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Previous work, using fluorescent adenosine receptor agonists and antagonists, has provided novel insights into the allosteric regulation of adenosine A3 (A3AR) and A1 (A1AR) receptors by allosteric ligands and receptor dimerization in single living cells [1, 2]. We have also used a fluorescent analogue of {"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 to investigate ligand binding to the human β1-adrenoceptor. This work has demonstrated that there is negative cooperativity between the two different ligand-binding conformations of the β1-adrenoceptor activated by catecholamines and {"type":"entrez-protein","attrs":{"text":"CGP12177","term_id":"877152897","term_text":"CGP12177"}}CGP12177 respectively [3]. Finally, we have used fluorescence correlation spectroscopy (FCS) to investigate ligand binding to A1AR and A3AR in small 0.2 µm2 microdomains of single living cells [4]. FCS studies with a fluorescent A3-agonist have enabled high affinity labeling of the active conformation (R*) of the receptor [4]. We have also used a fluorescent adenosine A3-antagonist ({"type":"entrez-nucleotide","attrs":{"text":"CA200645","term_id":"35234116","term_text":"CA200645"}}CA200645) to study the binding characteristics of antagonist-occupied receptor conformations (R) in membrane microdomains of single cells [5]. Investigation of the dissociation kinetics of {"type":"entrez-nucleotide","attrs":{"text":"CA200645","term_id":"35234116","term_text":"CA200645"}}CA200645 provided further support for allosteric regulation of this receptor by homodimerization [5].

Perinatal complications are a serious clinical problem, in particular hypoxic-ischemic (HI) episodes, caused by birth asphyxia or uterine and fetal blood flow 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 beneficial 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 effect. 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). 24h 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.

MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3’-untranslated region (3’-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets. The long 3’-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs. Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer’s disease (AD). Focusing on evolutionarily conserved and primate-specific miRNA controllers of cholinergic signaling (‘CholinomiRs’), we find modified CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson’s disease, with treatment-related differences in their levels and prominent impact on the cognitive and anti-inflammatory consequences of cholinergic signals. Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain. Furthermore, we found that interruption of AChE mRNA’s interaction with the primate-specific CholinomiR-608 in carriers of a SNP in the AChE’s miR-608 binding site induces domino-like effects that reduce the levels of many other miR-608 targets. Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially affecting the responsiveness to AD’s anti-AChE therapeutics, and show elevated trait anxiety, inflammation and hypertension. Non-coding regions affecting miRNA-target interactions in neurodegenerative brains thus merit special attention.

Proximal Spinal Muscular Atrophy (SMA) is caused by an insufficient supply of the Survival of Motor Neurons (SMN) protein. It is characterized by the selective degradation of the alpha motor neurons in the spinal cord. Analysis of the mouse model, which faithfully mimics the SMN insufficiency and the motor neuron phenotype, shows that motor neuron degeneration starts in the axons, specifically 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 insufficient supply of the SMN protein affects 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 find that one such LSm protein is significantly depleted in the SMA mouse model before the onset of the disease, possibly indicating a causal involvement.

The major focus of ALS research has recently moved to RNA control of motor neuron functions, as most of the newly identified 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 dys-metabolism is likely to represent a central issue in ALS pathogenesis. Yet, whether a specific step of RNA processing is particularly affected in ALS motor neurons is unclear. We have recently obtained evidence that a prominent effect 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 effects and their potential relevance in ALS pathogenesis.

The effect of galanin is mediated through three GPCR subtypes, GalR1-3. The limited number of specific ligands to the galanin receptor subtypes has hindered the understanding of the individual effects of each receptor subtype. This review summarizes the current data of the importance of the galanin receptor subtypes and receptor subtype specific agonists and antagonists and their involvement in different biological and pathological functions.

