff ectively increases dopamine levels. Furthermore, our proteomics analysis shows that PACAP treatment also counteracts the 6-OHDA-induced decrease in PARK-7 protein, eff ective against oxidative stress. Studying the role of endogenous PACAP, we found that PACAP-defi cient mice show higher susceptibility to toxic agents causing degeneration of the substantia nigra dopaminergic neurons. Using proteomic analysis we revealed that the expression of numerous proteins is altered in the mesencephalon and striatum of knockout mice. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, beta-synuclein and aspartate amino transferase. The altered expression of these proteins might partially account for the decreased antioxidant, cytoprotective and detoxifying capacity of PACAP-defi cient mice. The described changes may provide further explanation for the neuroprotective potency of PACAP.
galanin (GAL1, GAL2, GAL3) we found GAL3 to be the most abundantly expressed on the vasculature and GAL2 on diff erent types of immune cells including polymorphonuclear neutrophils and natural killer cells. Therefore, we evaluated if galanin exerts direct or indirect eff ects on these immune cells. Our data revealed that galanin can be regarded as an immunomodulatory peptide as it can sensitize polymorphonuclear neutrophils and natural killer cells towards proinfl ammatory cytokines.
Since there are only scarce in vivo data concerning the role of GAL3 in infl ammatory disease conditions, we analysed its involvement in the K/BxN serum transfer model of autoimmune arthritis and the oxazolone-model of allergic contact dermatitis, employing GAL3 gene-defi cient mice. After arthritis induction, GAL3-knockout mice demonstrated increased clinical disease severity and earlier hindlimb edema than wildtype mice. Vascular hyperpermeability was also elevated compared to wildytpes, but neutrophil myeloperoxidase activity and arthritic hyperalgesia were not signifi cantly diff erent. In contrast, disease severity, vascular, and immune components were not aff ected in allergic contact dermatitis in GAL3 knockouts in comparison with wildtypes. Our fi ndings suggest GAL3 activation as a substantial anti-infl ammatory pathway in neutrophil-dominated autoimmune arthritis, modulating the early neurogenic vascular hyperpermeability and consequent edema formation. However, the involvement of GAL3 activation in the T-cell dependent allergic contact dermatitis remains unsupported.
Keywords Galanin, infl ammation, immune cells During cerebellar development, granule cell precursors are produced from a secondary germinative zone forming the external granule cell layer (EGL). Immature granule neurons from the inner part of the EGL then start a tangential migration followed by a centripetal inward radial migration across the molecular and Purkinje cell layers to reach their fi nal destination at the bottom of the forming internal granule cell layer (IGL). This complex migratory process is highly regulated and takes about 2 days in rodents and it is essential for the proper formation of the cortical layers forming the mature cerebellum. In the IGL, granule cells diff erentiate to establish functional excitatory synapses with GABAergic neurons including Purkinje, basket, stellate and Golgi cells, or die. Some neurotrophins and neurotransmitters have been shown to be involved in the control of cerebellar granule cell survival, migration and diff erentiation. Initially, when I started my carrier as a researcher, we used to claim that very few neuropeptides were produced in the cerebellum. Nevertheless, we now know that this was wrong as we have recently identifi ed by mass spectrometry over 70 peptides expressed in the cerebellum during development. Over the years, the involvement of some of these peptides such as somatostatin, PACAP or ODN, has been established in the control of cerebellar granule cell survival, migration and diff erentiation as will illustrate my presentation. Orexins A and B (hypocretins 1 and 2) are two closely related peptides produced mainly by hypothalamic neurons that project to numerous brain structures. Orexins exert their biological activity by binding to two subtypes of GPCR receptors, OX1R and OX2R. The orexin system has been shown to orchestrate multifaceted physiological functions, including vigilance and the sleep/wake cycle, energy homeostasis, endocrine, visceral functions and pathological states, such as narcolepsy and drug abuse [1]. In addition, a neuroprotective potential of orexin A has been recently demonstrated in rats using a model of focal cerebral ischemia [2]. In our studies we investigated eff ects of orexins on survival of cultured neurons from the rat cerebral cortex. Quantitative Dopamine receptors are G-protein-coupled receptors (GPCRs), which are involved in a wide variety of physiological processes. Abnormalities in dopaminergic signal transduction are associated with many diff erent diseases. Therefore, dopamine receptors are targets for variety of drugs involved in disorders like schizophrenia, Parkinson's disease, depression and many others. In order to develop drugs with less side eff ects and better effi cacy it is necessary to understand and characterize receptor-ligand interactions in further detail. In addition, measuring the on-and off -rates of diff erent ligands provides important information about the kinetic profi les of potential drug candidates. Previous studies of our group indicate that genetic deletion and pharmacological antagonism of P2Y12 receptors alleviate mechanical allodynia in acute infl ammatory pain. In this project we investigated the role of P2Y12 receptors in chronic infl ammation. For this study we have introduced a CFA-model in wild type and P2Y12R gene defi cient (P2ry12-/-) mice. By the injection of Complete Freund's Adjuvant (CFA) in the plantar surface of the right hind paw, we could induce local infl ammation persisting at least 14 days. During this 2 week period, mechanical sensitivity was evaluated at several time points using dynamic plantar von Frey aesthesiometer. Mechanical allodynia could be observed 3 days after CFA injection. In P2Y12 receptor gene defi cient mice the decline in the paw withdrawal threshold (PWT) was signifi cantly lower than in wild type mice. Both intrathecal and intraplantar administration of PSB-0739, a selective and potent P2Y12 receptor antagonist had robust antihyperalgesic eff ect in wild type mice, whereas treatment did not aff ect the PWT in the P2ry12-/-group. Intraperitoneal injection of A-803467, a potent and selective NaV1.8 sodium channel blocker evoked antihyperalgesic eff ect similar to the PSB-0739. When these two compounds added together, no additive eff ect was observed, i.e. A-803467 occluded the eff ect of PSB-0739. To investigate whether P2Y12 receptors on thrombocytes contribute to this eff ect, we used anti-mouse CD41 antibodies to induce depletion of platelets. Mice were submitted to intraperitoneal injection of this antibody at the end of the fi rst week and measurement of the mechanical allodynia was performed on the second week. Our fi ndings indicate that P2Y12 receptor inhibition is a potential therapeutic approach in chronic infl ammatory pain, although its exact mechanism of action needs further investigation. Keywords Infl ammation, pain, purin Tyrphostin 23 (T23) is a well-known inhibitor of protein tyrosine kinases. To investigate potential eff ects of T23 on the viability and the glucose metabolism of brain cells, we exposed cultured primary rat astrocytes to T23. While the viability and the morphology of the cells were not acutely aff ected during an incubation of the cultures for up to 4 hours with T23 in concentrations of up to 200 μM, the presence of T23 rapidly stimulated glycolytic fl ux as demonstrated by a timeand concentration-dependent increase in glucose consumption and lactate release. Maximal eff ects were observed for incubations with 100 μM T23 which caused a doubling of glucose consumption and lactate production. The stimulation of glycolytic fl ux by T23 was fully reversible upon removal of the compound. In contrast to T23, the structurally related tyrosin kinase inhibitor tyrphostin 25 did not aff ect glycolytic fl ux, nor was the stimulation by T23 substantially aff ected by the trichloracetate-induced activation of pyruvate dehydrogenase. Further experiments are now required to elucidate the mechanism of T23-induced stimulation of astrocytic glycolysis. Keywords Tyrphostin 23, astrocytes, glycolysis The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. New fi ndings show existence of FGFR1-5-HT1A heteroreceptor complexes in the rat hippocampus with a partial characterization of their interface and in midbrain raphe 5-HT nerve cells. With in situ Proximity Ligation Assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague-Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also established. After combined FGF-2 and 8-OH-DPAT treatment, a marked and signifi cant increase in PLA positive clusters was found in the RN33B cells. Synergistic receptor-receptor interactions in these receptor complexes indicated their enhancing role in hippocampal plasticity. The existence of FGFR1-5-HT1A heteroreceptor complexes also in midbrain raphe 5-HT nerve cells open up the possibility that antidepressant drugs by increasing extracellular 5-HT levels, can cause an activation of the FGF-2/FGFR1 mechanism in these nerve cells as well. Therefore, the agonist modulation of the FGFR1-5-HT1A heteroreceptor complexes and their specifi c role is now determined in rat medullary raphe RN33B cells and in the caudal midline raphe area of the midbrain rich in 5-HT nerve cells. The combined icv treatment with FGF-2 and the 5-HT1A agonist 8-OHDPAT synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and in the RN33B cells. Cotreatment with FGF2 and the 5-HT1A agonist induced RN33B cell diff erentiation as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and diff erentiation events were dependent on the receptor interface since they were blocked by incubation with TMV but not by TMII of the 5-HT1A receptor. Taken together, the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells appears to have also a trophic role in the central 5-HT neuron systems besides playing a key role in reducing the fi ring of these neurons. To test for potential consequences of an exposure of brain cells to copper oxide nanoparticles (CuO-NPs), we have synthesized dimercaptosuccinate-coated CuO-NPs. These particles had a diameter of around 5 nm as determined by transmission electron microscopy but were dispersed as aggregate as demonstrated by their average hydrodynamic diameter in aqueous dispersion of 136 ± 4 nm. Exposure of cultured primary astrocytes to CuO-NPs increased the cellular copper levels and compromised the cell viability in a time-and concentrationdependent manner. CuO-NPs in concentrations above 100 μM (6.3 μg SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 copper/mL) severely aff ected the viability of the cells, as demonstrated by a lowered tetrazolium dye reduction capacity, a lowered cellular lactate dehydrogenase activity, a increased membrane permeability and the generation of reactive oxygen species. In contrast, exposure of astrocytes for 24 h with 100 μM CuO-NPs did hardly aff ect the viability of astrocytes but stimulated the glycolytic fl ux, increased the cellular glutathione content, stimulated the release of glutathione and elevated the level of the metal storing proteins metallothioneins. Presence of the intracellular copper chelator tetrathiomolybdate throughout the incubation with CuO-NPs protected the cells against the toxicity of CuO-NPs and prevented the stimulation of the glycolytic fl ux as well as the increased levels of metallothioneins. These data demonstrate that CuO-NPs can severely damage cultured astrocyes and that copper ions derived from sub-toxic concentrations of CuO-NPs strongly aff ected the metabolism of astrocytes. Motor neuronal system and muscle tissue are two districts diff erently aff ected at onset and during the progression of diseases like amyotrophic lateral sclerosis (ALS) or spinal and bulbar muscular atrophy (SBMA). The bases of these diseases are linked to mutated proteins: fALS is commonly caused by mutations in the SOD1 or the TDP43 genes and SBMA is caused by a of CAG repeat in the AR gene. A fraction of these proteins can not reach a mature conformation and misfold. The Protein Quality Control (PQC) system is responsible for the correct protein homeostasis: the chaperones maintain proteins in their correct conformations. If they fail, mutated proteins are directed to the proteasome or the macroautophagy. When misfolded proteins are not correctly removed, they aggregate in nucleus and cytoplasm.

