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We thank Dr. Michele Dipalo (Istituto Italiano di Tecnologia, Genova, Italy) for help with Raman measurements. We also thank Ilaria Dallorto, Rossana Ciancio, Diego Moruzzo, and Arta Mehilli for administrative and technical help. The project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 881603 Graphene Flagship Core3 (to F.B.), the Italian Ministry of Foreign Affairs and International Cooperation (Grant Agreement No. MAE00694702021-05-20 to F.B.), and IRCCS Ospedale Policlinico San Martino, Genova, Italy (Ricerca Corrente and “5x1000” to F.B and V.C.)., Materials and methods and additional figures on materials characterization, in vitro experiments, imaging and mass spectrometry analysis. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nano- lett.3c00377. Materials and methods and additional figures on materials characterization, in vitro experiments, imaging and mass spectrometry analysis (PDF) https://pubs.acs.org/doi/suppl/10.1021/acs.nanolett.3c00377/suppl_file/nl3c00377_si_001.pdf Supplementary File P1: Full list of quantified proteins (XLSX) Supplementary File P2: Full sets of altered proteins (XLSX) Supplementary File P3: Full sets of altered proteins (XLSX), Thanks to their biocompatibility and high cargo capability, graphene-based materials (GRMs) might represent an ideal brain delivery system. The capability of GRMs to reach the brain has mainly been investigated in vivo and has highlighted some controversy. Herein, we employed two in vitro BBB models of increasing complexity to investigate the bionano interactions with graphene oxide (GO) and few-layer graphene (FLG): a 2D murine Transwell model, followed by a 3D human multicellular assembloid, to mimic the complexity of the in vivo architecture and intercellular crosstalk. We developed specific methodologies to assess the translocation of GO and FLG in a label-free fashion and a platform applicable to any nanomaterial. Overall, our results show good biocompatibility of the two GRMs, which did not impact the integrity and functionality of the barrier. Sufficiently dispersed subpopulations of GO and FLG were actively uptaken by endothelial cells; however, the translocation was identified as a rare event., European Union's Horizon 2020 Research and Innovation Programme 881603, Ministry of Foreign Affairs and International Cooperation (Italy) MAE00694702021-05-20, IRCCS Ospedale Policlinico San Martino, Genova, Italy

MECP2 mutations cause Rett syndrome (RTT), a severe and progressive neurodevelopmental disorder mainly affecting females. Although RTT patients exhibit delayed onset of symptoms, several evidences demonstrate that MeCP2 deficiency alters early development of the brain. Indeed, during early maturation, Mecp2 null cortical neurons display widespread transcriptional changes, reduced activity, and defective morphology. It has been proposed that during brain development these elements are linked in a feed‐forward cycle where neuronal activity drives transcriptional and morphological changes that further increase network maturity. We hypothesized that the enhancement of neuronal activity during early maturation might prevent the onset of RTT‐typical molecular and cellular phenotypes. Accordingly, we show that the enhancement of excitability, obtained by adding to neuronal cultures Ampakine CX546, rescues transcription of several genes, neuronal morphology, and responsiveness to stimuli. Greater effects are achieved in response to earlier treatments. In vivo, short and early administration of CX546 to Mecp2 null mice prolongs lifespan, delays the disease progression, and rescues motor abilities and spatial memory, thus confirming the value for RTT of an early restoration of neuronal activity., Neuronal activity drives transcriptional and morphological changes that ensure maturation. Such mechanism is affected by Mecp2 absence. We show the rescue effects produced by enhancing Mecp2 null neurons activity and propose new therapeutic time windows for the treatment of Rett syndrome.