We analyzed the pathological consequences of abnormal Wnt/β-catenin 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 cell-autonomous, 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 sulfide and sulindac sulfone, which attenuate β-catenin transcription activity, significantly reduce the number and dimension of vascular lesions in the central nervous system of mice with endothelial cell CCM3 knockout. These NSAIDs thus represent pharmacological tools for inhibition of the formation of vascular lesions, particularly with a view to patients affected by the genetic variant of CCM, who continue to develop new malformations over time.

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 differentiating 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 fine-tuned regulation of microtubule dynamics and stability during neuronal differentiation.

Ginkgo biloba extract is a neuroactive agent that is widely used for correction of age-associated impairment of memory, attention deficit and other cognitive functions. It has been shown that ginkgolides and bilobalides, Ginkgo biloba extract components, are potent blockers of glycine receptors (Kondratskaya et al., 2002, Hawthorne et al., 2006). However, the effect 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 different subunits in CHO cells we have shown that glycine receptors (GlyRs) are modulated by ginkgolic acid in a subunit-specific 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 significant effect 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-specific effect of ginkgolic acid we have focused on possible interaction sites for this compound inside different 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 effect, 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 effect of ginkgolic acid from inhibition to potentiation.

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.

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 affects 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 confirmed 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 effective 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 pro-epileptogenic inflammatory 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.

G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor and the dopamine D3 receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved FRET studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI and VII, as well as intracellular helix VIII, to the overall organization. Molecular modelling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally-resolved, multi-photon FRET. Modelling studies suggest, furthermore, an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

Post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (PSA) is crucial for nervous system development and brain plasticity. PSA attachment is catalyzed by the two polysialyltransferases, ST8SiaII and ST8SiaIV. ST8SiaII dominates during embryonic and early postnatal development while ST8SiaIV is the prevailing enzyme of the juvenile and adult brain. A growing body of evidence links aberrant levels of NCAM and PSA to neuropsychiatric disorders, including schizophrenia. To investigate whether polysialyltransferase deficiency might cause a schizophrenia-like phenotype, ST8SiaII-/- mice, ST8SiaIV-/- mice and their wild-type littermates were assessed neuroanatomically and subjected to tests of cognition and sensory functions. ST8SiaII-/- but not ST8SiaIV-/- mice displayed enlarged lateral ventricles and a size reduction of the thalamus accompanied by a smaller internal capsule and a highly disorganized pattern of thalamocortical and corticothalamic fibers. Loss of ST8SiaII was associated with reduced phosphorylation of fibroblast growth factor receptor 1 in the frontal cortex of newborn mice and retarded neuronal differentiation of newly generated cells in the dentate gyrus of adults. ST8SiaII-/- and ST8SiaIV-/- mice were both impaired in short- and long-term recognition memory, but only ST8SiaII-/-mice displayed impaired working memory and a deficit in prepulse inhibition, which could be attenuated by clozapine treatment. Furthermore, ST8SiaII-/- mice exhibited anhedonic behavior and increased sensitivity to amphetamine-induced hyperlocomotion. These data reveal that reduced polysialylation in ST8SiaII-/- mice leads to pathological brain development and schizophrenia-like behavior. We therefore propose that ST8SiaII deficiency has the potential to cause a neurodevelopmental predisposition to schizophrenia.

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 effects of orexins on survival of cultured neurons from the rat cerebral cortex. Quantitative real-time PCR revealed the presence of OX1R and OX2R in cortical neurons. Orexins and [Ala11-D-Leu15]orexin B (a selective agonist of OX2R), used at 0.001-1 microM concentrations, markedly increased neuronal cells viability, an effect associated with an attenuation of caspase-3 activity. Comparable potency of the three peptides suggests a predominant role of OX2R in the studied phenomenon. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons from oxidative stress. The pro-survival effect of orexins was blocked by U0126 and 10-DEBC, inhibitors of MEKK and Akt, respectively. In addition, orexins A and B stimulated Akt and ERK1/2 in cortical neurons in a time- and concentration-dependent manner. It is suggested that both Akt and ERK1/2 play an important role in the pro-survival effects of orexins in neurons.