Increase of the FGFR1 signaling in the FGFR1-5-HT1A heteroreceptor complex in midbrain raphe 5-HT neuron systems via allosteric receptor-receptor interaction
To understand cellular behavior in presence of misfolded toxic proteins we investigate the diff erent activation of the PQC system in the two mayor tissues involved. Initially we investigated the diff erences in PQC activation between NSC34 motor neuronal and C2C12 muscular cell models. Using RTq PCR, western blot and immunocytochemical analysis for p62 and LC3 expression, localization, and turnover we demonstrated that C2C12 cells have a more active autophagic system than NSC34 cells. Then, we compared the two models in presence of misfolded protein inhibiting degradative systems. With Filter Retardation Assay, we observed that these proteins tend to aggregate when PQC system is impaired. Then, we potentiated the PQC response to reduce the insoluble species. By overexpressing the small heat shock protein B8 in both systems we demonstrated that AR polyQ and SODG93A insoluble species were reduced. Also autophagy activation by trehalose caused a reduction in protein aggregation in both cell models. In conclusion misfolded protein aggregates can be reduced by modulating macroautophagy and this could represent a new therapeutical strategy for disease like SBMA and ALS. Keywords Neurodegeneration, misfolded proteins, PQC system Nerve Growth Factor (NGF) plays a key role in development and function of specifi c neuronal populations of the CNS. Decreased NGF availability is also responsible for neuronal vulnerability in neurodegenerative disorders, such as Alzheimer's disease (AD). We used PC12 cells and primary neurons to investigate the potential role of NGF in modulating neuronal autophagy, a cellular process whose deregulation has been linked to the loss of neuronal. The mammalian target of rapamycin has been recently proposed as a therapeutic target for AD due to the role of the autophagy pathway in improving cognitive function by reducing Aβ and Tau pathology. We here show that NGF treatment induces LC3 conversion (LC3-I to LC3-II), a marker of autophagy, through the activation of AMP-activated protein kinase (AMPK). We show that NGF increases the autophagic fl ux (the dynamic process of autophagosome synthesis, delivery to the lysosome and degradation) as indicated by western blot and fl uorescence microscopy analyses in the presence of autophagy inhibitors. These data were confi rmed by RT-PCR array analysis and RNA interference-mediated knockdown of autophagyrelated genes: Atg9b, Atg12 and AMBRA1, a positive regulator of the BECLIN 1-dependent program of autophagy. In addition, fl ow cytometry analysis by DCF-DA showed that treatment with autophagy inhibitors determined a strong increase of reactive oxygen species (ROS) followed by decreased cell survival. These changes were fully reversed by NGF treatment, suggesting its potential role in clearing dysfunctional mitochondria. This hypothesis was further confi rmed by fl uorescence microscopy studies using LC3 and Cox-IV antibodies, showing co-localization of autophagosomes and mitochondria. Overall these data identify a novel aspect of the neuroprotective function of NGF in promoting the clearance of dysfunctional mitochondria (or mitophagy), thus further supporting its therapeutic potential in neurodegenerative pathologies. Keywords NGF, Alzheimer disease, autophagy  Cerebral ischemic injuries and neurodegenerative disorders lead to death or impairment of neurons in the central nervous system. Application of stem cell based therapies, namely stimulation of endogenous neurogenesis or cell transplantation, are promising strategies and currently under investigation. Carbon monoxide (CO) is an endogenous product of heme degradation by heme oxygenase. Although there is no published data reporting CO as a factor involved in stem cell diff erentiation, several evidences support this hypothesis. This gasotransmitter induces mitochondrial biogenesis, which is also broadly described to be involved in metabolic shifts during neuronal diff erentiation process. Likewise, CO-induced pathways can occur via generation of small amounts of ROS, which are also important signaling molecules in neuronal diff erentiation. The CO eff ect on modulation of neuronal diff erentiation is assessed in three diff erent models with increasing complexity: human neuroblastome SH-S5Y5 cell line, human teratocarcinome NT2 cell line and hippocampal organotypic slice cultures (HOSC). CO does increase the fi nal yield of post-mitotic neurons. During neuronal diff erentiation, CO promotes an increase on precursor cell proliferation and in parallel CO inhibits cell death. Furthermore, cell mitochondrial population is increased by CORM-A1 supplementation. Further work is needed for assessing the mechanisms underlying CO eff ects in neuronal diff erentiation, namely by targeting modulation of cellular metabolic pathways, redox alterations and autophagy related pathways. In conclusion, CO appears as a promising therapeutic molecule to stimulate endogenous neurogenesis or to improve in vitro neuronal production for cell therapy strategies. Oxygen starvation is observed in a variety of pathological states and serves as one of the urgent problems in medicine. A decrease in oxygen supply to tissues is accompanied by the inhibition of metabolic processes (primarily of energy metabolism), which impairs functional activity of the brain. The main source of energy for brain is adenosine triphosphate (ATP). It was shown that the components of adenylate pool can be used as early predictors of hypoxia.

Neuroprotection by Nerve Growth Factor (NGF) involves modulation of neuronal autophagy
Aim of the study The aim of our study was analysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) experimental concentrations and integral coeffi cient K= in intact animals brain tissue and in disturbances of the oxygen regime by methods of mathematical analysis, as well as detection of some regularity in the character of their changes under the impact of hypoxia for the assessment and prediction of direction of production and utilization energy in metabolic pathways. Methodology In this study empirical dependencies and criteria of statistical signifi cance of mathematical modeling of quantitative relation between specifi ed brain nucleotide stock indicators for the assessment and prognostication of brain energy state in extreme conditions were used.

