Biomimetic graphene for enhanced interaction with the external membrane of astrocytes

© The Royal Society of Chemistry. Graphene and graphene substrates display huge potential as material interfaces for devices and biomedical tools targeting the modulation or recovery of brain functionality. However, to be considered reliable neural interfaces, graphene-derived substrates should properly interact with astrocytes, favoring their growth and avoiding adverse gliotic reactions. Indeed, astrocytes are the most abundant cells in the human brain and they have a crucial physiological role to maintain its homeostasis and modulate synaptic transmission. In this work, we describe a new strategy based on the chemical modification of graphene oxide (GO) with a synthetic phospholipid (PL) to improve interaction of GO with brain astroglial cells. The PL moieties were grafted on GO sheets through polymeric brushes obtained by atom-transfer radical-polymerization (ATRP) between acryloyl-modified PL and GO nanosheets modified with a bromide initiator. The adhesion of primary rat cortical astrocytes on GO-PL substrates increased by about three times with respect to that on glass substrates coated with standard adhesion agents (i.e. poly-d-lysine, PDL) as well as with respect to that on non-functionalized GO. Moreover, we show that astrocytes seeded on GO-PL did not display significant gliotic reactivity, indicating that the material interface did not cause a detrimental inflammatory reaction when interacting with astroglial cells. Our results indicate that the reported biomimetic approach could be applied to neural prosthesis to improve cell colonization and avoid glial scar formation in brain implants. Additionally, improved adhesion could be extremely relevant in devices targeting neural cell sensing/modulation of physiological activity

Fabrication of biocompatible free-standing nanopatterned films for primary neuronal cultures

Devising and constructing biocompatible devices for nervous system regeneration is an extremely challenging task. Besides tackling the issue of biocompatibility, biomaterials for neuroscience applications should mimic the complex environment of the extracellular matrix, which in vivo provides neurons with a series of cues and signals to guide cells towards their appropriate targets. In this work, a novel nanopatterned biocompatible poly-ε-caprolactone (PCL) film is realized to assist the attachment and growth of primary hippocampal neurons. Costly and time-consuming processes can be avoided using plasma-surface nanotexturing obtained by a mixed gas SF6/Ar at -5 °C. The intrinsic composition and line topography of nanopatterned PCL ensure healthy development of the neuronal network, as shown by confocal microscopy, by analysing the expression of a range of neuronal markers typical of mature cultures, as well as by scanning electron microscopy. In addition, we show that surface nanopatterning improves differentiation of neurons compared to flat PCL films, while no neural growth was observed on either flat or nanopatterned substrates in the absence of a poly-d-lysine coating. Thus, we successfully optimized a nanofabrication protocol to obtain nanostructured PCL layers endowed with several mechanical and structural characteristics that make them a promising, versatile tool for future tissue engineering studies aimed at neural tissue regeneration

Graphene Oxide Upregulates the Homeostatic Functions of Primary Astrocytes and Modulates Astrocyte-to-Neuron Communication

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 endolysosomal 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 with astrocyte dysfunctions

Membrane Environment Enables Ultrafast Isomerization of Amphiphilic Azobenzene

G.M.P. and E.C. contributed equally to this work. G.M.P. acknowledges the financial support from Fondazione Cariplo, grant no. 2018-0979. The authors thank the financial support from the EU Horizon 2020 Research and Innovation Programme under Grant Agreement No. 643238 (SYNCHRONICS). The authors also thank Dr. Daniele Viola for helping with the analysis of the TA data.The non‐covalent affinity of photoresponsive molecules to biotargets represents an attractive tool for achieving effective cell photo‐stimulation. Here, an amphiphilic azobenzene that preferentially dwells within the plasma membrane is studied. In particular, its isomerization dynamics in different media is investigated. It is found that in molecular aggregates formed in water, the isomerization reaction is hindered, while radiative deactivation is favored. However, once protected by a lipid shell, the photochromic molecule reacquires its ultrafast photoisomerization capacity. This behavior is explained considering collective excited states that may form in aggregates, locking the conformational dynamics and redistributing the oscillator strength. By applying the pump probe technique in different media, an isomerization time in the order of 10 ps is identified and the deactivation in the aggregate in water is also characterized. Finally, it is demonstrated that the reversible modulation of membrane potential of HEK293 cells via illumination with visible light can be indeed related to the recovered trans→cis photoreaction in lipid membrane. These data fully account for the recently reported experiments in neurons, showing that the amphiphilic azobenzenes, once partitioned in the cell membrane, are effective light actuators for the modification of the electrical state of the membrane.Fondazione Cariplo. Grant Number: 2018‐0979EU Horizon 2020 Research and Innovation Programme. Grant Number: 64323

An Increase in Membrane Cholesterol by Graphene Oxide Disrupts Calcium Homeostasis in Primary Astrocytes

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, the molecular changes induced in cortical astrocytes by few-layer graphene (FLG) and graphene oxide (GO) flakes are addressed. The results show that exposure to FLG/GO does not affect cell viability or proliferation. However, proteomic and lipidomic analyses unveil alterations in several cellular processes, including intracellular Ca2+ ([Ca2+ ]i ) homeostasis and cholesterol metabolism, which are particularly intense in cells exposed to GO. Indeed, GO exposure impairs spontaneous and evoked astrocyte [Ca2+ ]i signals and induces a marked increase in membrane cholesterol levels. Importantly, cholesterol depletion fully rescues [Ca2+ ]i dynamics in GO-treated cells, indicating a causal relationship between these GO-mediated effects. The results indicate that exposure to GNMs alters intracellular signaling in astrocytes and may impact astrocyte-neuron interactions