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 fiber. 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 specific 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 beneficial in regeneration of the peripheral nervous tissue.

The nucleus accumbens has long been a major target for studies on the rewarding effects 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), differentially 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 affected. 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.

Autism spectrum disorders (ASD) are heterogeneous, heritable neurodevelopmental conditions, affecting ~0.5% of the population across cultures, with a ~4:1 male/female ratio. ASD are characterized by social interaction and communication deficits, restricted interests, repetitive behaviors, and reduced cognitive flexibility. 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 identified ‘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.

Diabetic retinopathy (DR) is a neovascular, inflammatory and neurodegenerative disease. The neovascular component is treatable but no therapeutic agents are available for the other two components. Early changes in the diabetic retina include neuronal death of amacrine and retinal ganglion cells (RGC)(Barber et al., J Clin Invest,1998), and elevated levels of inflammatory mediators (Yoshimura et al., Plos One,2009). Nerve Growth factor (NGF) receptors, namely TrkA and p75NTR, are expressed in RGC. The TrkA receptor activates prosurvival, while p75NTR activates inflammatory and apoptotic pathways (Mysona et al., Expert Rev Ophthalmol,2014). Dehydroepiandrosterone (DHEA) binds to both receptors, mimicking NGF. It affords anti-apoptotic, neuroprotective and anti-inflammatory effects in the retina (Kokona et al., Neuro-pharmacol, 2012, Straub et al., J Clin Endocrinol, 1998). The therapeutic use of DHEA is restricted due to its metabolic products. The main objective of this study was to investigate and compare the neuroprotective and anti-inflammatory effects of DHEA and its spiro-epoxy 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, 50mg/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 effective. 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-inflammatory cytokines (IL10). These results suggest that NGF TrkA receptor is involved in the neuroprotective and anti-inflammatory effects of BNN27 and is a valuable target via which BNN27 could afford efficacious therapeutics for the treatment of DR.

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 final 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 differentiate 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 differentiation. 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 identified 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 differentiation as will illustrate my presentation.

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. Efficient protein quality control (PQC) is required for the maintenance of physiological and soluble protein pool in affected motoneuron. The balance between autophagy and ubiquitin-proteasome system (UPS) prevents protein aggregation and increases degradation of SBMA and ALS misfolded proteins. Dynein binds the co-chaperone 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 flux. 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 filter retardation assay (FRA). Moreover, dynein silencing drastically altered autophagic markers localization (LC3 and p62) by immunofluorescence. 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.

A number of different 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 effects and their potential relevance in the pathogenesis of ALS.

G protein–coupled receptors (GPCRs) are intensely studied as drug targets and for their role in signaling. High-resolution crystal structures of GPCRs capturing different 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 first 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 different 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 specific 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 sufficiently 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.

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 defined 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 effects 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 different effects on RNA metabolism caused by the TDP43 loss and gain of function.

Neurogenic inflammatory components mediated by peptidergic sensory nerves have a crucial impact on the symptoms of inflammatory diseases. Galanin is a regulatory sensory neuropeptide, which was shown to attenuate neurogenic inflammation, but our current understanding about its endogenous targets, and physiologic importance is incomplete. Among the endogenous receptors of galanin (GAL1, GAL2, GAL3) we found GAL3 to be the most abundantly expressed on the vasculature and GAL2 on different types of immune cells including polymorphonuclear neutrophils and natural killer cells. Therefore, we evaluated if galanin exerts direct or indirect effects 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 proinflammatory cytokines. Since there are only scarce in vivo data concerning the role of GAL3 in inflammatory 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-deficient 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 significantly different. In contrast, disease severity, vascular, and immune components were not affected in allergic contact dermatitis in GAL3 knockouts in comparison with wildtypes. Our findings suggest GAL3 activation as a substantial anti-inflammatory 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.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of midbrain dopamine (mDA) neurons. Clinical trials using human embryonic midbrain tissue for transplantation have provided proof of concept that cell replacement therapy (CRT) can lead to not only symptomatic relief, but also changes in the course of disease and 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 differentiated into mDA neurons capable of inducing functional recovery in animal models of PD. However, protocols for mDA differentiation 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]. Different 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 differentiation of postmitotic mDA neuroblasts [2]. We are currently applying this knowledge to improve protocols for the differentiation of human stem cells into mDA neurons suitable for transplantation and functional recovery in animal models for PD [3].

