Showing papers by "Fabio Benfenati published in 2017"

A fully organic retinal prosthesis restores vision in a rat model of degenerative blindness

Abstract: The degeneration of photoreceptors in the retina is one of the major causes of adult blindness in humans. Unfortunately, no effective clinical treatments exist for the majority of retinal degenerative disorders. Here we report on the fabrication and functional validation of a fully organic prosthesis for long-term in vivo subretinal implantation in the eye of Royal College of Surgeons rats, a widely recognized model of retinitis pigmentosa. Electrophysiological and behavioural analyses reveal a prosthesis-dependent recovery of light sensitivity and visual acuity that persists up to 6–10 months after surgery. The rescue of the visual function is accompanied by an increase in the basal metabolic activity of the primary visual cortex, as demonstrated by positron emission tomography imaging. Our results highlight the possibility of developing a new generation of fully organic, highly biocompatible and functionally autonomous photovoltaic prostheses for subretinal implants to treat degenerative blindness. A retinal prosthesis fabricated from an organic photovoltaic polymer can restore degenerative blindness in vivo, and over long implantation periods.

Intersectin associates with synapsin and regulates its nanoscale localization and function

Abstract: 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.

The Knockout of Synapsin II in Mice Impairs Social Behavior and Functional Connectivity Generating an ASD-like Phenotype.

Abstract: Autism spectrum disorders (ASD) and epilepsy are neurodevelopmental conditions that appear with high rate of co-occurrence, suggesting the possibility of a common genetic basis. Mutations in Synapsin (SYN) genes, particularly SYN1 and SYN2, have been recently associated with ASD and epilepsy in humans. Accordingly, mice lacking Syn1 or Syn2, but not Syn3, experience epileptic seizures and display autistic-like traits that precede the onset of seizures. Here, we analyzed social behavior and ultrasonic vocalizations emitted in 2 social contexts by SynI, SynII, or SynIII mutants and show that SynII mutants display the most severe ASD-like phenotype. We also show that the behavioral SynII phenotype correlates with a significant decrease in auditory and hippocampal functional connectivity as measured with resting state functional magnetic resonance imaging (rsfMRI). Taken together, our results reveal a permissive contribution of Syn2 to the expression of normal socio-communicative behavior, and suggest that Syn2-mediated synaptic dysfunction can lead to ASD-like behavior through dysregulation of cortical connectivity.

Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

Abstract: Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-β (Aβ), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aβ-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

A novel SYN1 missense mutation in non-syndromic X-linked intellectual disability affects synaptic vesicle life cycle, clustering and mobility.

Abstract: Intellectual Disability is a common and heterogeneous disorder characterized by limitations in intellectual functioning and adaptive behaviour, whose molecular mechanisms remain largely unknown. Among the numerous genes found to be involved in the pathogenesis of intellectual disability, 10% are located on the X-chromosome. We identified a missense mutation (c.236 C > G; p.S79W) in the SYN1 gene coding for synapsin I in the MRX50 family, affected by non-syndromic X-linked intellectual disability. Synapsin I is a neuronal phosphoprotein involved in the regulation of neurotransmitter release and neuronal development. Several mutations in SYN1 have been identified in patients affected by epilepsy and/or autism. The S79W mutation segregates with the disease in the MRX50 family and all affected members display intellectual disability as sole clinical manifestation. At the protein level, the S79W Synapsin I mutation is located in the region of the B-domain involved in recognition of highly curved membranes. Expression of human S79W Synapsin I in Syn1 knockout hippocampal neurons causes aberrant accumulation of small clear vesicles in the soma, increased clustering of synaptic vesicles at presynaptic terminals and increased frequency of excitatory spontaneous release events. In addition, the presence of S79W Synapsin I strongly reduces the mobility of synaptic vesicles, with possible implications for the regulation of neurotransmitter release and synaptic plasticity. These results implicate SYN1 in the pathogenesis of non-syndromic intellectual disability, showing that alterations of synaptic vesicle trafficking are one possible cause of this disease, and suggest that distinct mutations in SYN1 may lead to distinct brain pathologies.

A refined model of claudin-15 tight junction paracellular architecture by molecular dynamics simulations.

Abstract: Tight-junctions between epithelial cells of biological barriers are specialized molecular structures that regulate the flux of solutes across the barrier, parallel to cell walls. The tight-junction backbone is made of strands of transmembrane proteins from the claudin family, but the molecular mechanism of its function is still not completely understood. Recently, the crystal structure of a mammalian claudin-15 was reported, displaying for the first time the detailed features of transmembrane and extracellular domains. Successively, a structural model of claudin-15-based paracellular channels has been proposed, suggesting a putative assembly that illustrates how claudins associate in the same cell (via cis interactions) and across adjacent cells (via trans interactions). Although very promising, the model offers only a static conformation, with residues missing in the most important extracellular regions and potential steric clashes. Here we present detailed atomic models of paracellular single and double pore architectures, obtained from the putative assembly and refined via structural modeling and all-atom molecular dynamics simulations in double membrane bilayer and water environment. Our results show an overall stable configuration of the complex with a fluctuating pore size. Extracellular residue loops in trans interaction are able to form stable contacts and regulate the size of the pore, which displays a stationary radius of 2.5-3.0 A at the narrowest region. The side-by-side interactions of the cis configuration are preserved via stable hydrogen bonds, already predicted by cysteine crosslinking experiments. Overall, this work introduces an improved version of the claudin-15-based paracellular channel model that strengthens its validity and that can be used in further computational studies to understand the structural features of tight-junctions regulation.

Optogenetic Modulation of Intracellular Signalling and Transcription: Focus on Neuronal Plasticity

Abstract: 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.

Synapsins Are Downstream Players of the BDNF-Mediated Axonal Growth.

Abstract: Synapsins (Syns) are synaptic vesicle-associated phosphoproteins involved in neuronal development and neurotransmitter release. While Syns are implicated in the regulation of brain-derived neurotrophic factor (BDNF)-induced neurotransmitter release, their role in the BDNF developmental effects has not been fully elucidated. By using primary cortical neurons from Syn I knockout (KO) and Syn I/II/III KO mice, we studied the effects of BDNF and nerve growth factor (NGF) on axonal growth. While NGF had similar effects in all genotypes, BDNF induced significant differences in Syn KO axonal outgrowth compared to wild type (WT), an effect that was rescued by the re-expression of Syn I. Moreover, the significant increase of axonal branching induced by BDNF in WT neurons was not detectable in Syn KO neurons. The expression analysis of BDNF receptors in Syn KO neurons revealed a significant decrease of the full length TrkB receptor and an increase in the levels of the truncated TrkB.t1 isoform and p75NTR associated with a marked reduction of the BDNF-induced MAPK/Erk activation. By using the Trk inhibitor K252a, we demonstrated that these differences in BDNF effects were dependent on a TrkB/p75NTR imbalance. The data indicate that Syn I plays a pivotal role in the BDNF signal transduction during axonal growth.

Pavlovian conditioning of larval drosophila: an illustrated, multilingual, hands-on manual for odor-taste associative learning in maggots

Abstract: Larval Drosophila offer a study case for behavioral neurogenetics that is simple enough to be experimentally tractable, yet complex enough to be worth the effort. We provide a detailed, hands-on manual for Pavlovian odor-reward learning in these animals. Given the versatility of Drosophila for genetic analyses, combined with the evolutionarily shared genetic heritage with humans, the paradigm has utility not only in behavioral neurogenetics and experimental psychology, but for translational biomedicine as well. Together with the upcoming total synaptic connectome of the Drosophila nervous system and the possibilities of single-cell-specific transgene expression, it offers enticing opportunities for research. Indeed, the paradigm has already been adopted by a number of labs and is robust enough to be used for teaching in classroom settings. This has given rise to a demand for a detailed, hands-on manual directed at newcomers and/or at laboratory novices, and this is what we here provide. The paradigm and the present manual have a unique set of features: • the paradigm is cheap, easy, and robust; • the manual is detailed enough for newcomers or laboratory novices; • it briefly covers the essential scientific context; • it includes sheets for scoring, data analysis, and display; • it is multilingual: in addition to an English version we provide German, French, Japanese, and Spanish language versions as well. The present manual can thus foster science education at an earlier age and enable research by a broader community than has been the case to date.

Neuronal hyperactivity causes Na+/H+ exchanger-induced extracellular acidification at active synapses.

Abstract: Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H+ concentration. The aim of this study was 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 that is 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 located at active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle H+ into the cleft nor to the activity of membrane-exposed H+ V-ATPase, 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, emphasizing 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.

Imaging and structural studies of DNA–protein complexes and membrane ion channels

Abstract: 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 A, 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.

Synapsin-antibodies in psychiatric and neurological disorders: Prevalence and clinical findings.