Astroglial cells are key to maintain nervous system homeostasis, as they are able to perceive a wide variety of extracellular signals and to transduce them into responses that may be protective or disruptive toward neighboring neurons through the activation of distinct signaling pathways. Neurotrophins are a family of growth factors known for their pleiotropic effects on neuronal survival, maturation and plasticity. In this work, we investigated: (i) the signaling competence of embryonic and postnatal primary cortical astrocytes exposed to brain-derived neurotrophic factor (BDNF); and (ii) the role of the scaffold protein Kinase D interacting substrate/ankyrin repeat-rich membrane spanning (Kidins220/ARMS), a transmembrane protein that mediates neurotrophin signaling in neurons, in the astrocyte response to BDNF. We found a shift from a kinase-based response in embryonic cells to a predominantly [Ca2+]i-based response in postnatal cultures associated with the decreased expression of the full-length BDNF receptor TrkB. We also found that Kidins220/ARMS contributes to the BDNF-activated intracellular signaling in astrocytes by potentiating both kinase and [Ca2+]i pathways. Finally, Kidins220/ARMS contributes to astrocytes’ homeostatic function by controlling the expression of the inwardly rectifying potassium channel (Kir) 4.1. Overall, our data contribute to the understanding of the complex role played by astrocytes within the central nervous system and identify Kidins220/ARMS as a novel actor in the increasing number of pathologies characterized by astrocytes’ dysfunctions.

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.

The synthesis of new bis-deoxy-coelenterazine (1) derivatives bearing ester protective groups (acetate, propionate and butyrate esters) was accomplished. Moreover, their hydrolytic stability at room temperature was evaluated in dimethylsulfoxide (DMSO) as solvent, using the nuclear magnetic resonance (NMR) spectra of the key products at different time intervals. The results showed an increasing hydrolysis rate according to longest aliphatic chain, with a half-life of 24 days of the more stable acetate derivative (4a). Furthermore, the analysis of the experimental data revealed the greater stability of the enol tautomer in this aprotic polar solvent. This result was confirmed by theoretical calculations using the density functional theory (DFT) approach, which gave us the opportunity to propose a detailed decomposition mechanism. Additionally, the derivatives obtained were tested by bioluminescence luciferase assays to evaluate their potential use as extracellular pH-sensitive reporter substrates of luciferase. The biological data support the idea that further structural modifications of these molecules may open promising perspectives in this field of research.

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression.

Synapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient’s autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by FcγII/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid–protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA–protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

In recent years, the scientific community has witnessed an exponential increase in the use of nanomaterials for biomedical applications. In particular, the interest of graphene and graphene-based materials has rapidly risen in the neuroscience field due to the properties of this material, such as high conductivity, transparency and flexibility. As for any new material that aims to play a role in the biomedical area, a fundamental aspect is the evaluation of its toxicity, which strongly depends on material composition, chemical functionalization and dimensions. Furthermore, a wide variety of three-dimensional scaffolds have also started to be exploited as a substrate for tissue engineering. In this application, the topography is probably the most relevant amongst the various properties of the different materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation.This chapter discusses the in vitro approaches, ranging from microscopy analysis to physiology measurements, to investigate the interaction of graphene with the central nervous system. Moreover, the in vitro use of three-dimensional scaffolds is described and commented.

In recent years, the scientific community has witnessed an exponential increase in the use of nanomaterials for biomedical applications. In particular, the interest of graphene and graphene-based materials has rapidly risen in the neuroscience field due to the properties of this material, such as high conductivity, transparency and flexibility. As for any new material that aims to play a role in the biomedical area, a fundamental aspect is the evaluation of its toxicity, which strongly depends on material composition, chemical functionalization and dimensions. Furthermore, a wide variety of three-dimensional scaffolds have also started to be exploited as a substrate for tissue engineering. In this application, the topography is probably the most relevant amongst the various properties of the different materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation. This chapter discusses the in vitro approaches, ranging from microscopy analysis to physiology measurements, to investigate the interaction of graphene with the central nervous system. Moreover, the in vitro use of three-dimensional scaffolds is described and commented.