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse occurrence and functions. One of the most well-known effects of PACAP is its strong neuroprotective effect. 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 effectively counteracts the dopamine-decreasing effect of rotenone, a mitochondrial neurotoxin. Similarly, in a 6-hydroxydopamine-induced rat model of Parkinson’s disease, we found that PACAP effectively increases dopamine levels. Furthermore, our proteomics analysis shows that PACAP treatment also counteracts the 6-OHDA-induced decrease in PARK-7 protein, effective against oxidative stress. Studying the role of endogenous PACAP, we found that PACAP-deficient 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-deficient mice. The described changes may provide further explanation for the neuroprotective potency of PACAP.

WNT signaling in microglia and the glioma microenvironment Gunnar Schulte WNT signaling is important during embryonic development and organogenesis having specific 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 inflammatory 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 differential proinflammatory modulation of the cells. Interestingly, the net effect of WNT stimulation on the inflammatory potential of mouse microglia is context dependent. While WNTs increase inflammatory markers when giving to microglia alone, they are able to act in an anti-inflammatory manner when microglia are activated by prestimulation with lipopolysaccharides. Our findings 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 inflammatory signature of the tumor as well as a substantial invasion of microglia.

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 different 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 effective agents may still be a promising approach in clinical neurooncology.

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 offer protection in permanent ischemic stroke or what mechanism underlies the effect. 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 effects of CO, we exposed wildtype and Nrf2-/- mice to 250ppm CO or control air for 18h immediately after permanent middle cerebral artery occlusion. Infarct volume and neurological deficits 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 7d. Additionally, 18h CO treatment led to Nrf2 dissociation from Keap1, nuclear translocation, increased binding activity of Nrf2 to HO1 antioxidant response elements, and elevated HO1 expression 6-48h 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 beneficial effect would be at least partially mediated by activation of the endogenous Nrf2 pathway.

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 significantly decreasing in the serum of Alzheimer’s disease (AD) patients. Our most recent results revealed sex-related learning/memory differences in mice, reflecting hippocampal expression changes in ADNP and ADNP-controlled 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 significant 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 neurofibrillary 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 ASD-like phenotype in mice and we have shown that hippocampal eIF4E expression was specifically 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.

Myelin is a membrane characterized by high lipid content to facilitate impulse propagation. Changes in myelin fatty acid (FA) composition have been associated with peripheral neuropathy [1], but the specific 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 fibers, 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 findings reveal the importance of FA synthesis to sustain peripheral nerve structure and function.

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 inflammation and oxidative stress play a critical role. Our team is studying the relevance of the transcription factor NRF2 (Nuclear factor (erythroid-derived 2)-like 2), a master regulator of oxidant and inflammatory defense, as a new therapeutic target in PD. The pro-inflammatory activation of microglia and astroglia in response to LPS, MPTP and human α-synuclein over-expression is exacerbated in Nrf2-deficient 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 fulfill criteria of safety, tolerability and efficacy. The use of repurposing drugs, such as dimethyl fumarate used nowadays for treatment of remitting relapsing multiple sclerosis, may provide excellent candidates.

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 different regions of the brain. Differentiation from the early progenitor to the mature multiprocessed oligodendrocyte is characterized by different 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 specifically prominent in the branching points of the cellular processes. Downregulation of tau impairs cell differentiation and the process of early myelination. In neurodegenerative diseases collectively termed tauopathies, fibrillary 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.

