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.

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.

Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests.

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.

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.

Spatial segregation of BDNF transcripts enables BDNF to differentially shape distinct dendritic compartments.

BDNF is produced from many transcripts that display distinct subcellular localization, suggesting that spatially restricted effects occur as a function of genetic and physiological regulation. Different BDNF 5' splice variants give a restricted localization in the cell body or the proximal and distal compartments of dendrites; however, the functional consequences are not known. Silencing individual endogenous transcripts or overexpressing BDNF-GFP transcripts in cultured neurons demonstrated that whereas some transcripts (1 and 4) selectively affected proximal dendrites, others (2C and 6) affected distal dendrites. Moreover, segregation of BDNF transcripts resulted in a highly selective activation of the BDNF TrkB receptor. These studies indicate that spatial segregation of BDNF transcripts enables BDNF to differentially shape distinct dendritic compartments.

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.

24-h continuous non-invasive multiparameter home monitoring of vitals in patients with Rett syndrome by an innovative wearable technology: evidence of an overlooked chronic fatigue status.

Background: Sleep is disturbed in Rett syndrome (RTT), a rare and progressive neurodevelopmental disorder primarily affecting female patients (prevalence 7.1/100,000 female patients) linked to pathogenic variations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Autonomic nervous system dysfunction with a predominance of the sympathetic nervous system (SNS) over the parasympathetic nervous system (PSNS) is reported in RTT, along with exercise fatigue and increased sudden death risk. The aim of the present study was to test the feasibility of a continuous 24 h non-invasive home monitoring of the biological vitals (biovitals) by an innovative wearable sensor device in pediatric and adolescent/adult RTT patients. Methods: A total of 10 female patients (mean age 18.3 ± 9.4 years, range 4.7-35.5 years) with typical RTT and MECP2 pathogenic variations were enrolled. Clinical severity was assessed by validated scales. Heart rate (HR), respiratory rate (RR), and skin temperature (SkT) were monitored by the YouCare Wearable Medical Device (Accyourate Group SpA, L'Aquila, Italy). The average percentage of maximum HR (HRmax%) was calculated. Heart rate variability (HRV) was expressed by consolidated time-domain and frequency-domain parameters. The HR/LF (low frequency) ratio, indicating SNS activation under dynamic exercise, was calculated. Simultaneous continuous measurement of indoor air quality variables was performed and the patients' contributions to the surrounding water vapor partial pressure [PH2O (pt)] and carbon dioxide [PCO2 (pt)] were indirectly estimated. Results: Of the 6,559.79 h of biovital recordings, 5051.03 h (77%) were valid for data interpretation. Sleep and wake hours were 9.0 ± 1.1 h and 14.9 ± 1.1 h, respectively. HRmax % [median: 71.86% (interquartile range 61.03-82%)] and HR/LF [median: 3.75 (interquartile range 3.19-5.05)] were elevated, independent from the wake-sleep cycle. The majority of HRV time- and frequency-domain parameters were significantly higher in the pediatric patients (p ≤ 0.031). The HRV HR/LF ratio was associated with phenotype severity, disease progression, clinical sleep disorder, subclinical hypoxia, and electroencephalographic observations of multifocal epileptic activity and general background slowing. Conclusion: Our findings indicate the feasibility of a continuous 24-h non-invasive home monitoring of biovital parameters in RTT. Moreover, for the first time, HRmax% and the HR/LF ratio were identified as potential objective markers of fatigue, illness severity, and disease progression.

Regulation of the spatial code for BDNF mRNA isoforms in the rat hippocampus following pilocarpine-treatment: a systematic analysis using laser microdissection and quantitative real-time PCR.

Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and plasticity and is one of those genes that generate multiple mRNAs with different alternatively spliced 5'UTRs. The functional significance of many BDNF transcripts, each producing the same protein, is emerging. On the basis of the analysis of the four most abundant brain BDNF transcripts, we recently proposed the "spatial code hypothesis of BDNF splice variants" according to which the BDNF transcripts, through their differential subcellular localization in soma or dendrites, represent a mechanism to synthesize the protein at distinct locations and produce local effects. In this study, using laser microdissection of hippocampal laminae and reverse transcription-quantitative real-time PCR (RT-qPCR), we analyzed all known BDNF mRNA variants at resting conditions or following 3 h pilocarpine-induced status epilepticus. In untreated rats, we found dendritic enrichment of BDNF transcripts encoding exons 6 and 7 in CA1; exons 1, 6, and 9a in CA3; and exons 5, 6, 7, and 8 in DG. Considering the low abundance of the other transcripts, exon 6 was the main transcript in dendrites under resting conditions. Pilocarpine treatment induced an increase of BDNF transcripts encoding exons 4 and 6 in all dendritic laminae and, additionally, of exon 2 in CA1 stratum radiatum and exons 2, 3, 9a in DG molecular layer while the other transcripts were decreased in dendrites, suggesting restriction to the soma. These results support the hypothesis of a spatial code to differentially regulate BDNF in the somatic or dendritic compartment under conditions of pilocarpine-induced status epilepticus and, furthermore, highlight the existence of subfield-specific differences.