Through their ability to modulate synaptic transmission, glial cells are key regulators of neuronal circuit formation and activity. Kidins220/ARMS (kinase-D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning) is one of the key effectors of the neurotrophin pathways in neurons where it is required for differentiation, survival, and plasticity. However, its role in glial cells remains largely unknown. Here, we show that ablation of Kidins220 in primary cultured astrocytes induced defects in calcium (Ca(2+)) signaling that were linked to altered store-operated Ca(2+) entry and strong overexpression of the transient receptor potential channel TRPV4. Moreover, Kidins220(−/−) astrocytes were more sensitive to genotoxic stress. We also show that Kidins220 expression in astrocytes is required for the establishment of proper connectivity of cocultured wild-type neurons. Altogether, our data reveal a previously unidentified role for astrocyte-expressed Kidins220 in the control of glial Ca(2+) dynamics, survival/death pathways and astrocyte–neuron communication.

The use of graphene nanomaterials (GNMs) for biomedical applications targeted to the central nervous system is exponentially increasing, although precise information on their effects on brain cells is lacking. In this work, we addressed the molecular changes induced in cortical astrocytes by few-layer graphene (FLG) and graphene oxide (GO) flakes. Our results show that exposure to FLG/GO does not affect cell viability or proliferation. However, proteomic and lipidomic analyses unveiled alterations in several cellular processes, including intracellular Ca2+ ([Ca2+]i) homeostasis and cholesterol metabolism, which were particularly intense in cells exposed to GO. Indeed, GO exposure impaired spontaneous and evoked astrocyte [Ca2+]i signals and induced a marked increase in membrane cholesterol levels. Importantly, cholesterol depletion fully rescued [Ca2+]i dynamics in GO-treated cells, indicating a causal relationship between these GO-mediated effects. Our results indicate that exposure to GNMs alters intracellular signaling in astrocytes and may impact on astrocyte-neuron interactions., Comment: This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in Small after peer review. To access the final edited and published work see https://onlinelibrary.wiley.com/doi/10.1002/smll.201900147 40 pages, 6 main figures and 1 supplementary figure

In recent years, the scientific community has witnessed an exponential increase in the use of graphene for biomedical applications. For what concerns neuroscience, the interest raised by this material is given by the fact that graphene nanosheets can be used as carriers for biomolecule delivery to the central nervous system. In this case, an important aspect is the evaluation of their toxicity, which strongly depends on flake composition, chemical functionalization and dimensions. Furthermore, graphene can be exploited as a substrate for tissue engineering. In this application, conductivity is probably the most relevant amongst the various properties of the different graphene materials, as it may allow to instruct and interrogate neural networks, as well as to drive neural growth and differentiation. This chapter discusses the engineering of graphene nanosheets able to cross the blood-brain-barrier to reach neural cells, and to achieve on-demand delivery of specific drugs. Moreover, the use of graphene to develop three-dimensional scaffolds, or as a component of hybrid composites/multi-layer organic electronics devices, is described. The need of an accurate theoretical modeling of the interface between graphene and biological material is also addressed, by describing the interaction of graphene with proteins and cell membranes at the nanoscale.

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Biopolymers are increasingly employed for neuroscience applications as scaffolds to drive and promote neural regrowth, thanks to their ability to mediate the upload and subsequent release of active molecules and drugs. Synthetic degradable polymers are characterized by different responses ranging from tunable distension or shrinkage to total dissolution, depending on the function they are designed for. In this paper we present a biocompatible microfabricated poly-e-caprolactone (PCL) scaffold for primary neuron growth and maturation that has been optimized for the in vitro controlled release of brain-derived neurotrophic factor (BDNF). We demonstrate that the designed morphology confers to these devices an enhanced drug delivery capability with respect to monolithic unstructured supports. After incubation with BDNF, micropillared PCL devices progressively release the neurotrophin over 21 days in vitro. Moreover, the bioactivity of released BDNF is confirmed using primary neuronal cultures, where it mediates a consistent activation of BDNF signaling cascades, increased synaptic density, and neuronal survival. These results provide the proof-of-principle on the fabrication process of micropatterned PCL devices, which represent a promising therapeutic option to enhance neuronal regeneration after lesion and for neural tissue engineering and prosthetics.