Abstract: Objective To study the prevalence of autoantibodies to synapsin in patients with psychiatric and neurological disorders and to describe clinical findings in synapsin antibody positive patients. Methods Sera of 375 patients with different psychiatric and neurological disorders and sera of 97 healthy controls were screened (dilution 1:320) for anti-synapsin IgG using HEK293 cells transfected with rat synapsin Ia. Positive sera were further analyzed by immunoblots with brain tissue from wild type and synapsin knock out mice and with HEK293 cells transfected with human synapsin Ia and Ib. Binding of synapsin IgG positive sera to primary neurons was studied using murine hippocampal neurons. Results IgG in serum from 23 (6.1%) of 375 patients, but from none of the 97 healthy controls (p = 0.007), bound to rat synapsin Ia transfected cells with a median (range) titer of 1:1000 (1:320–1:100,000). Twelve of the 23 positive sera reacted with a protein of the molecular size of synapsin I in immunoblots of wild type but not of synapsin knock out mouse brain tissue. Out of 19/23 positive sera available for testing, 13 bound to human synapsin Ia and 16 to human synapsin Ib transfected cells. Synapsin IgG positive sera stained fixed and permeabilized murine hippocampal neurons. Synapsin IgG positive patients had various psychiatric and neurological disorders. Tumors were documented in 2 patients (melanoma, small cell lung carcinoma); concomitant anti-neuronal or other autoantibodies were present in 8 patients. Conclusions Autoantibodies to human synapsin Ia and Ib are detectable in a proportion of sera from patients with different psychiatric and neurological disorders, warranting further investigation into the potential pathophysiological relevance of these antibodies.

PRRT2, a network stability gene

Abstract: Specific paroxysmal disorders, namely benign infantile epilepsy (BFIE), kinesigenic dyskinesia (PKD), infantile convulsions and choreoathetosis (ICCA) and hemiplegic migraine (HM), are associated with mutations in the gene encoding for PRoline-Rich Transmembrane protein 2 (PRRT2; Figure 1A, 1B). PRRT2 is a neuronspecific protein expressed on neuronal membranes and at synapses, with a prevalent presynaptic location [1, 2]. Several nonsense, missense and frame-shift mutations were identified in the PRRT2 gene, but the vast majority of patients (80%) carry the same frameshift single-nucleotide duplication (c.649dupC) that leads to a premature stop codon and results in a loss-of-function pathogenetic mechanism [3]. Despite the extensive characterization of PRRT2 mutations, no clear evidence for genotypephenotype correlation exists and the three main diseases (BFIE, PKD and PKD/ICCA) form a continuous spectrum, starting from BFIE in the first months of life and evolving to PKD or PKD/ICCA often in association with HM during adolescence [4-5]. To model the disorders and investigate the underlying neurobiological alterations, we recently characterized a PRRT2 knock out mouse (PRRT2 KO; Figure 1C), carrying a constitutive inactivation of the PRRT2 gene [6]. The promoterless lacZ gene integrated into the PRRT2 locus allowed mapping the PRRT2 regional expression, that is not widespread, but rather concentrated at restricted brain areas. Interestingly, the expression is high in neurons of the lower hindbrain particularly in the cerebellum, a brain area involved in the generation of motor/epileptic phenotype and in which altered synaptic plasticity at the parallel fibersPurkinje cells synapse was found [6] (Figure 1D). A selective staining was also identified in the cerebral cortex, claustrum and dorsal horns of the spinal cord. Moreover, in the hippocampus, PRRT2 is particularly expressed in the hilus of the dentate gyrus, where mossy cells control the excitability of granule cells, playing a role in preventing hippocampal seizures (Figure 1D). Notably, all the brain regions positive for PRRT2 are involved in processing sensory information, motor disorders and epilepsy, all neurological traits present in patients with PRRT2 mutations. The PRRT2 KO mouse recapitulates many of the phenotypic features of the human PRRT2-linked disorders, showing abnormal motor behaviors and a motor/epileptic phenotype in response to environmental stimuli [6]. The motor problems are represented by gait abnormalities and a peculiar paroxysmal backwalking appearing early in the postnatal life and persisting in the adult [4]. The motor/ epileptic phenotype of PRRT2 KO mice becomes dramatic in response to audiogenic stimuli (Figure 1E), which trigger wild running, backwalking and jumping, and to the administration of convulsants, such as pentylentetrazole (Figure 1F). In both kinds of provocations, however, the seizure propensity was not very severe, consistent with the mild epileptic phenotype of patients bearing PRRT2 mutations [4, 5]. Overall, the PRRT2 KO mouse reproduces the paroxysmal traits described in PRRT2 patients and characterized by sudden and short attacks that usually occur periodically in response to a sensory trigger. The episodic nature of these events suggests that they result from network instability caused by changes in the excitation/inhibition balance. Interestingly, data obtained in primary neurons showed that PRRT2 silencing is associated with alterations of synaptic transmission and short-term plasticity [1]. PRRT2 silencing gives rise to a decreased number of excitatory synapses and a marked impairment of fast synchronous neurotransmitter release. These effects are associated with a decrease in Ca2+ sensitivity and release probability, revealing a role for Editorial