Results and area of their application
The use of empirical dependencies methods allowed to create multiregression models, subtly enough to unite experimental indicators ATP and AMP in hypobaric hypoxia and ischemia with diff erent-term exposure. Obtained models can be used for prognostication of ATP and AMP concentrations in disturbances of the oxygen regime in a short or over a long period of time, as well as to receive information of indicator K= changing depending on brain hypoxia. Conclusions functional dependencies are presented in this study to analyze shape, closeness and stability of relations between adenine nucleotides characterizing coupling of production and utilization energy processes, and also to predict the direction of these processes under hypoxic condition. Brain ischemia is accompanied by lowering of pHo and pHi. We investigated an infl uence of of acidosis on free radical formation in synaptosomes. Three models were used: 1) Strong extracellular acidifi cation down to pHo 6.0; 2) Moderate extracellular acidifi cation down to pHo 7.0; 3) Intracellular acidifi cation induced by addition of 1 mM amiloride corresponding to pHi decrease down to 6.65. We have shown that both types of extracellular acidifi cation, but not intracellular acidifi cation, increase DCFDA fl uorescence by calcium-independent way that refl ects free radical formation. These three treatments induce the rise of the dihydroethidium fl uorescence that reports synthesis of superoxide anion. However, the impact of low pHi on superoxide anion synthesis was less than induced by moderate extracellular acidifi cation. Mitochondrial uncoupler CCCP did not inhibit an increase of fl uorescence of both dyes at pHo 6.0. In contrast, superoxide anion synthesis at pHo 7.0 was almost completely eliminated by CCCP. Furthermore, using fl uorescent dyes JC-1 and rhodamine-123, we confi rmed that decrease of pHo leads to mitochondria depolarization. Low pHi was not eff ective. Iron chelator deferoxamine and antioxidant ionol are inhibits pH-induced increase of DCFDA fl uorescence, but does not infl uenced mitochondria depolarization. We are failed to found sodium infl ux monitored by fl uorescent dye Sodium Green SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 in case of low pHo. Involving of plasma membrane receptor which is distinct from acid-sensitive ion channels (ASIC) and electron transport chain of mitochondria for moderate acidifi cation can be suggested. Action of strong acidifi cation seems to be mediated by release of iron from proteins. We have shown that low pHo led to oxidative stress in neuronal presynaptic endings that might underlie the long term irreversible changing in synaptic transmission.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive loss of brain tissue and accumulation of amyloid-β(Aβ) and tau. Aggregation of Aβ peptides into amyloid plaques is considered to be a causative factor in AD, however the precise mechanism behind the AD onset has remained elusive. Oxidative stress (OS) is also characteristic to AD, but it is not known whether OS is a risk factor or a consequence of AD. It is assumed that Aβ aggregates generate free radicals in the presence of copper ions by participation of the Met35 residue, which can increase the OS levels. The aim of our study was to establish the role of Met35 residue in the oxidation of Aβ and peptide aggregation processes. Oxidation of Aβ was studied in the presence of two redox-active compounds: H2O2 and copper ions. In the absence of copper ions the Met35 residue was readily oxidized by H2O2 in a two electron process. The fi brillization of Aβ with Met35 oxidized to sulfoxide was threefold slower compared to that of the native peptide. TEM analysis showed that the fi brils of native and oxidized peptides are similar. The relatively small inhibitory eff ect of Met35 oxidation on the fi brillization suggests that the possible variation in the Met oxidation state should not aff ect the in vivo plaque formation. In the presence of copper ions (one-electron process) the oxidation was more complex: addition of the fi rst oxygen was still the fastest process, however, it was accompanied by multiple unspecifi c modifi cations of several amino acid residues. Addition of copper ions to the already oxidized Aβ Met35 by H2O2, resulted in a similar pattern of nonspecifi c modifi cations, suggesting that the one-electron oxidation processes in Aβ do not depend on the oxidation state of Met35. Thus, it can be concluded that Met35 residue is not a part of the radical generating mechanism of Aβ-Cu(II) complex. Keywords β-Amyloid, copper(II)ion, methionine oxidation

P14
The eff ects of optical, electrical and chemical stimulation on serotonin release from median raphe and hippocampus of mice Flora Goloncser 1,2 , Romeo Ando 1 , Dora Zelena 1 , Jozsef Haller 1 , Beata Sperlagh 1 The present study has examined several characteristics of the release of [3H]5-HT from the median raphe nucleus (MRN) and hippocampus in terms of its dependence of nerve impulse. We used electrical stimulation and the sodium channel opener veratridine, which excite all of the neuronal processes in the stimulation fi eld, and optogenetics to selectively stimulate those terminals which express channelrhodopsin-2 (ChR2) and compared 5-HT release evoked by electrical and chemical depolarization and by light. We injected an adeno-associated virus containing DNA construct encoding ChR2 into the MRN of mice and investigated [3H]5-HT release from MRN and hippocampal slices. Serotonergic nerve terminals was locally stimulated with 473 nm light (blue laser diode) and electrically by bipolar electrode and by veratridine and transmitter release was monitored by collecting the effl uent in a fraction collector. Electrical fi led stimulation and veratridine resulted in a signifi cantly increase in the effl ux of 5-HT, whereas optical stimulation of ChR2 expressing nerve terminals at various frequencies (10, 20, 50, 100 Hz) elicited only a negligible increase in 5-HT release either from the hippocampus or from the MRN itself. The electrically induced release of radioactive neurotransmitter was completely inhibited by perfusion with tetrodotoxin. We have also applied the 5-HT transporter inhibitor, fl uoxetine and the GABAA blocker bicuculline to relieve released 5-HT from re-uptake and any endogenous inhibition. Nevertheless, the eff ect of optical stimulation remained closed to the detection limit under these condition. In conclusion, whereas our method is suitable to detect [3H]5-HT effl ux in response to ongoing neuronal sodium channel activity its sensitivity is too low to detect transmitter effl ux evoked by focal optogenetic stimulation. The most likely reason for the failure of detection of 5-HT effl ux is that ChR2 is expressed only by a small subpopulation of nerve terminals. Keywords Channelrhodopsin-2, median raphe nucleus, serotonin Recombinant human IgM22 (rHIgM22) binds to myelin and to oligodendrocytes, and promotes remyelination in a mouse model of multiple sclerosis [1]. rHIgM22 preferentially reacts with sulfatidepositive (O4-positive) oligodendrocytes [1]. Moreover, binding of rHIgM22 is abolished in CNS tissue slices from Cst(-/-) mice [2], suggesting that its binding to myelin requires the presence of a product of cerebroside sulfotransferase, possibly sulfatide, abundantly expressed in oligodendrocytes and in myelin. However the exact identity of the antigen recognized by this antibody remains to be elucidated. We have tested the binding of rHIgM22 to purifi ed lipids and to lipid extracts prepared from mouse brain, brain myelin, mixed glial cultures, and O4-positive oligodendrocytes using TLC immunostaining and ELISA using liposomes and lipid monolayers with diff erent composition. Our preliminary results show that rHIgM22 binds to sulfatide in vitro, while it does not bind to other myelin sphingolipids, including galactosylceramide and sphingomyelin, suggesting that sulfatide at the oligodendrocyte surface might be important for the binding of rHIgM22 to the surface of these cells and to myelin. However, IgM22 does not bind structures expressing sulfatide outside the nervous system, thus additional factors are likely relevant for the immunoreactivity of IgM22 in CNS. Indeed, we have observed in lipid extracts from diff erent sources another lipid molecule selectively recognized by rHIgM22, whose identity is still under investigation.