Graphene-based materials are the focus of intense research efforts to devise novel theranostic strategies for targeting the central nervous system. In this work, we have investigated the consequences of long-term exposure of primary rat astrocytes to pristine graphene (GR) and graphene oxide (GO) flakes. We demonstrate that GR/GO interfere with a variety of intracellular processes as a result of their internalization through the endo-lysosomal pathway. Graphene-exposed astrocytes acquire a more differentiated morphological phenotype associated with extensive cytoskeletal rearrangements. Profound functional alterations are induced by GO internalization, including the upregulation of inward-rectifying K+ channels and of Na+-dependent glutamate uptake, which are linked to the astrocyte capacity to control the extracellular homeostasis. Interestingly, GO-pretreated astrocytes promote the functional maturation of co-cultured primary neurons by inducing an increase in intrinsic excitability and in the density of GABAergic synapses. The results indicate that graphene nanomaterials profoundly affect astrocyte physiology in vitro, with consequences for neuronal network activity. This work supports the view that GO-based materials could be of great interest to address pathologies of the central nervous system associated to astrocyte dysfunctions., Comment: This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in Nano Letters. To access the final edited and published work see https://pubs.acs.org/articlesonrequest/AOR-wvjrggcBp7kC8NwEImXr

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220(-/-) mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220(-/-) hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na(+) current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na(+) current alterations reproduced the firing phenotype observed in Kidins220(-/-) neurons. These results identify Kidins220 as a novel modulator of Nav channel activity, broadening our understanding of the molecular mechanisms regulating network excitability.

Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H+ concentration. The aim of this study is to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.E2GFP, a membrane-targeted extracellular ratiometric pH indicator exquisitely sensitive to acidic shifts. By monitoring ex.E2GFP fluorescence in real time in primary cortical neurons we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or high frequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and localized to active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle protons into the cleft nor to the activity of membrane-exposed H+-vATPase, but rather to the activity of the Na+/H+-exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, underlying the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.

Several fields in neuroscience have been revolutionized by the advent of optogenetics, a technique that offers the possibility to modulate neuronal physiology in response to light stimulation. This innovative and far-reaching tool provided unprecedented spatial and temporal resolution to explore the activity of neural circuits underlying cognition and behaviour. With an exponential growth in the discovery and synthesis of new photosensitive actuators capable of modulating neuronal networks function, other fields in biology are experiencing a similar re-evolution. Here, we review the various optogenetic toolboxes developed to influence cellular physiology as well as the diverse ways in which these can be engineered to precisely modulate intracellular signalling and transcription. We also explore the processes required to successfully express and stimulate these photo-actuators in vivo before discussing how such tools can enlighten our understanding of neuronal plasticity at the systems level.

Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H

The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis.

The correct functioning of the nervous system depends on the exquisitely fine control of neuronal excitability and synaptic plasticity, which relies on an intricate network of protein-protein interactions and signaling that shapes neuronal homeostasis during development and in adulthood. In this complex scenario, Kinase D interacting substrate of 220 kDa/ankyrin repeat-rich membrane spanning (Kidins220/ARMS) acts as a multi-functional scaffold protein with preferential expression in the nervous system. Engaged in a plethora of interactions with membrane receptors, cytosolic signaling components and cytoskeletal proteins, Kidins220/ARMS is implicated in numerous cellular functions including neuronal survival, neurite outgrowth and maturation and neuronal activity, often in the context of neurotrophin (NT) signaling pathways. Recent studies have highlighted a number of cell- and context-specific roles for this protein in the control of synaptic transmission and neuronal excitability, which are at present far from being completely understood. In addition, some evidence has began to emerge, linking alterations of Kidins220 expression to the onset of various neurodegenerative diseases and neuropsychiatric disorders. In this review, we present a concise summary of our fragmentary knowledge of Kidins220/ARMS biological functions, focusing on the mechanism(s) by which it controls various aspects of neuronal activity. We have tried, where possible, to discuss the available evidence in the wider context of NT-mediated regulation, and to outline emerging roles of Kidins220/ARMS in human pathologies.

Graphene has the potential to make a very significant impact on society, with important applications in the biomedical field. The possibility to engineer graphene-based medical devices at the neuronal interface is of particular interest, making it imperative to determine the biocompatibility of graphene materials with neuronal cells. Here we conducted a comprehensive analysis of the effects of chronic and acute exposure of rat primary cortical neurons to few-layers pristine graphene (GR) and monolayer graphene oxide (GO) flakes. By combining a range of cell biology, microscopy, electrophysiology and omics approaches we characterized the graphene neuron interaction from the first steps of membrane contact and internalization to the long-term effects on cell viability, synaptic transmission and cell metabolism. GR/GO flakes are found in contact with the neuronal membrane, free in the cytoplasm and internalized through the endolysosomal pathway, with no significant impact on neuron viability. However, GO exposure selectively caused the inhibition of excitatory transmission, paralleled by a reduction in the number of excitatory synaptic contacts, and a concomitant enhancement of the inhibitory activity. This was accompanied by induction of autophagy, altered Ca2+ dynamics and by a downregulation of some of the main players in the regulation of Ca2+ homeostasis in both excitatory and inhibitory neurons. Our results show that, although graphene exposure does not impact on neuron viability, it does nevertheless have important effects on neuronal transmission and network functionality, thus warranting caution when planning to employ this material for neuro-biological applications., This document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in ACS Nano. To access the final edited and published work see https://pubs.acs.org/articlesonrequest/AOR-MGXEfuAxY43fnrHfBEuQ

Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.

Objective: To report on the identification of intrathecally synthesized immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies to synapsin, a synaptic vesicle-associated protein, in a patient with limbic encephalitis. Methods: Methods included clinical characterization, indirect immunofluorescence, immunoprecipitation, mass spectrometry, immunoblots of wild-type and synapsin I/II/III knockout mice, and cell-based assays with synapsin Ia, Ib, IIa, and IIb plasmids. Results: A 69-year-old man presented with confusion, disorientation, seizures, and left hippocampal hyperintensities on MRI. CSF examinations revealed an intrathecal IgA and IgG synthesis. Except for IgG antibodies to voltage-gated potassium channels in CSF, screening for known neuronal autoantibodies in serum and CSF was negative. However, indirect immunofluorescence using the patient9s CSF showed binding of IgA to mouse hippocampus, amygdala, and cerebellum. Immunoprecipitation with CSF IgA followed by mass spectrometry identified synapsin as autoantigenic target. Knockout tissues and cell-based assays confirmed that IgA and IgG in the patient9s CSF and serum reacted with synapsin Ia, Ib, and IIa. Calculation of antibody indices proved intrathecal synthesis of anti-synapsin IgA and IgG. The patient responded clinically to immunotherapy but developed left hippocampal atrophy. CSF IgA or IgG of the patient did not bind to live, unfixed, and nonpermeabilized mouse hippocampal neurons, compatible with synapsin being an intracellular antigen. Conclusions: This report identifies isoforms of the synaptic vesicle-associated protein synapsin as targets of intrathecally produced IgA and IgG antibodies in a patient with limbic encephalitis. Future studies should clarify the prevalence and pathogenic relevance of anti-synapsin antibodies in limbic encephalitis.

Fiala et al. report that Kidins220/ARMS is a novel interactor of the B cell antigen receptor (BCR) and its deletion impairs B cell development and B cell functioning., B cell antigen receptor (BCR) signaling is critical for B cell development and activation. Using mass spectrometry, we identified a protein kinase D–interacting substrate of 220 kD (Kidins220)/ankyrin repeat–rich membrane-spanning protein (ARMS) as a novel interaction partner of resting and stimulated BCR. Upon BCR stimulation, the interaction increases in a Src kinase–independent manner. By knocking down Kidins220 in a B cell line and generating a conditional B cell–specific Kidins220 knockout (B-KO) mouse strain, we show that Kidins220 couples the BCR to PLCγ2, Ca2+, and extracellular signal-regulated kinase (Erk) signaling. Consequently, BCR-mediated B cell activation was reduced in vitro and in vivo upon Kidins220 deletion. Furthermore, B cell development was impaired at stages where pre-BCR or BCR signaling is required. Most strikingly, λ light chain–positive B cells were reduced sixfold in the B-KO mice, genetically placing Kidins220 in the PLCγ2 pathway. Thus, our data indicate that Kidins220 positively regulates pre-BCR and BCR functioning.

The use of implants that allow chronic electrical stimulation and recording in the brain of human patients is currently limited by a series of events that cause the deterioration over time of both the electrode surface and the surrounding tissue. The main reason of failure is the tissue inflammatory reaction that eventually causes neuronal loss and glial encapsulation, resulting in a progressive increase of the electrode-electrolyte impedance. Here, we describe a new method to create bio-inspired electrodes to mimic the mechanical properties and biological composition of the host tissue. This combination has a great potential to increase the implant lifetime by reducing tissue reaction and improving electrical coupling. Our method implies coating the electrode with reprogrammed neural or glial cells encapsulated within a hydrogel layer. We chose fibrin as a hydrogel and primary hippocampal neurons or astrocytes from rat brain as cellular layer. We demonstrate that fibrin coating is highly biocompatible, forms uniform coatings of controllable thickness, does not alter the electrochemical properties of the microelectrode and allows good quality recordings. Moreover, it reduces the amount of host reactive astrocytes - over time - compared to a bare wire and is fully reabsorbed by the surrounding tissue within 7 days after implantation, avoiding the common problem of hydrogels swelling. Both astrocytes and neurons could be successfully grown onto the electrode surface within the fibrin hydrogel without altering the electrochemical properties of the microelectrode. This bio-hybrid device has therefore a good potential to improve the electrical integration at the neuron-electrode interface and support the long-term success of neural prostheses.

Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.

Background: Syn I plays a key role at presynaptic terminals. Results: Sp1 binds to the SYN1 promoter, activating its transcription. Conclusion: Sp1 is a novel regulator of SYN1 transcription, whose activity is inhibited by REST and CpG methylation. Significance: Elucidating the mechanisms underlying basal activation of neuron-specific genes is fundamental to understand brain pathologies, where transcription is often dysregulated., The development and function of the nervous system are directly dependent on a well defined pattern of gene expression. Indeed, perturbation of transcriptional activity or epigenetic modifications of chromatin can dramatically influence neuronal phenotypes. The phosphoprotein synapsin I (Syn I) plays a crucial role during axonogenesis and synaptogenesis as well as in synaptic transmission and plasticity of mature neurons. Abnormalities in SYN1 gene expression have been linked to important neuropsychiatric disorders, such as epilepsy and autism. SYN1 gene transcription is suppressed in non-neural tissues by the RE1-silencing transcription factor (REST); however, the molecular mechanisms that allow the constitutive expression of this genetic region in neurons have not been clarified yet. Herein we demonstrate that a conserved region of human and mouse SYN1 promoters contains cis-sites for the transcriptional activator Sp1 in close proximity to REST binding motifs. Through a series of functional assays, we demonstrate a physical interaction of Sp1 on the SYN1 promoter and show that REST directly inhibits Sp1-mediated transcription, resulting in SYN1 down-regulation. Upon differentiation of neuroblastoma Neuro2a cells, we observe a decrease in endogenous REST and a higher stability of Sp1 on target GC boxes, resulting in an increase of SYN1 transcription. Moreover, methylation of Sp1 cis-sites in the SYN1 promoter region could provide an additional level of transcriptional regulation. Our results introduce Sp1 as a fundamental activator of basal SYN1 gene expression, whose activity is modulated by the neural master regulator REST and CpG methylation.