The Transcription Factors EBF1 and EBF2 Are Positive Regulators of Myelination in Schwann Cells

Abstract: Myelin formation by Schwann cells is tightly controlled by multiple pathways and regulatory molecules. The Ebf2 gene, belonging to the Ebf family of transcription factors regulating cell development and differentiation, is expressed in Schwann cells, and Ebf2 knockout mice show peripheral nerve defects. We also found that Ebf1 is expressed in Schwann cells. To investigate Ebf function in myelination, we silenced Ebf genes in myelinating dorsal root ganglia cultures. Combined downregulation of Ebf genes leads to a severe impairment of myelin formation that is completely rescued by their specific overexpression, suggesting that the expression level of Ebf genes strongly influences axon myelination. In addition, by profiling Ebf target genes, we found several transcripts belonging to pathways actively involved in peripheral myelination, including Gliomedin, a gene with a role in the formation of the nodes of Ranvier and recently implicated in the pathogenesis of the nodo-paranodopathies. Our results suggest that Ebf genes act as positive regulators of myelination and directly regulate the promoter of Gliomedin.

Impaired GABAB-mediated presynaptic inhibition increases excitatory strength and alters short-term plasticity in synapsin knockout mice.

Abstract: // Pierluigi Valente 1, * , Pasqualina Farisello 1, 2, * , Flavia Valtorta 3 , Pietro Baldelli 1, 2 and Fabio Benfenati 1, 2 1 Department of Experimental Medicine, Section of Physiology, University of Genoa, 16132 Genova, Italy 2 Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, 16132 Genova, Italy 3 S. Raffaele Scientific Institute and Vita-Salute University, 20132 Milano, Italy * These authors have contributed equally to this work Correspondence to: Fabio Benfenati, email: fabio.benfenati@iit.it Keywords: epilepsy, excitatory transmission, GABA receptors, facilitation, synaptic depression Received: June 22, 2017 Accepted: September 03, 2017 Published: September 30, 2017 ABSTRACT Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABA B receptors (GABA B Rs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABA B presynaptic inhibition. Exogenous stimulation of GABA B Rs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABA B Rs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABA B Rs on glutamate transmission, making the excitation/inhibition imbalance stronger.

Towards a magnetoresistive platform for neural signal recording

Abstract: A promising strategy to get deeper insight on brain functionalities relies on the investigation of neural activities at the cellular and sub-cellular level. In this framework, methods for recording neuron electrical activity have gained interest over the years. Main technological challenges are associated to finding highly sensitive detection schemes, providing considerable spatial and temporal resolution. Moreover, the possibility to perform non-invasive assays would constitute a noteworthy benefit. In this work, we present a magnetoresistive platform for the detection of the action potential propagation in neural cells. Such platform allows, in perspective, the in vitro recording of neural signals arising from single neurons, neural networks and brain slices.

Magnetic Tunnel Junction Based Chip to Detect the Magnetic Field of Neuronal Signals: A Platform for In Vitro Studies

Abstract: Magnetoencephalography (MEG) has revolutionized neuroscience, offering a tool with unprecedented spatial and temporal resolution. Today, MEG has clinical uses in detecting and localizing pathological activity in patients with brain tumors or intractable epilepsy [1]. Despite the wide clinical applications, the nature of MEG signals at local level is still not well understood [2]. In this context, there is evident crucial interest in developing a new generation of devices for local magnetic recording for an in vitro system. Several recent studies have implied that MagnetoResistive (MR) technologies can detect a biological magnetic field at local scale [3,4] (i.e., brain slice, muscle in vitro). However, to date, no attempts have been carried out for neurons in culture due to the long-term biocompatibility required. In this work, we will present a platform based on MR sensors array, namely magnetic tunneling junctions (MTJs) to detect the activity of neurons in culture from a magnetic point of view. We will show the biocompatibility of our devices and the preservation of the physical properties of the sensors. Murine embryonic hippocampal neurons were grown on top of the MR sensors array. We achieve a lifetime of the on-chip neuronal networks of longer than 20 days. Neurite growth was studied during development with immunostaining analysis. In conclusion, we achieved the biocompatibility conditions of a MR platform suitable for studying the magnetic field generated by the activity of in vitro neuronal networks.