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.

Dendritic trafficking of brain-derived neurotrophic factor mRNA: regulation by translin-dependent and -independent mechanisms.

Dendritic trafficking and translation of brain-derived neurotrophic factor (BDNF) transcripts play a key role in mediating synaptic plasticity. Recently, we demonstrated that siRNA-mediated knockdown of translin, an RNA-binding protein, impairs KCl-induced dendritic trafficking of BDNF mRNA in cultured hippocampal neurons. We have now assessed whether translin deletion impairs dendritic trafficking of BDNF mRNA in hippocampal neurons in vivo. We have found that translin and its partner protein, trax, undergo dendritic translocation in response to treatment with pilocarpine, a pro-convulsant muscarinic agonist that increases dendritic trafficking of BDNF mRNA in hippocampal neurons. In translin knockout mice, the basal level of dendritic BDNF mRNA is decreased in CA1 pyramidal neurons. However, translin deletion does not block pilocarpine's ability to increase dendritic trafficking of BDNF mRNA indicating that the requirement for translin in this process varies with the stimulus employed to drive it. Consistent with this inference, we found that dendritic trafficking of BDNF mRNA induced by bath application of recombinant BDNF in cultured hippocampal neurons, is not blocked by siRNA-mediated knockdown of translin. Taken together, these in vivo and in vitro findings indicate that dendritic trafficking of BDNF mRNA can be mediated by both translin-dependent and -independent mechanisms.

BDNF and TrkB in neuronal differentiation of Fmr1-knockout mouse.

Fragile X syndrome (FXS) is a common cause of inherited mental retardation and the best characterized form of autistic spectrum disorders. FXS is caused by the loss of functional fragile X mental retardation protein (FMRP), which leads to abnormalities in the differentiation of neural progenitor cells (NPCs) and in the development of dendritic spines and neuronal circuits. Brain-derived neurotrophic factor (BDNF) and its TrkB receptors play a central role in neuronal maturation and plasticity. We studied BDNF/TrkB actions in the absence of FMRP and show that an increase in catalytic TrkB expression in undifferentiated NPCs of Fmr1-knockout (KO) mice, a mouse model for FXS, is associated with changes in the differentiation and migration of neurons expressing TrkB in neurosphere cultures and in the developing cortex. Aberrant intracellular calcium responses to BDNF and ATP in subpopulations of differentiating NPCs combined with changes in the expression of BDNF and TrkB suggest cell subtype-specific alterations during early neuronal maturation in the absence of FMRP. Furthermore, we show that dendritic targeting of Bdnf mRNA was increased under basal conditions and further enhanced in cortical layer V and hippocampal CA1 neurons of Fmr1-KO mice by pilocarpine-induced neuronal activity represented by convulsive seizures, suggesting that BDNF/TrkB-mediated feedback mechanisms for strengthening the synapses were compromised in the absence of FMRP. Pilocarpine-induced seizures caused an accumulation of Bdnf mRNA transcripts in the most proximal segments of dendrites in cortical but not in hippocampal neurons of Fmr1-KO mice. In addition, BDNF protein levels were increased in the hippocampus but reduced in the cortex of Fmr1-KO mice in line with regional differences of synaptic plasticity in the brain of Fmr1-KO mice. Altogether, the present data suggest that alterations in the BDNF/TrkB signaling modulate brain development and impair synaptic plasticity in FXS.

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.

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.

Acute stress alters transcript expression pattern and reduces processing of proBDNF to mature BDNF in Dicentrarchus labrax.

BACKGROUND: Stress involves alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stress-specific mechanisms but also hormonal and emotional responses. For this reason we used, as an animal model of stress, a fish whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. RESULTS: We provide a comprehensive characterization of BDNF gene in the Dicentrarchus labrax and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF transcripts 1c and 1d resulted down regulated after acute stress. Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28Kda form, generated by the SKI-1 protease in mammals, is absent in fishes because the cleavage site has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%. CONCLUSION: The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man.

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 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.

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.