Trans-synaptic signaling at GABAergic connections: possible dysfunction in some forms of Autism Spectrum Disorders

Synapses are recognized as being highly plastic in structure and function, strongly influenced by their own histories of impulse traffic and by signals from nearby cells. Synaptic contacts are fundamental for the development, homeostasis and remodeling of complex neural circuits. Synapses are highly varied in their molecular composition. Understand this diversity is important because it sheds light on the way they function. In particular, this may be useful for understanding the mechanisms at the basis of synaptic dysfunctions associated with neurodevelopmental disorders, such as Autism Spectrum Disorders (ASD) in order to develop properly targeted therapeutic tools. During the first part of my Phd course I characterized the functional role of gephyrin at inhibitory synapses (paper N. 1). Gephyrin is a scaffold protein essential for stabilizing glycine and GABAA receptors at inhibitory synapses. Using recombinant intrabodies against gephyrin (scFv-gephyrin) I tested the hypothesis that this protein exerts a trans-synaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrintransfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NL2), a protein that, interacting with the neurexins, ensures the cross-talk between the post- and presynaptic sites. I also found that, hampering gephyrin function produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Over-expressing NLG2 in gephyrindeprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. These results suggest a key role of gephyrin in regulating trans-synaptic signaling at both inhibitory and excitatory synapses. Several lines of evidence suggest that proteins involved in synaptic function are altered in ASDs. In particular, in a small percentage of cases, ASDs have been found to be associated with single mutations in genes encoding for cell adhesion molecules of the neuroligin-neurexin families. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. In the second part of my PhD, I used transgenic mice carrying the human R451C mutation of Nlgn3, to study GABAergic and glutamatergic signaling in the hippocampus early in postnatal life (paper N. 2). I performed whole cell recordings from CA3 pyramidal neurons in hippocampal slices from NL3 R451C knock-in mice and I found an enhanced frequency of Giant Depolarizing Potentials, as compared to controls. This effect was probably dependent on an increased GABAergic drive to principal cells as demonstrated by the enhanced frequency of miniature GABAAmediated (GPSCs) postsynaptic currents, but not AMPA-mediated postsynaptic currents (EPSCs). The increase in frequency of mGPSCs suggest a presynaptic 9 type of action. This was further supported by the experiments with the fast-off GABAA receptor antagonist TPMPA that, as expected for an enhanced GABA transient in the cleft, showed a reduced blocking effect on miniature events. Although an increased number of available postsynaptic GABAA receptors, if these are not saturated by the content of a single GABA containing vesicle may account for these results, this was not the case since a similar number of receptor channels was revealed with peak-scaled non-stationary fluctuation analysis in both WT and NL3R451C knock-in mice, indicating that the observed effects were not postsynaptic in origin. Presynaptic changes in GABA release can be attributed to modifications in the probability of GABA release, in the number of release sites or in the content of GABA in single synaptic vesicles. Changes in probability of GABA release seem unlikely considering that we examined miniature events generated by the release of a single quantum. Our data do not allow distinguishing between the other two possibilities (changes in the number of release sites or in vesicle GABA content). However, in agreement with previous data from S\ufcdhof group showing an enhancement of the presynaptic GABAergic marker VGAT (but not VGlut1) in the hippocampus of NL3R451C KI mice (Tabuchi et al., 2007), it is likely that an increased GABAergic innervation may contribute to the enhancement of GABA release. In additional experiments I found that changes in frequency of miniature GABAergic events were associated with an acceleration of mGPSCs decay possibly of postsynaptic origin. The increased frequency of mEPSCs detected in adult, but not young NL3 R451C mice may represent a late form of compensatory homeostatic correction to counter the excessive GABAA-mediated inhibition. Therefore, it is reasonable to assume that alterations in the excitatory/inhibitory balance, crucial for the refinement of neuronal circuits early in postnatal development, accounts for the behavioral deficits observed in ASDs patients. Although also in the present case, a modification of gephyrin expression in R451C NL 3 knock-in mice was associated with changes in GABAergic innervations suggesting the involvement of a trans-synaptic signal, the role of NL3 mutation in this effect remains to be elucidated. Finally, I contribute in writing a review article (paper N. 3) that gives an up dated picture of alterations of GABAergic signaling present in different forms of Autism Spectrum Disorders

Alterations of GABAergic Signaling in Autism Spectrum Disorders

Autism spectrum disorders (ASDs) comprise a heterogeneous group of pathological conditions, mainly of genetic origin, characterized by stereotyped behavior, marked impairment in verbal and nonverbal communication, social skills, and cognition. Interestingly, in a small number of cases, ASDs are associated with single mutations in genes encoding for neuroligin-neurexin families. These are adhesion molecules which, by regulating transsynaptic signaling, contribute to maintain a proper excitatory/inhibitory (E/I) balance at the network level. Furthermore, GABA, the main inhibitory neurotransmitter in adult life, at late embryonic/early postnatal stages has been shown to depolarize and excite targeted cell through an outwardly directed flux of chloride. The depolarizing action of GABA and associated calcium influx regulate a variety of developmental processes from cell migration and differentiation to synapse formation. Here, we summarize recent data concerning the functional role of GABA in building up and refining neuronal circuits early in development and the molecular mechanisms regulating the E/I balance. A dysfunction of the GABAergic signaling early in development leads to a severe E/I unbalance in neuronal circuits, a condition that may account for some of the behavioral deficits observed in ASD patients