Intersectin associates with synapsin and regulates its nanoscale localization and function.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery

dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate

16siLoss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1−120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1–120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management.openopenFaustini G.; Longhena F.; Bruno A.; Bono F.; Grigoletto J.; La Via L.; Barbon A.; Casiraghi A.; Straniero V.; Valoti E.; Costantino G.; Benfenati F.; Missale C.; Pizzi M.; Spillantini M.G.; Bellucci A.Faustini, G.; Longhena, F.; Bruno, A.; Bono, F.; Grigoletto, J.; La Via, L.; Barbon, A.; Casiraghi, A.; Straniero, V.; Valoti, E.; Costantino, G.; Benfenati, F.; Missale, C.; Pizzi, M.; Spillantini, M. G.; Bellucci, A

Search for neutrino counterparts to the gravitational wave sources from LIGO/Virgo O3 run with the ANTARES detector

Since 2015 the LIGO and Virgo interferometers have detected gravitational waves from almost one hundred coalescences of compact objects (black holes and neutron stars). This article presents the results of a search performed with data from the ANTARES telescope to identify neutrino counterparts to the gravitational wave sources detected during the third LIGO/Virgo observing run and reported in the catalogues GWTC-2, GWTC-2.1, and GWTC-3. This search is sensitive to all-sky neutrinos of all flavours and of energies > 100 GeV, thanks to the inclusion of both track-like events (mainly induced by ¿µ chargedcurrent interactions) and shower-like events (induced by other interaction types). Neutrinos are selected if they are detected within ±500 s from the GW merger and with a reconstructed direction compatible with its sky localisation. No significant excess is found for any of the 80 analysed GW events, and upper limits on the neutrino emission are derived. Using the information from the GW catalogues and assuming isotropic emission, upper limits on the total energy Etot,¿ emitted as neutrinos of all flavours and on the ratio f¿ = Etot,¿/EGW between neutrino and GW emissions are also computed. Finally, a stacked analysis of all the 72 binary black hole mergers (respectively the 7 neutron star-black hole merger candidates) has been performed to constrain the typical neutrino emission within this population, leading to the limits: Etot,¿ < 4.0 × 1053 erg and f¿ < 0.15 (respectively, Etot,¿ < 3.2 × 1053 erg and f¿ < 0.88) for E-2 spectrum and isotropic emission. Other assumptions including softer spectra and non-isotropic scenarios have also been testedPeer ReviewedA. Albert, S. Alves, M. André, M. Ardid, S. Ardid, J.-J. Aubert, J. Aublin, B. Baret, S. Basa, Y. Becherini, B. Belhorma, M. Bendahman, F. Benfenati, V. Bertin, S. Biagi, M. Bissinger, J. Boumaaza, M. Bouta, M.C. Bouwhuis, H. Brânzaş, R. Bruijn, J. Brunner, J. Busto, B. Caiffi, D. Calvo, S. Campion, A. Capone, L. Caramete, F. Carenini, J. Carr, V. Carretero, S. Celli, L. Cerisy, M. Chabab, T.N. Chau, R. Cherkaoui El Moursli, T. Chiarusi, M. Circella, J.A.B. Coelho, A. Coleiro, R. Coniglione, P. Coyle, A. Creusot, A.S.M. Cruz, A.F. Díaz, B. De Martino, C. Distefano, I. Di Palma, A. Domi, C. Donzaud, D. Dornic, D. Drouhin, T. Eberl, T. van Eeden, D. van Eijk, S. El Hedri, N. El Khayati, A. Enzenhöfer, P. Fermani, G. Ferrara, F. Filippini, L. Fusco, S. Gagliardini, J. García, C. Gatius Oliver, P. Gay, N. Geißelbrecht, H. Glotin, R. Gozzini, R. Gracia Ruiz, K. Graf, C. Guidi, L. Haegel, S. Hallmann, H. van Haren, A.J. Heijboer, Y. Hello, J.J. Hernández-Rey, J. Hößl, J. Hofestädt, F. Huang, G. Illuminati, C.W. James, B. Jisse-Jung, M. de Jong, P. de Jong, M. Kadler, O. Kalekin, U. Katz, A. Kouchner, I. Kreykenbohm, V. Kulikovskiy, R. Lahmann, M. Lamoureux, A. Lazo, D. Lefèvre, E. Leonora, G. Levi, S. Le Stum, D. Lopez-Coto, S. Loucatos, L. Maderer, J. Manczak, M. Marcelin, A. Margiotta, A. Marinelli, J.A. Martínez-Mora, P. Migliozzi, A. Moussa, R. Muller, L. Nauta, S. Navas, E. Nezri, B. Ó Fearraigh, A. Păun, G.E. Păvălaş, M. Perrin-Terrin, V. Pestel, P. Piattelli, C. Poirè, V. Popa, T. Pradier, N. Randazzo, D. Real, S. Reck, G. Riccobene, A. Romanov, A. Sánchez-Losa, A. Saina, F. Salesa Greus, D.F.E. Samtleben, M. Sanguineti, P. Sapienza, J. Schnabel, J. Schumann, F. Schüssler, J. Seneca, M. Spurio, Th. Stolarczyk, M. Taiuti, Y. Tayalati, S.J. Tingay, B. Vallage, G. Vannoye, V. Van Elewyck, S. Viola, D. Vivolo, J. Wilms, S. Zavatarelli, A. Zegarelli, J.D. Zornoza, J. ZúñigaPostprint (published version

The Increased Activity of TRPV4 Channel in the Astrocytes of the Adult Rat Hippocampus after Cerebral Hypoxia/Ischemia

The polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult