Chemical LTP induces confinement of BDNF mRNA under dendritic spines and BDNF protein accumulation inside the spines.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays a key role in neuronal development and synaptic plasticity. The discovery that BDNF mRNA can be transported in neuronal dendrites in an activity-dependent manner has suggested that its local translation may support synapse maturation and plasticity. However, a clear demonstration that BDNF mRNA is locally transported and translated at activated synapses in response to long-term potentiation (LTP) is still lacking. Here, we study the dynamics of BDNF mRNA dendritic trafficking following the induction of chemical LTP (cLTP). Dendritic transport of BDNF transcripts was analyzed using the MS2 system for mRNA visualization, and chimeric BDNF-GFP constructs were used to monitor protein synthesis in living neurons. We found that within 15 min from cLTP induction, most BDNF mRNA granules become stationary and transiently accumulate in the dendritic shaft at the base of the dendritic spines, while at 30 min they accumulate inside the spine, similar to the control CamkIIα mRNA which also increased inside the spines at 60 min post-cLTP. At 60 min but not at 15 min from cLTP induction, we observed an increase in BDNF protein levels within the spines. Taken together, these findings suggest that BDNF mRNA trafficking is arrested in the early phase of cLTP, providing a local source of mRNA for BDNF translation at the base of the spine followed by translocation of both the BDNF mRNA and protein within the spine head in the late phase of LTP.

Neuroprotective Properties of Cardoon Leaves Extracts against Neurodevelopmental Deficits in an In Vitro Model of Rett Syndrome Depend on the Extraction Method and Harvest Time.

This study investigates the bioactive properties of different extracts of cardoon leaves in rescuing neuronal development arrest in an in vitro model of Rett syndrome (RTT). Samples were obtained from plants harvested at different maturity stages and extracted with two different methodologies, namely Naviglio® and supercritical carbon dioxide (scCO2). While scCO2 extracts more hydrophobic fractions, the Naviglio® method extracts phenolic compounds and less hydrophobic components. Only the scCO2 cardoon leaves extract obtained from plants harvested in spring induced a significant rescue of neuronal atrophy in RTT neurons, while the scCO2 extract from the autumn harvest stimulated dendrite outgrowth in Wild-Type (WT) neurons. The scCO2 extracts were the richest in squalene, 3ß-taraxerol and lupeol, with concentrations in autumn harvest doubling those in spring harvest. The Naviglio® extract was rich in cynaropicrin and exerted a toxic effect at 20 µM on both WT and RTT neurons. When cynaropicrin, squalene, lupeol and 3ß-taraxerol were tested individually, no positive effect was observed, whereas a significant neurotoxicity of cynaropicrin and lupeol was evident. In conclusion, cardoon leaves extracts with high content of hydrophobic bioactive molecules and low cynaropicrin and lupeol concentrations have pharmacological potential to stimulate neuronal development in RTT and WT neurons in vitro.

Mirtazapine treatment in a young female mouse model of Rett syndrome identifies time windows for the rescue of early phenotypes.

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder, mainly caused by mutations in the MECP2 gene. Reduction in monoamine levels in RTT patients and mouse models suggested the possibility to rescue clinical phenotypes through antidepressants. Accordingly, we tested mirtazapine (MTZ), a noradrenergic and specific-serotonergic tetracyclic antidepressant (NaSSA). In previous studies, we showed high tolerability and significant positive effects of MTZ in male Mecp21m1.1Bird-knock-out mice, adult female Mecp2tm1.1Bird-heterozygous (Mecp2+/-) mice, and adult female RTT patients. However, it remained to explore MTZ efficacy in female Mecp2+/- mice at young ages. As RTT-like phenotypes in young Mecp2+/- mice have been less investigated, we carried out a behavioural characterization to analyze Mecp2+/- mice in "early adolescence" (6 weeks) and "young adulthood" (11 weeks) and identified several progressive phenotypes. Then, we evaluated the effects of either a 15- or a 30-day MTZ treatment on body weight and impaired motor behaviours in 11-week-old Mecp2+/- mice. Finally, since defective cortical development is a hallmark of RTT, we performed a histological study on the maturation of perineuronal nets (PNNs) and parvalbuminergic (PV) neurons in the primary motor cortex. The 30-day MTZ treatment was more effective than the shorter 15-day treatment, leading to the significant rescue of body weight, hindlimb clasping and motor learning in the accelerating rotarod test. Behavioural improvement was associated with normalized PV immunoreactivity levels and PNN thickness. These results support the use of MTZ as a new potential treatment for adolescent girls affected by RTT and suggest a possible mechanism of action.

Amyloid-ß Impairs Dendritic Trafficking of Golgi-Like Organelles in the Early Phase Preceding Neurite Atrophy: Rescue by Mirtazapine.

Neurite atrophy with loss of neuronal polarity is a pathological hallmark of Alzheimer's disease (AD) and other neurological disorders. While there is substantial agreement that disruption of intracellular vesicle trafficking is associated with axonal pathology in AD, comparatively less is known regarding its role in dendritic atrophy. This is a significant gap of knowledge because, unlike axons, dendrites are endowed with the complete endomembrane system comprising endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus, post-Golgi vesicles, and a recycling-degradative route. In this study, using live-imaging of pGOLT-expressing vesicles, indicative of Golgi outposts and satellites, we investigate how amyloid-ß (Aß) oligomers affect the trafficking of Golgi-like organelles in the different dendritic compartments of cultured rat hippocampal neurons. We found that short-term (4 h) treatment with Aß led to a decrease in anterograde trafficking of Golgi vesicles in dendrites of both resting and stimulated (with 50 mM KCl) neurons. We also characterized the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi dynamics in dendrites. Mirtazapine treatment (10 µM) increased the number and both anterograde and retrograde motility, reducing the percentage of static Golgi vesicles. Finally, mirtazapine reverted the neurite atrophy induced by 24 h treatment with Aß oligomers, suggesting that this drug is able to counteract the effects of Aß by improving the dendritic trafficking of Golgi-related vesicles.

Distinct role of 5'UTR sequences in dendritic trafficking of BDNF mRNA: additional mechanisms for the BDNF splice variants spatial code.

The neurotrophin Brain-derived neurotrophic factor (BDNF) is encoded by multiple bipartite transcripts. Each BDNF transcript is composed by one out of 11 alternatively spliced exons containing the 5'untranslated region (UTR), and one common exon encompassing the coding sequence (CDS) and the 3'UTR with two variants (short and long). In neurons, BDNF mRNA variants have a distinct subcellular distribution, constituting a "spatial code", with exon 1, 3, 5, 7 and 8 located in neuronal somata, exon 4 extending into proximal dendrites, and exon 2 and 6 reaching distal dendrites. We previously showed that the CDS encodes constitutive dendritic targeting signals (DTS) and that both the 3'UTR-short and the 3'UTR-long contain activity-dependent DTS. However, the role of individual 5'UTR exons in mRNA sorting remains unclear. Here, we tested the ability of each different BDNF 5'UTRs to affect the subcellular localization of the green fluorescent protein (GFP) reporter mRNA. We found that exon 2 splicing isoforms (2a, 2b, and 2c) induced a constitutive dendritic targeting of the GFP reporter mRNA towards distal dendritic segments. The other isoforms did not affect GFP-mRNA dendritic trafficking. Through a bioinformatic analysis, we identified five unique cis-elements in exon 2a, 2b, and 2c which might contribute to building a DTS. This study provides additional information on the mechanism regulating the cellular sorting of BDNF mRNA variants.

Protective role of mirtazapine in adult female Mecp2+/- mice and patients with Rett syndrome.

BACKGROUND: Rett syndrome (RTT), an X-linked neurodevelopmental rare disease mainly caused by MECP2-gene mutations, is a prototypic intellectual disability disorder. Reversibility of RTT-like phenotypes in an adult mouse model lacking the Mecp2-gene has given hope of treating the disease at any age. However, adult RTT patients still urge for new treatments. Given the relationship between RTT and monoamine deficiency, we investigated mirtazapine (MTZ), a noradrenergic and specific-serotonergic antidepressant, as a potential treatment. METHODS: Adult heterozygous-Mecp2 (HET) female mice (6-months old) were treated for 30 days with 10 mg/kg MTZ and assessed for general health, motor skills, motor learning, and anxiety. Motor cortex, somatosensory cortex, and amygdala were analyzed for parvalbumin expression. Eighty RTT adult female patients harboring a pathogenic MECP2 mutation were randomly assigned to treatment to MTZ for insomnia and mood disorders (mean age = 23.1 ± 7.5 years, range = 16-47 years; mean MTZ-treatment duration = 1.64 ± 1.0 years, range = 0.08-5.0 years). Rett clinical severity scale (RCSS) and motor behavior assessment scale (MBAS) were retrospectively analyzed. RESULTS: In HET mice, MTZ preserved motor learning from deterioration and normalized parvalbumin levels in the primary motor cortex. Moreover, MTZ rescued the aberrant open-arm preference behavior observed in HET mice in the elevated plus-maze (EPM) and normalized parvalbumin expression in the barrel cortex. Since whisker clipping also abolished the EPM-related phenotype, we propose it is due to sensory hypersensitivity. In patients, MTZ slowed disease progression or induced significant improvements for 10/16 MBAS-items of the M1 social behavior area: 4/7 items of the M2 oro-facial/respiratory area and 8/14 items of the M3 motor/physical signs area. CONCLUSIONS: This study provides the first evidence that long-term treatment of adult female heterozygous Mecp2tm1.1Bird mice and adult Rett patients with the antidepressant mirtazapine is well tolerated and that it protects from disease progression and improves motor, sensory, and behavioral symptoms.

BDNF impact on synaptic dynamics: extra or intracellular long-term release differently regulates cultured hippocampal synapses.

Brain Derived Neurotrophic Factor (BDNF) signalling contributes to the formation, maturation and plasticity of Central Nervous System (CNS) synapses. Acute exposure of cultured brain circuits to BDNF leads to up-regulation of glutamatergic neuro-transmission, by the accurate tuning of pre and post synaptic features, leading to structural and functional synaptic changes. Chronic BDNF treatment has been comparatively less investigated, besides it may represent a therapeutic option to obtain rescue of post-injury alterations of synaptic networks. In this study, we used a paradigm of BDNF long-term (4 days) incubation to assess in hippocampal neurons in culture, the ability of such a treatment to alter synapses. By patch clamp recordings we describe the augmented function of excitatory neurotransmission and we further explore by live imaging the presynaptic changes brought about by long-term BDNF. In our study, exogenous long-term BDNF exposure of post-natal neurons did not affect inhibitory neurotransmission. We further compare, by genetic manipulations of cultured neurons and BDNF release, intracellular overexpression of this neurotrophin at the same developmental age. We describe for the first-time differences in synaptic modulation by BDNF with respect to exogenous or intracellular release paradigms. Such a finding holds the potential of influencing the design of future therapeutic strategies.

In vitro modeling of dendritic atrophy in Rett syndrome: determinants for phenotypic drug screening in neurodevelopmental disorders.

Dendritic atrophy, defined as the reduction in complexity of the neuronal arborization, is a hallmark of several neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, affecting 1:10,000 girls worldwide, is mainly caused by mutations in the MECP2 gene and has no cure. We describe here an in vitro model of dendritic atrophy in Mecp2-/y mouse hippocampal primary cultures, suitable for phenotypic drug-screening. Using High-Content Imaging techniques, we systematically investigated the impact of culturing determinants on several parameters such as neuronal survival, total dendritic length, dendritic endpoints, soma size, cell clusterization, spontaneous activity. Determinants included cell-seeding density, glass or polystyrene substrates, coating with poly-Ornithine with/without Matrigel and miniaturization from 24 to 96-half surface multiwell plates. We show that in all plate-sizes at densities below 320 cells/mm2, morphological parameters remained constant while spontaneous network activity decreased according to the cell-density. Mecp2-/y neurons cultured at 160 cells/mm2 density in 96 multiwell plates, displayed significant dendritic atrophy and showed a marked increase in dendritic length following treatment with Brain-derived neurotrophic factor (BDNF) or Mirtazapine. In conclusion, we have established a phenotypic assay suitable for fast screening of hundreds of compounds, which may be extended to other neurodevelopmental diseases with dendritic atrophy.

BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo.

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for B DNF- l ive- e xon- v isualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications.

Distinct CCL2, CCL5, CCL11, CCL27, IL-17, IL-6, BDNF serum profiles correlate to different job-stress outcomes.

Chronic psychosocial stress at workplace is an important factor in the development of physical and mental illness. Objective biological measures of chronic stress are still lacking, but inflammatory response and growth factors are increasingly considered as potential stress biomarkers. Therefore, we investigated the relationship between psychophysical strain and serum levels of 48 chemokines, cytokines and growth factors measured using a multiplex immunoassay, and serum brain-derived neurotrophic factor (BDNF) measured by ELISA. Severity of psychophysical strain was scored in 115 healthy hospital workers using specific scales for anxiety, depression-like emotion, gastrointestinal or cardiac disturbances, and ergonomic dysfunction. Multivariate analysis revealed that higher anxiety scale scores were correlated with lower serum chemokine C-C motif ligand-2 (CCL2/MCP-1), chemokine C-C motif ligand-5 (CCL5/RANTES), chemokine C-C motif ligand-27 (CCL27/CTACK), chemokine C-C motif ligand-11 (CCL11/Eotaxin) and interleukin-6 (IL-6); gastrointestinal disturbances correlated with increased levels of interleukin-17 (IL-17) and reduced CCL11/Eotaxin, CCL27/CTACK and CCL2/MCP-1; while cardiac dysfunctions associate only to reduced CCL27/CTACK, and ergonomic dysfunction correlated with increased BDNF and reduced CCL11/Eotaxin and CCL5/RANTES. Thus, these 7 serum factors may provide a distinct signature for each different stress-related psychophysical outcome giving indications on individual vulnerabilities.

Signaling pathways controlling activity-dependent local translation of BDNF and their localization in dendritic arbors.

Brain-derived neurotrophic factor (BDNF) is encoded by multiple mRNA variants whose differential subcellular distribution constitutes a 'spatial code' for local translation of BDNF and selective morphological remodeling of dendrites. Here, we investigated where BDNF translation takes place and what are the signaling pathways involved. Cultured hippocampal neurons treated with KCl showed increased BDNF in the soma, proximal and distal dendrites, even in quaternary branches. This activity-dependent increase of BDNF was abolished by cycloheximide, suggesting local translation, and required activation of glutamate and Trk receptors. Our data showed that BDNF translation was regulated by multiple signaling cascades including RAS-Erk and mTOR pathways, and CaMKII-CPEB1, Aurora-A-CPEB1 and Src-ZBP1 pathways. Aurora-A, CPEB1, ZBP1 (also known as IGF2BP1), eiF4E, S6 (also known as rpS6) were present throughout the dendritic arbor. Neuronal activity increased the levels of Aurora-A, CPEB1 and ZBP1 in distal dendrites whereas those of eiF4E and S6 were unaffected. BDNF-6, the main dendritic BDNF transcript, was translated in the same subcellular domains and in response to the same pathways as total BDNF. In conclusion, we identified the signaling cascades controlling BDNF translation and we describe how the translational machinery localization is modulated in response to electrical activity.

Combined cisplatin and aurora inhibitor treatment increase neuroblastoma cell death but surviving cells overproduce BDNF.

Drug-resistance to chemotherapics in aggressive neuroblastoma (NB) is characterized by enhanced cell survival mediated by TrkB and its ligand, brain-derived neurotrophic factor (BDNF); thus reduction in BDNF levels represent a promising strategy to overcome drug-resistance, but how chemotherapics regulate BDNF is unknown. Here, cisplatin treatment in SK-N-BE neuroblastoma upregulated multiple BDNF transcripts, except exons 5 and 8 variants. Cisplatin increased BDNF mRNA and protein, and enhanced translation of a firefly reporter gene flanked by BDNF 5'UTR exons 1, 2c, 4 or 6 and 3'UTR-long. To block BDNF translation we focused on aurora kinases inhibitors which are proposed as new chemotherapeutics. NB cell survival after 24 h treatment was 43% with cisplatin, and 22% by cisplatin+aurora kinase inhibitor PHA-680632, while the aurora kinases inhibitor alone was less effective; however the combined treatment induced a paradoxical increase of BDNF in surviving cells with strong translational activation of exon6-3'UTR-long transcript, while translation of BDNF transcripts 1, 2C and 4 was suppressed. In conclusion, combined cisplatin and aurora kinase inhibitor treatment increases cell death, but induces BDNF overproduction in surviving cells through an aurora kinase-independent mechanism.

Pharmacological treatment with mirtazapine rescues cortical atrophy and respiratory deficits in MeCP2 null mice.

Loss of MeCP2 (Methyl CpG binding protein 2) in Rett syndrome (RTT) causes brain weight decrease, shrinkage of the cortex with reduced dendritic arborization, behavioral abnormalities, seizures and cardio-respiratory complications. The observed monoamine neurotransmitters reduction in RTT suggested antidepressants as a possible therapy. We treated MeCP2-null mice from postnatal-day 28 for two weeks with desipramine, already tested in RTT, or mirtazapine, an antidepressant with limited side-effects, known to promote GABA release. Mirtazapine was more effective than desipramine in restoring somatosensory cortex thickness by fully rescuing pyramidal neurons dendritic arborization and spine density. Functionally, mirtazapine treatment normalized heart rate, breath rate, anxiety levels, and eliminated the hopping behavior observed in MeCP2-null mice, leading to improved phenotypic score. These morphological and functional effects of mirtazapine were accompanied by reestablishment of the GABAergic and glutamatergic receptor activity recorded in cortex and brainstem tissues. Thus, mirtazapine can represent a new potential pharmacological treatment for the Rett syndrome.

A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays.

Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKine(TM), Promega-Emax(®), R&D-System-Quantikine(®)) and one multiplexing assay (Millipore-Milliplex(®)). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.

Dendritic targeting of short and long 3' UTR BDNF mRNA is regulated by BDNF or NT-3 and distinct sets of RNA-binding proteins.

Sorting of mRNAs in neuronal dendrites relies upon inducible transport mechanisms whose molecular bases are poorly understood. We investigated here the mechanism of inducible dendritic targeting of rat brain-derived neurotrophic factor (BDNF) mRNAs as a paradigmatic example. BDNF encodes multiple mRNAs with either short or long 3' UTR, both hypothesized to harbor inducible dendritic targeting signals. However, the mechanisms of sorting of the two 3' UTR isoforms are controversial. We found that dendritic localization of BDNF mRNAs with short 3' UTR was induced by depolarization and NT3 in vitro or by seizures in vivo and required CPEB-1, -2 and ELAV-2, -4. Dendritic targeting of long 3' UTR was induced by activity or BDNF and required CPEB-1 and the relief of soma-retention signals mediated by ELAV-1, -3, -4, and FXR proteins. Thus, long and short 3' UTRs, by using different sets of RNA-binding proteins provide a mechanism of selective targeting in response to different stimuli which may underlay distinct roles of BDNF variants in neuronal development and plasticity.

Brain-derived neurotrophic factor serum levels in genetically isolated populations: gender-specific association with anxiety disorder subtypes but not with anxiety levels or Val66Met polymorphism.

Anxiety disorders (ADs) are disabling chronic disorders with exaggerated behavioral response to threats. This study was aimed at testing the hypothesis that ADs may be associated with reduced neurotrophic activity, particularly of Brain-derived neurotrophic factor (BDNF), and determining possible effects of genetics on serum BDNF concentrations. In 672 adult subjects from six isolated villages in North-Eastern Italy with high inbreeding, we determined serum BDNF levels and identified subjects with different ADs subtypes such as Social and Specific Phobias (PHSOC, PHSP), Generalized Anxiety Disorder (GAD), and Panic Disorder (PAD). Analysis of the population as a whole or individual village showed no significant correlation between serum BDNF levels and Val66Met polymorphism and no association with anxiety levels. Stratification of subjects highlighted a significant decrease in serum BDNF in females with GAD and males with PHSP. This study indicates low heritability and absence of any impact of the Val66Met polymorphism on circulating concentrations of BDNF. Our results show that BDNF is not a general biomarker of anxiety but serum BDNF levels correlate in a gender-specific manner with ADs subtypes.

The mediating role of interpersonal conflict at work in the relationship between negative affectivity and biomarkers of stress.

This study examined the association between interpersonal conflict at work (ICW) and serum levels of three possible biomarkers of stress, namely the pro-inflammatory cytokines Interleukin 1 beta (IL-1ß), Interleukin 12 (IL-12), and Interleukin 17 (IL-17). Additionally, this study investigated the role of negative affectivity (NA) in the relationship between ICW and the pro-inflammatory cytokines. Data from 121 employees in an Italian healthcare organization were analyzed using structural equation modeling. Results showed that ICW was positively associated with IL-1ß, IL-12, and IL-17, after controlling for the effect of gender. Moreover, ICW completely mediated the relationship between NA and the pro-inflammatory cytokines IL-1ß, IL-12, and IL-17. This mediating effect was significant after controlling for the effect of gender. Overall, this study suggests that work-related stress may be associated with biomarkers of inflammation, and that negative affectivity may influence the stress process affecting the exposure to psychosocial stressors.

Expression and Dendritic Trafficking of BDNF-6 Splice Variant are Impaired in Knock-In Mice Carrying Human BDNF Val66Met Polymorphism.

BACKGROUND: The human Val66Met polymorphism in brain-derived neurotrophic factor (BDNF), a key factor in neuroplasticity, synaptic function, and cognition, has been implicated in the pathophysiology of neuropsychiatric and neurodegenerative disorders. BDNF is encoded by multiple transcripts with distinct regulation and localization, but the impact of the Val66Met polymorphism on BDNF regulation remains unclear. METHODS: In BDNF Val66Met knock-in mice, which recapitulate the phenotypic hallmarks of individuals carrying the BDNF(Met) allele, we measured expression levels, epigenetic changes at promoters, and dendritic trafficking of distinct BDNF transcripts using quantitative PCR, chromatin immunoprecipitation (ChIP), and in situ hybridization. RESULTS: BDNF-4 and BDNF-6 transcripts were reduced in BDNF(Met/Met) mice, compared with BDNF(Val/Val) mice. ChIP for acetyl-histone H3, a marker of active gene transcription, and trimethyl-histone-H3-Lys27 (H3K27me3), a marker of gene repression, showed higher H3K27me3 binding to exon 5, 6, and 8 promoters in BDNF(Met/Met). The H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) is involved in epigenetic regulation of BDNF expression, because in neuroblastoma cells BDNF expression was increased both by short interference RNA for EZH2 and incubation with 3-deazaneplanocin A, an inhibitor of EZH2. In situ hybridization for BDNF-2, BDNF-4, and BDNF-6 after pilocarpine treatment showed that BDNF-6 transcript was virtually absent from distal dendrites of the CA1 and CA3 regions in BDNF(Met/Met) mice, while no changes were found for BDNF-2 and BDNF-4. CONCLUSIONS: Impaired BDNF expression and dendritic targeting in BDNF(Met/Met) mice may contribute to reduced regulated secretion of BDNF at synapses, and may be a specific correlate of pathology in individuals carrying the Met allele.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.