Background Alzheimer’s Disease (AD) Research has long been focusing on Ab-containing amyloid plaque deposition and tau-containing tangles. However, recent clinical trial outcomes by Ab-lowering approaches have been disappointing. As an alternative approach, the present study focuses on mechanisms that prevent neuronal loss, another pathological hallmark of AD. Specifically, we have uncovered an unexpected role of glial cell line-derived neurotrophic factor (GDNF) in AD.

Background Presenilin (PS) is the catalytic subunit of g-secretase and mutations in this protein cause familial Alzheimer’s Disease (FAD). Moreover, g-secretase has emerged as an appealing drug target for Alzheimer’s disease (AD) and cancer due to its central role in the generation of Ab peptides and the regulation of Notch signaling. g-Secretase is composed of at least four subunits: PS, Nicastrin, Aph1 and Pen2; with a total of 19 putative transmembrane domains.

The role of nitric oxide in cellular signaling in the past three decades has become one of the most rapidly growing areas in biology. Nitric oxide is a gas and a free radical with an unshared electron that can regulate an ever-growing list of biological processes. Nitric oxide is formed from L-arginine by a family of enzymes called nitric oxide synthases. These enzymes have a complex requirement for a number of cofactors and regulators including NADPH, tetrahydrobioterin, flavins, calmodulin and heme. The enzymes are present in most cells and tissues. In many instances, nitric oxide mediates its biological effects by activating the soluble isoform of guanylyl cyclase and increasing cyclic GMP synthesis from GTP. Cyclic GMP, in turn, can activate cyclic GMP-dependent protein kinase (PKG) and can cause smooth muscles and blood vessels to relax, decrease platelet aggregation, alter neuron function, etc. These effects can decrease blood pressure, increase blood flow to tissues, alter memory and behavior, decrease blood clotting, etc. The list of effects of nitric oxide that are independent of cyclic GMP formation is also growing at a rapid rate. For example, nitric oxide can interact with transition metals such as iron, thiol groups, other free radicals, oxygen, superoxide anion, unsaturated fatty acids, and other molecules. Some of these reactions result in the oxidation of nitric oxide to nitrite and nitrate to terminate the effect, while other reactions can lead to altered protein structure function and/or catalytic capacity. These effects probably regulate bacterial infections, inflammation of tissues, tumor growth, and other disorders. These diverse effects of nitric oxide that are cyclic GMP dependent or independent can alter and regulate numerous important physiological events in cell regulation and function. Nitric oxide can function as an intracellular messenger, an antacoid, a paracrine substance, a neurotransmitter, or as a hormone that can be carried to distant sites for effects. Thus, it is a unique molecule with an array of signaling functions. However, with any messenger molecule, there can be too little or too much of the substance, resulting in pathological events. Some of the methods to regulate either nitric oxide formation metabolism, or function have been in clinical use for more than a century, as with the use of organic nitrates and nitroglycerin in angina pectoris that was initiated in the 1870s. Inhalation of low concentrations of nitric oxide can be beneficial in premature infants with pulmonary hypertension and increase survival rates. Ongoing clinical trials with nitric oxide synthase inhibitors and nitric oxide scavengers are examining the effects of these agents in septic shock, hypotension with dialysis, inflammatory disorders, cancer therapy, etc. Recognition of additional molecular targets in the areas of nitric oxide and cyclic GMP research will continue to promote drug discovery and development programs in the field. Current and future research will undoubtedly expand the clinician’s therapeutic armamentarium to manage a number of important diseases by perturbing nitric oxide formation and metabolism. Such promise and expectations have obviously fueled the interests in nitric oxide research for a growing list of potential therapeutic applications. There have been and will continue to be many opportunities from nitric oxide and cyclic GMP march to develop novel and important therapeutic agents. There are presently more than 80,000 publications in the area of nitric oxide research. The lecture will discuss our discovery of the first biological effects of nitric oxide and how the field has evolved since our original reports in 1977. The possible utility of this signaling pathway to facilitate novel drug development and the creation of numerous projects in the Pharmaceutical and biotechnology industrials will also be discussed.