In the central nervous system, most synapses show a fast mode of neurotransmitter release known as synchronous release followed by a phase of asynchronous release, which extends over tens of milliseconds to seconds. Synapsin II (SYN2) is a member of the multigene synapsin family (SYN1/2/3) of synaptic vesicle phosphoproteins that modulate synaptic transmission and plasticity, and are mutated in epileptic patients. Here we report that inhibitory synapses of the dentate gyrus of Syn II knockout mice display an upregulation of synchronous neurotransmitter release and a concomitant loss of delayed asynchronous release. Syn II promotes γ-aminobutyric acid asynchronous release in a Ca2+-dependent manner by a functional interaction with presynaptic Ca2+ channels, revealing a new role in synaptic transmission for synapsins., The arrival of action potentials at nerve terminals often leads to synchronous neurotransmitter release. Medrihan and colleagues use electrophysiology on mouse hippocampal neurons to show that the vesicle protein Synapsin II promotes GABAergic asynchronous release by interacting with calcium channels.

An increasing body of evidence suggests that several membrane receptors – in addition to activating distinct signalling cascades – also engage in substantial crosstalk with each other, thereby adjusting their signalling outcome as a function of specific input information. However, little is known about the molecular mechanisms that control their coordination and integration of downstream signalling. A protein that is likely to have a role in this process is kinase-D-interacting substrate of 220 kDa [Kidins220, also known as ankyrin repeat-rich membrane spanning (ARMS), hereafter referred to as Kidins220/ARMS]. Kidins220/ARMS is a conserved membrane protein that is preferentially expressed in the nervous system and interacts with the microtubule and actin cytoskeleton. It interacts with neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, and is a common downstream target of several trophic stimuli. Kidins220/ARMS is required for neuronal differentiation and survival, and its expression levels modulate synaptic plasticity. Kidins220/ARMS knockout mice show developmental defects mainly in the nervous and cardiovascular systems, suggesting a crucial role for this protein in modulating the cross talk between different signalling pathways. In this Commentary, we summarise existing knowledge regarding the physiological functions of Kidins220/ARMS, and highlight some interesting directions for future studies on the role of this protein in health and disease., This study was supported by research grants from: Cancer Research UK (to G.S.); the Italian Institute of Technology (to F.C. and F.B.); the Italian Ministry of University and Research [2008T4ZCNL grant number 2008T4ZCNL to F.B.]; the Compagnia di San Paolo, Torino (to F.B.); Telethon-Italy [grant number GGP09134 to F.B.] and the Spanish Ministry of Science and Innovation [grant number JCI-2008-01843 to V.N.].

Synapsins (Syn I, Syn II, and Syn III) are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes have been identified as major epilepsy susceptibility genes in humans and synapsin I/II/III triple knockout (TKO) mice are epileptic. However, excitatory and inhibitory synaptic transmission and short-term plasticity have never been analyzed in intact neuronal circuits of TKO mice. To clarify the generation and expression of the epileptic phenotype, we performed patch-clamp recordings in the CA1 region of acute hippocampal slices from 1-month-old presymptomatic and 6-month-old epileptic TKO mice and age-matched controls. We found a strong imbalance between basal glutamatergic and γ-aminobutyric acid (GABA)ergic transmission with increased evoked excitatory postsynaptic current and impaired evoked inhibitory postsynaptic current amplitude. This imbalance was accompanied by a parallel derangement of short-term plasticity paradigms, with enhanced facilitation of glutamatergic transmission in the presymptomatic phase and milder depression of inhibitory synapses in the symptomatic phase. Interestingly, a lower tonic GABA(A) current due to the impaired GABA release is responsible for the more depolarized resting potential found in TKO CA1 neurons, which makes them more susceptible to fire. All these changes preceded the appearance of epilepsy, indicating that the distinct changes in excitatory and inhibitory transmission due to the absence of Syns initiate the epileptogenic process.

The generation of 3D networks of primary neurons is a big challenge in neuroscience. Here, a novel method is presented for a 3D neuronal culture on superhydrophobic (SH) substrates. How nano-patterned SH devices stimulate neurons to build 3D networks is investigated. Scanning electron microscopy and confocal imaging show that soon after plating neurites adhere to the nanopatterned pillar sidewalls and they are subsequently pulled between pillars in a suspended position. These neurons display an enhanced survival rate compared to standard cultures and develop mature networks with physiological excitability. These findings underline the importance of using nanostructured SH surfaces for directing 3D neuronal growth, as well as for the design of biomaterials for neuronal regeneration.