Identifi cation of the antigen recognized by rHIgM22, a remyelination-promoting human monoclonal antibody
Remarkably, this lipid is also present in the extracts from mixed glial cultures, which do not contain mature O4-positive oligodendrocytes, suggesting that other glial cells in addition to oligodendrocytes might be important in the response to rHIgM22. and increased repetitive behaviours with limited interests of environment. Recent studies have revealed that purinergic signalling (hyperpurinergia) is one of a key features of autism. Our aim was to establish a reliable model of ASD in our lab utilizing a broad range of behavioural experiments in order to investigate the role of P2X7 in autism. We injected Poly(I:C) (PIC) in two doses to pregnant C57Bl/6 mice: 3 mg/kg on E12.5 and 1.5 mg/kg on E17.5 respectively. Off springs were weaned 4 weeks of age and behavioural studies started from 8 weeks of age. We performed social preference test, measured the body temperature and sensorymotor coordination (rotarod). We used self grooming and marble burying test in order to investigate manifestation of repetitive behaviour and measured the sensorymotor gating with Prepulse Inhibition (PPI). After behavioural experiments animals were sacrifi ced. Para-sagittal sections of the cerebellar vermis were cut and Purkinje cells were counted. Synaptosome fractions were made from half brains of animals and examined by electron microscopy. Striatum and Hippocampus monoamine content were measured by HPLC. We compared PIC treated off springs with naive animals (n = 10-16 animals/group). PIC treated animals showed decreased sociability and sensorymotoric coordination but we did not fi nd change in body temperature of PIC animals. MIA animals showed increased repetitive behaviour in the marble burying and self grooming test. Quantitative Purkinje cell dropout was found in PIC mice and electron microscopy of half brain revealed ultrastructural abnormalities in them. Higher level of monoamins were detected in ASD mice compared to the control group. Based on these results this model seem to be suitable to measure the eff ect of diff erent compounds or genetic deletion on PIC induced ASD symptoms in rodents. Keywords Poly(I:C), autism, P2X7R Alzheimer's disease (AD) is a neurodegenerative disorder. The pathohistological features in AD are intracellular accumulation of neurofi brillary tangles and extracellular senile plaques. Plaque deposition leads to recruitment and activation of microglial cells, which induces neuroinfl ammation and drives neurodegeneration.Recent evidence show that soluble pre-fi brillar Aβ species, rather than insoluble fi brils, are highly neurotoxic and correlate with disease severity.Hence, preventing formation of soluble Aβ and its interaction with neurons is a major goal in AD. Despite massive eff orts, how extracellular factors regulate assembly of Aβ peptide and neurotoxic activity of Aβ species is still largely undefi ned. Recent studies indicate that Extracellular Vesicles(EVs),including exosomes and PM-derived microvesicles(MVs), may infl uence Aβ neurotoxicity. Our fi ndings reveal that production of microglial MVs(m-MVs) is strikingly high in patients with mild cognitive impairment and AD as compared to healthy controls and positively correlates with markers of neurodegeneration and hippocampal atrophy. Furthermore we found that MVs isolated from the CSF of AD patients are toxic to cultured hippocampal neurons. Through in vitro studies we demonstrate that the m-MVs promote generation of neurotoxic soluble species from almost inert Aβ aggregates, which is mediated by lipid components of MVs. Our fi ndings suggest that m-MVs favor formation of neurotoxic Aβspecies throughout the brain, possibly representing the mechanism behind transynaptic spread of In recent years the study of age-related derangements of the gonadotropin releasing hormone (GnRH) synthesis and secretion resulting from both the GnRH gene expression changes and interaction of glia with GnRH-ergic neurons of the hypothalamus is a focus of attention. It was demonstrated earlier that this could result from the decreased activity of monoaminergic and peptidergic systems that control the GnRH preovulatory secretion surge initiation, specifi cally from the loss of the signal coming from the suprachiasmatic nuclei (SCN) of the hypothalamus. This signal is critical for the emerging of the GnRH regular cyclic secretion, which is mediated prominently by vasoactive intestinal peptide (VIP). We have studied age-related changes in the biogenic amines and VIP content in the hypothalamic structures responsible for the GnRH synthesis and secretion. It has been shown by us that the GnRH level in the median eminence with the arcuate nuclei (ME-Arc) of the hypothalamus of 22-month-old rats is half as high compared to that of 7-8-month-old animals. Beside that, the VIP level in the SCN tended to decrease, with the norepinephrine, dopamine, and 5-hydroxyindoleacetic acid levels decreased signifi cantly in the median preoptic area of the hypothalamus responsible for the GnRH synthesis and in the ME-Arc exercising its secretion into the portal vein of the pituitary. It has been shown that the initial phase of the reproductive failure with 13-14-month-old animals having irregular estrous cycles is characterized by gradual disappearance of the normal biogenic amine diurnal dynamics in the studied hypothalamic structures, which could be due to the loss of regulatory signals coming from the SCN. Keywords GnRH, biogenic amines, hypothalamus Objectives Amyloid beta plaques are primary hallmark of Alzheimer's disease, which is characterized by specifi c neurodegeneration. Amyloid beta peptide -the main plaque component-was shown to be neurotoxic in animal models, primary neuronal cultures and immortalized cell lines. However, the results are often controversial and there is no good human cell line model for evaluation of the toxicity of amyloid peptides. Here we studied the eff ect of amyloid beta 1-40 and 1-42 on undiff erentiated and diff erentiated human neuroblastoma cell line SH-SY5Y. Results Undiff erentiated cell culture was too diverse and unstable to reveal a toxic eff ect of amyloid beta peptides quantitatively. Diff erentiated cells established more neuron-like phenotype and were more identical and stable in culture suggesting potential susceptibility to amyloid beta as a neurotoxic agent. Amyloid peptides are prone to form diff erent aggregates with diverse toxic properties, in current study, monomeric amyloid beta 1-40 and 1-42 were applied to the cells. Viability test WST-1 and propidium iodide (PI) uptake tests showed that undiff erentiated cells are not susceptible to amyloid beta, however, diff erentiated cells showed reduced viability and increased PI uptake in case of amyloid beta 1-42, but not in case of amyloid beta 1-40.

Eff ect of extracellular vesicles derived from distinct brain cells on Aβ toxicity and assembly: focus on microglia derived vesicles
SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 Conclusions Current study revealed that amyloid beta has no remarkably toxic eff ect on undiff erentiated SH-SY5Y cell line whereas viability of the neuron-like diff erentiated cell culture is signifi cantly decreased by the amyloid beta 1-42 peptide that is known to form spontaneously toxic aggregates. Keywords Amyloid beta, toxicity, SH-SY5Y

P20
Arsenic induced oxidative stress and mitochondrial dysfunction in rat brain Vijay Kumar, Chandra Prakash Maharshi Dayanand University, India SpringerPlus 2015, 4(Suppl 1):P20 The present study was undertaken to reveal the eff ects of chronic arsenic exposure (25 ppm intragastrically for 12 weeks) on mitochondrial functions and oxidative stress in male Wister rats. Chronic arsenic exposure resulted in decrease in the activities of the mitochondrial complexes. There was increased generation of ROS followed by decrease in MnSOD activity. The generation of oxidative stress was associated with increased protein oxidation and lipid peroxidation in rat brain as evident by FTIR spectra. The RT-PCR analysis of NRF 1, NRF 2 and PGC 1α revealed decrease in gene expression suggesting decreased biogenesis following chronic exposure in rat brain. Thus, the fi ndings of the present study reveal that arsenic induced decrease in mitochondrial biogenesis may be responsible for the decreased metabolic response that may be further involved in the generation of oxidative stress and neurodegeneration in rat brain. Keywords Arsenic, oxidative stress, mitochondria Ichemic-reperfusion injury induced by four vessel occlusion aff ects vulnerable hippocampal CA1 (cornus ammonis 1) pyramidal neurons. Ischemic tolerance evoked by preconditioning (IPC) represents a phenomenon of CNS adaptation to any subsequent ischemia. We refer here for the changes in the external signal receptor protein kinase pathways of the hippocampal area following by IPC. Ischemia was induced by a 4-vessels occlusion (4VO) and the rats were preconditioned by a non-injurious ischemia. Apoptotic markers were used to follow the degeneration process. Western blot and immunohistochemistry identifi ed phosphorylated extracellular signal-regulated protein kinase and p38 proteins in injured hippocampal areas. P-ERK quantifi cation increased after IPC and reached the highest level at 24 h after ischemia. Interestingly, neuroprotection induced by IPC lead to the opposite eff ect on MAPK/p38, where the level was lowest at 24 h after ischemia.
The study clearly shows that phosphorylated form takes part in complex cascades triggered by IPC in the selectively vulnerable hippocampal region. In addition, study reveals an interplay between p-ERK and p-p38 which participates in the tolerence mechanisminduced by preconditioning.
Wolfram syndrome 1 (WFS1) is a genetic disorder which has been asso ciated both with impaired early brain development and neurodegeneration. Recent studies have suggested regulation of Ca2+ homeostasis by wolframin (Wfs1) and have demonstrated the involvement of endoplasmatic reticulum (ER) stress in Wfs1 defi ciency.