Results Genetic studies suggest that PINK1 and parkin function in a common genetic pathway with PINK1 positively regulating parkin. The PINK1/parkin pathway regulates mitochondrial dynamics by positively regulating drp1 and negatively regulating mitofusin. Mammalian cell-based studies suggest that the PINK1/parkin may regulate mitochondrial quality control via mitophagy. How autophagy intersects with mitochondrial dynamics in vivo will be discussed.

Background Amyloid b (Ab) peptides, the primary constituents of senile plaques and a hallmark in Alzheimer’s disease pathology, are generated through the sequential cleavage of amyloid precursor protein (APP) by BACE1 and g-secretase. Evidence is accumulating that the early endosome constitutes a major compartment for APP processing; however, the mechanisms of how BACE1 encounters APP are largely unknown.

Background ApoER2, a member of the LDL receptor family, binds to reelin, triggering a signaling pathway that regulates neuronal migration and positioning in the developing brain and dendritic ramification. In the adult, reelin participates in neuronal survival, synaptic plasticity and neurogenesis. Our interest has been to study cell biology aspects of ApoER2, including the molecular and cellular determinants of its endocytic trafficking, how these features regulate the receptor signaling properties and ApoER2 participation in neurodegenerative conditions, such as Alzheimer’s Disease and in the Niemann Pick Type C disease (NPC), a lipid storage and neurodegenerative disorder caused by genetic mutations in npc1 gene that causes cholesterol accumulation in late endosome/lysosomes. In NPC there are alterations in the trafficking and signaling of some receptors however this remains unexplored for ApoER2. As APP, ApoER2 is also processed by proteases and these events are regulated by ligand binding and some cytosolic proteins such as Dab1. By yeast two-hybrid it was found that ApoER2 binds to SNX17, a cytosolic protein that regulates trafficking of membrane proteins in the early endosomal pathway. However SNX17 role in ApoER2 trafficking and signaling has not been evaluated. Methods Endocytic trafficking (internalization and/or recycling) and processing of ApoER2 was evaluated in cell lines transfected with different forms of the receptor to determine the role of 1) its NPxY motif and proline rich insert present in the cytoplasmic domain, 2) Lipid rafts association, 3) dominant negative expression or silencing of adaptor proteins such as Dab2 and SNX17 4) Intracellular cholesterol accumulation. ApoER2 signaling in different conditions (silencing of SNX17; late endosome/lysosome cholesterol accumulation mimicking the NPC condition) were performed in primary cultured neurons activated by reelin, analyzing dendritic branching and determinations of signaling proteins by Western blot. Results ApoER2 internalization is clathrin mediated, determined by its NPXY motif and the adaptor protein Dab2. Lipid rafts association or the presence of the proline-rich insert do not modulate the receptor internalization. The NPXY motif is also responsible of the recycling of ApoER2 from the early endosome, which is mediated by SNX17. Abolishing SNX17 affects ApoER2 signaling in neurons and the receptor processing. ApoER2 processing is stimulated by its own ligand reelin but also by activation of other signaling receptors. In cellular conditions miming NPC there was a decrease in ApoER2 surface levels, explained by a more efficient internalization and less recycling. NPC neurons, exhibited less dendritic ramification while the number of apoptotic cells was increased. Both effects were partial but significantly rescued when exogenous Reelin was added. We found relevant differences in the expression of ApoER2 and its ligand reelin in the brains of the NPC KO mice. Conclusions