Despite the ER dysfunction WFS1 shows several characteristics of pathologies related to mitochondrial dynamics. Therefore our aim was to examine the hypothesis that Wfs1 defi ciency could disturb mitochondrial dynamics contributing to impaired neuronal functioning. First we show that Wfs1 defi ciency induces mild ER stress leading to Inositol 1,4,5-Trisphosphate Receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn alters mitochondrial traffi cking, inhibits mitochondrial fusion and augments mitophagy. The overexpression of the active IP3R fragment restores IP3R-mediated Ca2+ release and corrects all perturbations in mitochondrial dynamics suggesting that these events are causally linked. We further demonstrate that suppressing the expression of two Parkinson disease-related proteins, Pink1 and Parkin, leads to reduced Wfs1 defi ciency-induced mitophagy and also to the correction of the fusion-fi ssion dynamics and mitochondrial motility. These data suggest that Wfs1 defi ciency may over-activate Pink1 and Parkin pathways. Our most important discovery is that Wfs1 defi ciency delays neuronal development and axonal growth in primary rat cortical neurons. According to our data, the link between Wfs1 defi ciency and delayed neuronal development appears to be mediated by impaired mitochondrial dynamics because suppression of the Pink1-Parkin pathway corrected also the developmental delay. Our data shed light on the mechanisms of neuronal abnormalities in WFS1 and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases. Keywords Mitochondrial dynamics, ER stress, neuronal development Melanocortin receptors (MCRs) are seven transmembrane G proteincoupled receptors that are known for their broad physiological relevance. The subtype 4 melanocortin receptor (MC4R) has emerged as a central element in the regulation of energy homeostasis, eating behavior and regulation of sexual functions. MCRs are governed by a complex dynamic homotropic regulation [1]. There is an increasing trend towards using fl uorescence anisotropy (FA) for studying the aforementioned complex receptor-ligand interactions. FA allows the characterization of ligand binding dynamics [2]. The quality of the FA assay can be greatly increased with budded baculovirus particles that display the receptors of interest on their surfaces [3]. The use of a fl uorescent ligand Cy3B-NDP-α-MSH has made it possible to study MC4Rs with higher precision and sampling rate [3]. However, this ligand has relatively slow kinetics. Modifi cation of the structure of a MC4R antagonist revealed two new reporter ligands (UTBC101 and UTBC102) for fl uorescence labeling. These new reporter ligands selectively bind to MC4Rs and exhibit improved kinetic properties. The association and dissociation rate constants of UTBC101 and UTBC102 are kon = ((2.0 ± 0.6) × 10 7 M -1 min -1 ), koff = ((4.6 ± 0.3) × 10 -3 min -1 ) and kon = ((1.9 ± 0.5) × 10 7 M -1 min -1 )), koff = ((1.0 ± 0.2) × 10 -1 min -1 ), accordingly. UTBC101 and UTBC102 enable the characterization of both labelled and non-labelled ligand binding dynamics in regard to the MC4R. UTBC102 could be especially valuable for ligand screening, because of its very high dissociation rate, which makes it possible to achieve equilibrium conditions. During early development neurons undergo complex morphological rearrangements. This active axo-dendritic growth must be supported by suffi cient cellular energy, thus, cellular energy status could be a key factor in initiating this process. However, the precise mechanisms of how cells adapt their energetic status to physiological demand and upcoming energy needs and how they are coupled to adaptive energy generation, e.g., increased ATP production by mitochondria, remain unclear. Previous studies have shown that peroxisome-proliferatoractivated receptor gamma co-activator 1 (PGC-1α) is a master regulator of mitochondrial biogenesis and cellular energy metabolism. Our aim was to examine whether neuronal growth depends on mitochondrial biogenesis and whether activation of cell growth pathways could promote mitochondrial biogenesis to support the energetic need of neuronal development. Over-expression of PGC-1α in cortical neurons increased mitochondrial density in the periphery of axonal tree and was two-fold higher in axonal tips compared to control group. Moreover, induction of mitochondrial biogenesis by PGC-1α facilitated the axonal growth and neuronal development. Activation of PGC-1α upstream kinases such as Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), transforming growth factor-β-activated kinase (TAK1) and STE-related adaptor (STRAD) increased PGC-1α transcriptional activity and mitochondrial density in axons. Most importantly, they promoted neuronal development through mitochondrial biogenesis. This study shows that mitochondrial biogenesis itself is limiting factor for axonal growth and AMPK-PGC-1α upstream pathways sensing cellular energy status could signal not only the energy defi cit but also upcoming energy need to generate new mitochondria. Keywords Mitochondrial biogenesis, PGC-1α, neuronal development GPR37 is a G protein-coupled receptor that is abundantly expressed in the brain and has been implicated in dopaminergic signaling [1]. The receptor has been identifi ed as a substrate of the ubiquitin ligase parkin and it has been linked to the autosomal recessive juvenile parkinsonism (AR-JP), an early onset familial Parkinson's disease. The loss of parkin function and defi cits in the ubiquitin proteasome pathway were proposed to cause intracellular accumulation of unfolded GPR37 leading to the AR-JP pathogenesis [2]. Here, we found that while GPR37 appears to mature normally in a heterologous expression system, the receptor is subject to proteolytic cleavage at its large N-terminal extracellular region. To study this proteolytic processing, we used stably and transiently transfected human embryonic kidney (HEK) 293 and SH-SY5Y neuroblastoma cells that express N-and C-terminally epitope-tagged human GPR37. N-terminal sequencing of the cleaved C-terminal receptor fragment revealed that GPR37 is cleaved between Glu187and Gln188 and the metabolic pulse-chase data suggests that receptor cleavage is a rapid and effi cient process. Moreover, our results indicate that the receptor N-terminus is released from the cells by shedding, a phenomenon rarely described for GPCRs. Immunofl uorescence microscopy with subcellular markers indicates that GPR37 is still in the full-length form in the trans-Golgi network but is predominantly expressed in the cleaved form at the cell surface.
Additionally, experiments with various proteinase inhibitors imply that the receptor is cleaved by a metalloproteinase. As proteolytic processing is involved in the regulation of many cell surface receptors, our fi ndings provide valuable information about GPR37 that help to understand its function and role in AR-JP at the molecular level. Keywords GPR37, AR-JP, shedding Reference

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The S100B-RAGE pathway is dysregulated in the ALS-linked neuroinfl ammatory process The comprehension of the mechanisms at the basis of astrocytic dysfunction in ALS is crucial to limit neuronal injury. Most of the toxic astrocytic eff ects highlight the role of intracellular calcium. S100B is a Ca2+-binding protein particularly present in astrocytes, behaving as a neuroinfl ammatory mediator as it is secreted by astrocytes under pathological conditions and can display paracrine toxicity by binding to RAGE. During ALS progression S100B increases in patient astrocytes and, in a rat model of the disease, S100B is augmented in "aberrant astrocytes", characterized by their neurotoxic potential. The induction of S100B in astrocytes, its release and its interaction with RAGE in motoneurons could represent a hazardous mechanism that takes place during ALS. Main objectives of this work were to investigate 1) if the expression of S100B protein and RAGE change during the course of the disease in rodent models of ALS, 2) if the expression of mutant SOD1 protein per se is suffi cient to modify S100B levels in astrocytic cultures. We observed that S100B levels and localization are modulated in the spinal cord and in the brain cortex of rat and mouse models of ALS. We also demonstrated a diff erential expression of RAGE subunits in SOD1-G93A-derived CNS tissues. Moreover, we showed that the overexpression of mutant SOD1 in astrocytic cell line is suffi cient to increase the intracellular levels and release of S100B, while it is not enough to induce a diff erential expression of RAGE. Thus, the expression of mutant SOD1 interferes with the physiological expression of S100B and RAGE and reveals that in astrocytes S100B modulation is an early event related to the mere expression of mutant SOD1, while the dysregulation of RAGE might be a phenomenon possibly requiring a more complex interplay between diff erent cell types and pathways.
Overall, these data suggest that S100B might be a toxic mediator released by astrocytes in the ALS-linked neuroinfl ammatory process. Keywords S100B, RAGE, ALS Toxic eff ects of ammonia in the brain are partly related to impaired NO production which depending on the dose/time of ammonia exposure, may be either increased or decrease. Asymmetric dimethylarginine (ADMA), is endogenous NOSs inhibitor and symmetric dimethylarginine (SDMA) is arginine (Arg) transport inhibitor (Teerlink et al., 2009). Previously we reported an increase of ADMA and SDMA concentration in brain of rats with acute liver failure (Milewski et al., 2014), but distribution of the ADMA/SDMA surplus between the particular intra and extracellular compartments has not been studied. Here, we measured the intracellular concentration of ADMA, SDMA and ADMA/ SDMA/NO precursor Arg, in cultured cortical astrocytes and rat brain endothelium cells (RBE-4) treated or not with ammonia. In RBE-4 cells not treated with ammonia the ADMA concentration was twice higher and the Arg/ADMA ratio was much lower than in astrocytes, confi rming the well documented role of ADMA in endothelial NOS inhibition (Pope et al., 2009). Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell type. Since ammonia-dependent Arg transport in astrocytes is specifi cally mediated by the heteromeric Arg/Gln transporter y+LAT2 (Zielińska et al., 2012), we speculated that this may also hold for both Arg derivatives. Indeed, silencing of the y+LAT2 gene diminished the reduction of intracellular ADMA concentration caused by ammonia treatment in astrocytes. Moreover, the y+LAT2-dependent component of ammonia-evoked Arg uptake was reduced in the presence of ADMA in the medium. The results suggest that increased ADMA (and possibly SDMA) effl ux mediated by upregulated y+LAT2 may be one of the ways in which ammonia interferes with intra-astrocytic ADMA content and, subsequently, NO synthesis. Studies are underway to establish if the same sequence of changes holds for ammonia-treated cerebral endothelial cells. and autism spectrum disorder (ASD). IL1RAPL1 protein is localized at the postsynaptic compartment of excitatory synapses and plays a role in synapse formation and stabilization. Our project was to characterize IL1RAPL1 mutants identifi ed in patients with ID and ASD and to perform a behavioral and neuronal morphology analysis on IL1RAPL1 KO mice. Specifi cally, we studied the function of three novel mutations of IL1RAPL1 gene in patients presenting ID. We found that two of the studied mutants lead to a partial loss of function of IL1RAPL1 and we pointed out the important function of the extracellular domain for the trans-synaptic PTPδ/IL1RAPL1 interaction in synaptogenesis.
We also characterized the role of IL1RAPL1 wild type and mutants in regulating dendrite morphology using in vitro neuronal cultures and IL1RAPL1 KO mice. We identifi ed, associated to hippocampal cognitive impairment an increased number of dendrite branching points in CA1 and CA2 hippocampal neurons of IL1RAPL1 KO mice. In transfected hippocampal neurons the overexpression of full length IL1RAPL1 and mutants lacking part of C-terminal domains leads to a simplifi cation of neuronal arborisation. This eff ect is abolished when we overexpressed mutants lacking part of N-terminal domains. Our results indicate the importance of IL1RAPL1 extracellular domains not only in synaptogenesis but also in dendrite development. We also concluded that for this activity PTPδ interaction is not required, suggesting that an unknown IL1RAPL1 binding partner is involved in the eff ect on dendrite morphology. Keywords Synapse, intellectual disability, autism spectrum disorder L-carnitine is essential for translocation of fatty acids for their mitochondrial oxidation, a process shown in the brain to take place in astrocytes. However, it is not synthesized in the brain and has to be transported to brain cells. Organic cation/carnitine transporter novel family member 2 -OCTN2 (SLC22A5) presence was shown in endothelial cells forming the blood-brain barrier, as well as in neurons and astrocytes. We showed that OCTN2 activity in astrocytes and its presence in plasma membrane are higher upon activation of protein kinase C (PKC), but no phosphorylation of OCTN2 was detected. Therefore, we aimed to identify OCTN2-interacting partners and to defi ne their role in transporter regulation. Mass spectrometry analysis identifi ed several cytoskeletal, ribosomal, mitochondrial, and heatshock proteins as well as the proteins involved in signaling pathway and traffi cking. We focused on protein phosphatase PP2A subunits identifi ed in OCTN2 proteome and we observed co-precipitation of OCTN2 with PP2A structural (A) and catalytic (C) subunits, as well as with two regulatory subunits -striatin and SG2NA. Activation of PKC with phorbol ester (PMA) did not change the amount of coprecipitating subunits A and C but signifi cantly lowered the amount of SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 co-precipitating SG2NA. Immunocytochemistry analysis of astrocytes showed OCTN2 co-localization with PP2A C subunit and with SG2NA in vesicular structures in the cytoplasm. PMA treatment did not change this co-localization, although an augmented amount of OCTN2 was detected in plasma membrane. We postulate that interaction of OCTN2 with PP2A arrests the transporter in cytoplasm in dephosphorylated state, while PKC activation releases SG2NA subunit from the complex, resulting in transporter traffi cking to the cell surface.
Adenosine receptors play critical roles in cellular processes and signaling and have been shown to form heteromers with diverse biochemical and/or pharmacological activities that are diff erent from those of the corresponding homomers1. However, despite extensive experimental results supporting the formation of adenosine heteromers in heterologous systems, the existence of such heteroreceptor complexes in the brain remains largely unknown, mainly because of the lack of appropriate methodology. Also no systematic study was carried out on heteromers form by adenosine receptor subtypes alone. In this study, we used several experimental approaches2 to investigate whether adenosine receptor A2A and A1 subtypes can form heteromers among themselves. In situ PLA clearly demonstrated that adenosine receptors (A2A-A2A, A2A-A1) exist as homo/heteroreceptor complexes in rat brain. In the hippocampus, A2A-A1 heteroreceptor complexes are mainly localized to the pyramidal cell layer of the Ammon´s horn and the hilo (PoDG). The complex was also observed throughout the piriformis layer. Several distinct diff erences were apparent between the distribution of the A2A-A1 heteroreceptor complexes and that of the A2A-A2A homoreceptor complex, which could have important functional consequences. Furthermore, bioluminescence resonance energy transfer analysis of adenosine A2A receptors established that they can physically interact in HEK293T27 cells, as both homomers and heteromers. In addition, static/non-dynamical human GPCR data derived from this and other interaction studies were integrated in a large scale graph, called the GPCR heterodimer network (http://www. iiia.csic.es/~ismel/GPCR-Nets/index.html), which provides global insight into adenosine heteromer connectivity, topology and organization in the context of the adenosine receptor subfamily and the GPCR network as a whole. Keywords Science, microscope, histology Reference Quantitative real-time reverse transcription-polymerase (qRT-PCR) is a widely used technique to characterize changes in gene expression in complex cellular and tissue processes, such as the cytoprotection or infl ammation. The selection of an adequate internal reference gene for accurate and consistent analysis of gene expression is of major importance. Carbon monoxide (CO) aff ects several metabolic pathways and de novo protein synthesis is crucial in the cellular responses to the gasotransmitter. Herein a selection of commonly used reference genes was analyzed to identify the most suitable internal control genes to evaluate the eff ect of the CO on gene expression in cultured cortical astrocytes. The cells were exposed to CO by incubation with CORM-A1 (CO releasing molecule A1) and four diff erent algorithms (geNorm, NormFinder, Delta Ct and BestKeeper) were applied to better evaluate the stability of eight putative reference genes. Although depression is one of the prevailing central nervous system disorders, there is still not enough knowledge about the exact alterations underlying its pathophysiology and the treatment is often ineffi cient. Our study aims at exploring a potential relationship between major depressive disorder and the purinergic P2X7 receptor (P2X7R), since in previous behavioural tests P2rx7 knock out mice displayed an antidepressant-like phenotype. Among many other known symptoms, the loss of hippocampal spine synapses is a revealing feature of the disorder. Therefore we wanted to measure the density of spine synapses in the molecular layer of the dentate gyrus in the learned helplessness paradigm and later see if genetic inhibition of P2X7R could have infl uence on this condition. Wild type C57Bl/6 and P2rx7 knock out male mice were exposed to inescapable footshocks (IES) in shuttle boxes during 2 training days and on the 3rd day learned helplessness was tested, where helpless animals usually failed to escape. Control animals were also placed in shuttle boxes but did not receive footshocks until testing. Escape failures and the latency to escape were measured to determine helpless behaviour. Electron microscopy analysis was performed to determine spine synapse density in the diff erent groups. In wild type mice both average escape latency and the number of failed escapes were signifi cantly higher in the IES treated group, however, we could not fi nd such divergency in P2rx7 knock out mice. Electron microscopy analysis confi rmed alterations in spine synapse density in the molecular layer of the dentate gyrus subsequent to learned helplessness experiments, as results indicated a signifi cant decrease in spine synapse density in wild type mice, but not in knock out animals. These fi ndings may lead to presume a role of the P2X7 receptor in this disorder and further experiments will hopefully help get a better understanding and thus more eff ective treatment of major depression. Keywords p2x7, depression, spine synapse Maintaining a neurotrophic support after peripheral nerve injury can be a key point in reducing the plastic changes which neurons, microglia, and astrocytes in the ventral horn undergo due to the loss of aff erent synaptic and neurotrophic stimuli originating from the periphery. Therefore, intrathecal administration of trophic factors or the inhibition of the mechanisms responsible for their degradation could help prevent these changes. The purpose of our study was to analyze the changes in the ventral horn produced by gliopathy determined by the suff ering of the motor neurons after peripheral nerve injury following spared nerve injury (SNI) of the sciatic nerve and how the administration of NGF or its synthetic analogue BB14, as well as the increase of endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor modulate these events. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by decreased glial aminoacid transporters GLT1 and GlyT1, and increased levels of neuronal glutamate transporter EAAC1, this maladaptive behavior of neuronal and glial EAATs is paralleled by a net increase of the Glutamate/GABA ratio as measured by HPLC analysis. These molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF. The continuous i.t. NGF infusion or of its analogue BB14, or of the generic MMPs inhibitor GM6001 reduced reactive astrogliosis and normalized the expression of neuronal and glial glutamate and glycine transporters, restoring the reduction of the Glutamate/GABA ratio but it showed to be absolutely ineff ective in modifying the reactivity of microglia, demonstrating that the two glial populations have diff erent mechanisms of modulation associate to neuronal damage. Drug addiction is regarded as one of the most important neuropsychiatric diseases affl icting our society today. A prototypic drug of abuse is cocaine which directly acts on the brain reward system. In this work we present evidence on the existence of dopamine D2R-Sigma1R heteroreceptor complexes which may play a role in the etiology of cocaine addiction. By means of BRET D2R-Sigma1R heteromers were demonstrated in HEK293 cells after receptor cotransfection. The existence of D2R-Sigma1R heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assay. Through saturation binding assay it was clearly demonstrated that in membrane preparations of HEK293 cells co-expressing D2R-Sigma1R, cocaine (1 nM) signifi cantly increased the D2R Bmax values (998 ± 40 fmol/mg protein) over D2R alone cells (664 ± 37 fmol/mg protein). This eff ect was counteracted by the Sigma1R selective antagonist PD144418 (Bmax value: 728 ± 39 fmol/mg protein). Furthermore, CREB reporter luc-gene assay indicated that the presence of D2R-Sigma1R signifi cantly reduced the potency of the D2R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the presence of a low concentration of cocaine (100nM) was found to markedly increase the quinpirole potency to inhibit the forskolin induced increase of the CREB signal in the D2R-Sigma1R cells. These dynamic changes in D2R-Sigma1R signalling produced by cocaine maybe explained by synergistic allosteric receptor-receptor interactions in the D2R-Sigma1R heteroreceptor complexes at the plasma membrane level. An antagonistic allosteric receptor-receptor interaction between the dopamine D2R and the Sigma1R in absence of cocaine instead of can explain the reduced potency of quinpirole. These dual conformational changes in the D2R-Sigma1R heteroreceptor complexes could be associated with the redistribution of both protomers from the intracellular compartment to the plasma membrane as indicated by means of confocal analysis of agonist induced D2RSigma1R traffi cking and internalization. Overall, the dynamic of D2R-Sigma1R heteroreceptor complexes may represent a mechanism that shapes neuronal and addictive responses to cocaine. Ubiquitin-proteasome system (UPS) represents important intracellular system controlling protein quality and intracellular signalling. Overload or dysfunction of UPS leads to proteasome stress that is implicated in mechanisms of neurodegeneration associated with neurodegenerative diseases, e. g. Parkinson and Alzheimer disease. Proteasome stress is also considered as the main cause of delayed neuronal death observed after transient global brain ischemia. Despite signifi cant progress made to date, the exact mechanism and selectivity of cell death induced by proteasome stress after global brain ischemia is still not completely understood. The aim of our work was to study eff ect of proteasome stress on cell viability, stress response as well as on mechanism of death of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Proteasome stress was induced by treatment of cells with bortezomib, inhibitor of proteasome 26S complex. Neuroblastoma cells were more sensitive to bortezomib than glioblastoma cells and death of neuroblastoma cells occurred signifi cantly faster than death of glioblastoma cells. With respect to cellular response, treatment of both SH-SY5Y and T98G cells with bortezomib was associated with accumulation of polyubiquitinylated protein aggregates and increased expression of HSP70. With respect to cell death mechanism, we have documented bortezomib-induced release of cytochrome c from mitochondria and activation of caspase 3 in SH-SY5Y cells. In T98G cells, bortezomib induced activation of caspase 4 but not caspase 3 and did not induce release of cytochrome c from mitochondria. Our results indicate that proteasome stress aff ects neural cells in diff erent way but does not answer the question about selectivity and delay of cell after global brain ischemia. Mitophagy is a selective degradation of mitochondria that promotes the turnover of mitochondria and prevents the accumulation of damaged organelles. Pink1 and Parkin proteins are crucial in the removal of damaged mitochondria. Mutations in the corresponding PINK1 and PARK2 genes are associated with Parkinson's disease (PD), and mitochondrial impairments are central to PD pathogenesis. Pink1 has been shown to interact with the atypical Rho GTPases Miro, the outer mitochondrial membrane proteins involved in mitochondrial traffi cking. Miro1 is degraded shortly after mitochondrial damage in a Parkin-dependent manner. α-synuclein is a major component of Lewy bodies, the characteristic cellular inclusions in PD. Mutations of α-synuclein, including A53T, are linked to familial PD. α-synuclein has been shown to bind to the mitochondrial membrane, and increased membrane-bound α-synuclein in PD contributes to the functional disturbance of mitochondria. Overexpression of A53T-mutated α-synuclein has been shown to induce mitophagy in vitro and in vivo. We hypothesized that Miro1 function is disturbed in an α-synuclein (A53T)-overexpressing model, and the aim of this study was to elucidate the involvement of Miro1 in α-synuclein-induced mitophagy.

Miro1 overexpression protects against α-synuclein-induced mitochondrial loss in neuronal culture
We have found that α-synuclein is one component of the Miro1 interactome. Moreover, co-expression of Miro1 restored mitochondrial length and density in primary neuronal culture overexpressing A53Tmutated α-synuclein. Miro1 overexpression did not change the basal mitophagy, but decreased signifi cantly α-synuclein-induced mitochondrial removal. Together, our results suggest that Miro and α-synuclein may interact in the mitophagic pathway. Keywords Mitochondria, autophagy, Parkinson's disease Introduction Alzheimer's disease (AD) is an incurable neurodegenerative disease characterized by progressive dementia. Main neuropathological features of AD include extracellular β-amyloid (Aβ)containing plaques, intraneuronal aggregates of hyperphosphorylated τ-protein and neurofi laments, microglial activation and clustering around Aβ plaques and synaptic loss. 5XFAD transgenic mice are a model of AD, exhibiting rapid brain accumulation of Aβ and microgliosis. The aim of the study was to characterize the eff ects of streptozocin (STZ)-indced diabetes on learning and memory of 5XFAD and wild-type (WT) mice in Morris water maze (MWM) at ages 2 and 6 months and on brain amyloid load.

Methods and results
Mice were injected with STZ 90 mg/kg or vehicle i.p., once daily for 2 consecutive days. MWM was performed on week 9 and histological analysis of brains of mice injected with STZ or vehicle at 2 months of age was performed on week 16. STZ treatment did not aff ect locomotion or vision of mice in MWM. At both 2 and 6 months of age, STZ treatment impaired memory of both 5XFAD and WT. Learning SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 was signifi cantly impaired in STZ-treatedc 5XFAD mice at 2 months. Surprisingly, Congo Red-positive area fraction (%) of hippocampus and amygdala was decreased 5XFAD mice treated with STZ at 2 months. Plaque diameter was not diff erent between STZ treated and vehicle treated 5XFAD mice. Conclusions Insulin defi ciency could aff ect cognition through mechanisms unrelated to Aβ metabolism. Also diff erent mechanisms may underlie eff ects of STZ treatment on learning and memory in diff erent age groups, possibly including enhancement of brain amyloid deposition and inhibition of neural cell precursor proliferation. We also hypothesize that STZ treatment might increase the soluble brain amyloid fraction in this model, since it is currently acknowledged that oligomeric (soluble) rather than fi brillar Aβ species disrupt cognitive function in AD. Keywords Amyloid, diabetes, Alzheimer's Oxidative stress is a key mechanism of cellular damage during and after severe hypoxia. Accordingly, up-regulation of expression and activity of endogenous antioxidants is an important mechanism of cellular adaptation to hypoxia. Do endogenous antioxidants take part in postconditioning-induced neuroprotective mechanisms similarly to their participation in preconditioning-induced ones? In the present work the eff ect of postconditioning by 3-trial mild hypobaric hypoxia (360 Torr, 2 h, once a day) after 1 session of severe acute hypobaric hypoxia (180 Torr, 3 h) on the expression of Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was studied by immunocytochemical analysis in areas CA1, CA2, CA3, CA4 and DG of hippocampus and in frontoparietal neocortex (NC) of male Wistar rats. Two time points were examined: 3 h after the last session of postconditioning that was 3 days after severe hypoxia and 24 h after the last session of postconditioning that was 4 days after severe hypoxia. It has been shown that postconditioning signifi cantly increases the total number of Cu, Zn-SOD-immunoreactive cells (Nt) at least in two areas of hippocampus studied (CA2 and DG) compared to non-postconditioned rats at 3 days but not at 4 days after severe hypoxia. In contrast, in NC of postconditioned rats, Nt tends to increase compared to non-postconditioned animals at 4 days but not at 3 days after severe hypoxia. The eff ect of postconditioning on the number of intensely expressing Cu, Zn-SOD neurons diff ers in various areas and at various time points. The modifi cation of Cu, Zn-SOD expression in some areas of hippocampus and NC, induced by 3-trial hypoxic postconditioning, correlates with the prevention of massive delayed apoptotic neuronal death and amelioration of functional disorders caused by severe hypoxia. Thus, Cu, Zn-SOD and other endogenous antioxidants may play, apparently, an important role in the treatment of severe hypoxia/ ischemia stroke by postconditioning in brain neurons. Keywords Postconditioning, hypoxia, Cu, Zn-superoxide dismutase

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The brain acylcarnitine profi le depends on nutritional state Baiba Svalbe 1 , Marina Makrecka-Kuka 1,2 , Eduards Sevostjanovs 1 , Maija Dambrova 1,2 , Edgars Liepinsh 1 In addition to glucose, also fatty acids are important as an energy source in the brain where about 20% of the energy is gained from mitochondrial oxidation of fatty acids. Fatty acids are transported into mitochondria in the form of acylcarnitine. It is known that acylcarnitine concentration in the heart and blood plasma varies depending on the nutritional state, fed or fasted. The aim of the present study was to compare the concentration of short-chain (C2-C5), medium-chain (C6-C12) and long-chain (C14-C18) acylcarnitines in fed and fasted states in rat brain structures: cerebellum, cortex and hypothalamus. The total concentration of acylcarnitines was not aff ected by nutrient state in none of brain structures studied, but we found that cortex contained less acylcarnitines than cerebellum and hypothalamus. The nutritional state did not aff ect the overall concentration of shortchain acylcarnitines in the brain structures.In contrary, the nutritional state signifi cantly aff ected the concentration of total medium chain acylcarnitines in the cortex: 0.33 and 0.46 nmol/g tissues in fed and fasted state, respectively. The highest concentration of medium-chain acylcarnitines was found in hypothalamus in fed and fasted state compared to cortex and cerebellum. The nutritional state signifi cantly aff ected the concentration of total long-chain acylcarnitines in the cortex: 4.92 and 5.88 nmol/g tissues in fed and fasted state, respectively. In fed state the highest concentrations of long-chain acylcarnitines was found in hypothalamus compared to cortex and cerebellum. The results demonstrate that the nutrition state aff ects brain acylcarnitine concentration in diff erent brain structures, and cortex is the most aff ected brain structure. Further studies are needed to investigate the role of changes in acylcarnitine profi le in the brain signalling pathways.