Embryonic stem cells (ESCs) transplantation is a potential therapeutic approach for Parkinson’s disease. However, the key problems the therapy is facing are the efficiency of differentiation into dopaminergic (DA) neurons and the low survival of the transplanted DA neurons. In the present study, mouse ESC were effectively differentiated into DA neurons by serum free method and were transplanted into 6-OHDA lesioned striatum of PD rats. We found reduced viability of DA neurons after graft, being accompanied by activated microglia and high levels of TNF-α and iNOS. This suggested that inflammation might be an underlying mechanism for decreased cells viability. In the following in vitro assay, selenite, the source of essential micronutrient selenium, was tested to inhibit inflammatory activation of BV2 microglia cells. Furthermore, the anti-inflammatory effects of selenite in animals after cells transplantation were investigated. In PD rats treated by selenite, microglia activation after transplantation was inhibited in the graft niche, and the levels of TNF-α and iNOS were effectively abated nearly by 30% and 50%. The viability of implanted DA neurons was also remarkably improved after selenite treatment, with favored behavior recovery of PD rats. Therefore, selenite might benefit embryonic stem cells therapy in Parkinson’s disease through inhibiting inflammation.

Background Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Results To explore the pathogenic properties of mutant forms of TDP-43, we generated and characterized two mouse lines expressing human TDP-43 carrying the M337V mutation (hTDP-43M337V). We found hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, intranuclear and cytoplasmic phosphorylated TDP43 aggregates were frequently detected, and the levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased. Overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP-43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice. Conclusion While overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity.

Infectious diseases exert a major burden of disease in developing countries with 99% of the global burden of infectious diseases, as measured by DALYs, in low and middle income countries. While better use of existing vaccines would make an appreciable difference, the greatest burden is caused by diseases for which we currently have no vaccines. The picture, especially in children, is dominated by diarrheal and respiratory diseases. Paradoxically these diseases have relatively low priority for funding in absolute terms, and especially in relationship to the burden of disease. Thus, new vaccines for these neglected diseases need both innovative scientific solutions and innovative development schemes involving scientific institutes, public financing and industrial input. The industrial input is critical: not only will vaccine manufacture require an industrial partner, but the knowledge to efficiently undertake the technical and clinical development leading to vaccine production largely resides in industry. A potentially important development in this area has been the recent formation of Industry Linked Vaccine Institutes: For example, the Novartis Vaccines Institute for Global Health and the Hilleman Laboratories. These are an important conduit for applying industrial know how for developing commercial vaccines to the pressing need for vaccines for neglected diseases of developing countries.

The discovery of HIV in 1983 originated from a collective adventure, which mobilized clinicians, researchers and patients altogether. This collaboration was crucial to rapidly expand the knowledge of the virus and develop the first diagnostic tests and antiretroviral therapy (ART). More than 25 years after the discovery of the etiological agent responsible for AIDS, research priorities still remain care, treatment and prevention with the major objective of developing a preventive vaccine. Today, we have gained significant insight into the virus pathogenesis. The evolution and progression of the disease caused by HIV is closely linked to a number of determinants of the virus itself and the host. We also know today that, very early after exposure to the virus, a massive depletion of CCR5+ CD4+ T memory cells associated with microbial translocation occurs in the gastrointestinal tract of HIV infected patients. HIV infection is clearly inducing an inflammation and a generalized and persistent T cell activation, which may play a role in the persistence of HIV infection, resulting from the establishment of permanent reservoirs into host cells and in different host compartments. The reduction of the size of these reservoirs is representing one of the main challenges for the development of future therapeutic strategies. Among other challenges in therapy, we also need to better understand the mechanisms leading to the severe complications (cardiovascular diseases, accelerated aging, cancer...) observed in some patients on long-term ART. Again, whether the inflammatory response is contributing or not to these complications remains an opened question. The early acute phase of HIV infection appears therefore to be crucial in determining disease progression. Given the importance of the innate immune responses in this very early phase following infection and in driving adaptive immunity, further research on innate immunity in HIV infection are certainly among priorities for elaborating future therapeutic and vaccine strategies. New technologies are today available to address all these scientific challenges. But they will only be overcome with a multidisciplinary and translational research for the global benefit of humanity.