diabetes mellitus, optic nerve atrophy and deafness (DIDMOAD). WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene have disturbances in processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of wolframin protein are observed in the hippocampus, amygdala and limbic structures. The aim of this study was to investigate the eff ect of Wfs1 invalidation on the peptide processing in hippocampus of mice. Peptidomic approach was used to characterize individual peptides in the hippocampus of wild type and Wfs1 knock-out mice. We identifi ed 126 peptides in the hippocampal extracts and levels of 10 peptides were diff erent in Wfs1 and wild type mice at (p < 0.05). Largest alteration was found in the level of peptide little-LEN, which is processed (cleaved) from pro-SAAS (Pcsk1n) in prohormone convertase 2 (PC2) dependent ways. Results of this study reveal alterations of peptide processing in the hippocampus of Wfs1 defi cient mice. Keywords Peptide processing, Wfs1, pro-SAAS Development of the nervous system and its structural remodelling in the adult relies on molecules mediating the structural plasticity of neurons, e specially those involved in cell adhesion, cytoskeletal dynamics or synapse formation. Neural cell adhesion molecule (NCAM) is a membrane-associated glycoprotein that can be modifi ed by glycosylation with polysialic acid (PSA), attenuating NCAM-mediated cell interactions, thereby promoting structural plasticity [1]. It has been demonstrated that in conditions of neuroinfl ammation, NCAM can be cleaved extracellularly by metalloproteinases and other proteolytical enzymes. Prolyl endopeptidase (PREP) is a cytosolic serine protease, and alterations in PREP expression and activity have been associated with neuronal death and neuroinfl ammation [2]. Since the precise mechanisms and possible partners of PREP in neuroinfl ammation remain unclear, the aim of this study was to determine whether increased secretion and activation of PREP could impair NCAM expression and its polysialylation. SH-SY5H cell line overexpressing (o/e) PREP was used as an in vitro model for increased PREP expression and extracellular release. When measuring expression levels of NCAM and PSA-NCAM we found remarkable loss in PSA-NCAM and disrupted expression patterns of NCAM compared to wild-type cells. As matrix metalloproteinase 9 (MMP-9) has been indicated in processes regulating shedding of PSA-NCAM, MMP-9 expression level was measured. An increase in the level of the active form of MMP-9 was found, which was counteracted by a specifi c inhibitor of PREP, KYP-2047. As demonstrated, PREP might have an important role in processes involved in NCAM degradation and polysialylation, thereby inducing progression of pathologies associated with altered neuroplasticity. Keywords Neural cell adhesion molecule, prolyl endopeptidase, cellular plasticity Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads neurons to death. One key molecule of this pathway is c-Jun-N-terminal kinase (JNK), a MAP kinase, which plays both physiological and pathological roles in neurons. We have previously shown that JNK blockade by specifi c cell permeable peptide inhibitors signifi cantly reduces infarct size and neuronal death. On the other hand, JNK inhibition may have detrimental side eff ects due to blockade of its physiological function. Here we have designed a new inhibitor, which blocks MKK7, an upstream activator of JNK, which mediates its pathological activation. This inhibitor was designed taking advantage of the growth arrest and DNA damage inducible 45β (GADD45β) ability to bind MKK7, optimizing the essential domain of GADD45β and linking it with a spacer to TAT peptide sequence to penetrate cells. This inhibitor signifi cantly reduces neuronal death in two in vitro models of excitotoxic cell death, one induced by NMDA exposure and the other by oxygen glucose deprivation. We tested the MKK7 inhibitor in vivo, in two models of permanent ischemia, the one obtained by electrocoagulation, and the other by thromboembolic occlusion of the Middle Cerebral Artery. In both models, it blocked MKK7 activation and provided signifi cant protection, signifi cantly reducing the infarct size when injected 30' before the lesion. In the electrocoagulation model, we also tested the effi cacy of the peptide when injected 6h after lesion, obtaining similar protection. Therefore, we showed that it is possible to prevent JNK activation in excitotoxicity by specifi c inhibition of MKK7, preserving the physiological role of JNK driven by MKK4. Targeting MKK7 could represent a novel therapeutic strategy for several diseases involving JNK activation. Keywords Stroke, JNK, ischemia Postconditioning (PostC) is an exposure of the damaged organism to extreme factors of the mild intensity to mobilize endogenous protective mechanisms. In our laboratory method of PostC using three daily trials of mild hypobaric hypoxia (MHH) was developed. It has been found that such method of the PostC eff ectively prevents degeneration of the hippocampal and neocortical neurons in rats, subjected to severe hypoxia (SH). Present study has been aimed at examination of the impact of oxidative processes in the development of the neuroprotection acquired in the course of hypoxic PostC during fi rst three days of reoxygenation after SH in rats. The levels of thiobarbituric acid reactive substances (TBARS) and Schiff bases (SB) were used as markers of lipid peroxidation. In addition, the intensity of the apoptotic DNA fragmentation has been studied. During the three days after the SH a sustained increase of SB in the rat hippocampus was observed (700-1000% of the control value). After the fi rst PostC episode the SB levels decreased to 150% of the baseline. Subsequently this parameter did not diff er signifi cantly from the control values. TBARS showed accumulation on the fi rst day following the SH but afterwards its levels dropped to 40% of control and did not recover then to normal values. In the PostC animals, the levels of TBARS after each of three PostC episodes did not diff er from the control values.

Neuroprotection by MKK7 inhibition in excitotoxicity
These facts indicate that the PostC MHH balances the activity of proand antioxidant systems in vulnerable brain regions and promotes the eff ective utilization of components damaged by peroxidation. Fragments ladder typical for cells undergone apoptosis was obtained by the electrophoretic separation of the total DNA, extracted from a rat brain after one, two and three days after the SH. In the PostC group, the DNA fragmentation was revealed only after the fi rst PostC episode, demonstrating antiapoptotic action PostC MHH. Acknowledgements This work has been supported by RFBR (No. 13-04-00532 Extracellular matrix (ECM) forms the pericellular part of the tissue. Due to the ECM's constitution it defi nes the biophysical and biochemical properties of the tissue. In vertebrates' brain the neuronal ECM mainly consists of negatively charged chondroitin sulfate proteoglycans (CSPG), hyaluronan and glycoproteins. CSPG are supposed to be involved in cell adhesion, axonal pathfi nding and receptor binding. Therefore they are considered to play a role in neural development and plasticity as well as in several neurological and psychiatric disorders (e.g. schizophrenia and depression). One member of the CSPG family is brevican which was previously demonstrated by Blosa et al. (2013) to SpringerPlus 2015, Volume 4 Suppl 1 http://www.springerplus.com/supplements/4/S1 occur adjacent to the active zone of synapses. Accessorily, defi ciency in brevican was reported to lead to a reduction of hippocampal LTP and recruitment of local plasticity. In this study we therefore focused on the eff ect of brevican on the neuronal cell adhesion molecule NCAM and its polysialylated form PSA-NCAM. Both of them are known to be involved in the establishment and modulation of neuroplasticity which are essential for memory formation. Furthermore we analysed the expression of the prolyl endopeptidase PREP, the matrix metalloproteinase 9 (MMP9) and tissue inhibitors of MMPs (TIMP). Immunohistochemical and western blot analyses of hippocampus tissue of brevican knockout mice compared with wild type littermates did not reveal changes in the expression of MMP9, PREP, NCAM nor PSA-NCAM, but showed a reduction of TIMP1 and 3 by trend. Consequently, changes of LTP and local plasticity in brevican defi cient mice do not seem be mediated by an alteration of NCAM and PSA-NCAM, but might be supported by diff erent expression levels of TIMP 1 and 3 modulating several MMPs' activity. Keywords Extracellular matrix, brevican defi ciency, tissue inhibitor of matrix metalloproteinase