Molecular imaging of tau protein: new insights and future directions

Tau is a microtubule-associated protein (MAPT) that is highly expressed in neurons and implicated in several cellular processes. Tau misfolding and self-aggregation give rise to proteinaceous deposits known as neuro-fibrillary tangles. Tau tangles play a key role in the genesis of a group of diseases commonly referred to as tauopathies; notably, these aggregates start to form decades before any clinical symptoms manifest. Advanced imaging methodologies have clarified important structural and functional aspects of tau and could have a role as diagnostic tools in clinical research. In the present review, recent progresses in tau imaging will be discussed. We will focus mainly on super-resolution imaging methods and the development of near-infrared fluorescent probes

SRGAP2 and Its Human-Specific Paralog Co-Regulate the Development of Excitatory and Inhibitory Synapses.

The proper function of neural circuits requires spatially and temporally balanced development of excitatory and inhibitory synapses. However, the molecular mechanisms coordinating excitatory and inhibitory synaptogenesis remain unknown. Here we demonstrate that SRGAP2A and its human-specific paralog SRGAP2C co-regulate the development of excitatory and inhibitory synapses in cortical pyramidal neurons in vivo. SRGAP2A promotes synaptic maturation, and ultimately the synaptic accumulation of AMPA and GABAA receptors, by interacting with key components of both excitatory and inhibitory postsynaptic scaffolds, Homer and Gephyrin. Furthermore, SRGAP2A limits the density of both types of synapses via its Rac1-GAP activity. SRGAP2C inhibits all identified functions of SRGAP2A, protracting the maturation and increasing the density of excitatory and inhibitory synapses. Our results uncover a molecular mechanism coordinating critical features of synaptic development and suggest that human-specific duplication of SRGAP2 might have contributed to the emergence of unique traits of human neurons while preserving the excitation/inhibition balance

Tuning GABAergic Inhibition: Gephyrin Molecular Organization and Functions

To be highly reliable, synaptic transmission needs postsynaptic receptors (Rs) in precise apposition to the pre - synaptic release sites. At inhibitory synapses, the postsynaptic protein gephyrin self -assembles to form a scaffold that anchors glycine and GABA A Rs to the cytoskeleton, thus ensuring the accurate accumulation of postsynaptic receptors at the right place. This protein undergoes several post -translational modifications which control protein-protein interac- tion and downstream signaling pathways. In addition, through the constant exchange of scaffolding elements and recep- tors in and out of synapses, gephyrin dynamically regulates synaptic strength and plasticity.The aim of the present review is to highlight recent findings on the functional role of gephyrin at GABAergic inhibitory synapses. We will discuss different approaches used to interfere with gephyrin in order to unveil its function. In addition, we will focus on the impact of gephyrin structure and distribution at the nanoscale level on the functional properties of inhibitory synapses as well as the implications of this scaffold protein in synaptic plasticity processes. Finally, we will emphasize how gephyrin genetic mutations or alterations in protein expression levels are implicated in several neuropathological disorders, including aut- ism spectrum disorders, schizophrenia, temporal lobe epilepsy and Alzheimer's disease, all associated with severe def- icits of GABAergic signaling. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries. (c) 2019 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Insulin-like growth factor 2 (IGF-2) rescues social deficits in NLG3–/y mouse model of ASDs

Autism spectrum disorders (ASDs) comprise developmental disabilities characterized by impairments of social interaction and repetitive behavior, often associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASDs symptomatology; thus, identifying novel therapies is urgently needed. Here, we used the Neuroligin 3 knockout mouse (NLG3–/y), a model that recapitulates the social deficits reported in ASDs patients, to test the effects of systemic administration of IGF-2, a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-2 treatment reverses the typical defects in social interaction and social novelty discrimination reflective of ASDs-like phenotypes. This effect was not accompanied by any change in spontaneous glutamatergic synaptic transmission in CA2 hippocampal region, a mechanism found to be crucial for social novelty discrimination. However, in both NLG3+/y and NLG3–/y mice IGF-2 increased cell excitability. Although further investigation is needed to clarify the cellular and molecular mechanisms underpinning IGF-2 effect on social behavior, our findings highlight IGF-2 as a potential pharmacological tool for the treatment of social dysfunctions associated with ASDs

High-performance polyimide membranes for use in solar sail propulsion

Solar sailing is a propulsion technology that is capable of driving vehicles and artificial satellites in space without the use of chemical propellants or electrical systems. This propulsion utilizes the solar radiation pressure resulting from the momentum transfer of solar photons reflected off the sail membrane, which is made of an aluminum-coated thin polymer film. The choice of the polymeric material to be used as solar sail membrane is crucial aspect as it influences the correct deployment of the structure and its efficacy as propulsion system during the space mission. In this work, we synthesized several types of polyimides with aromatic chemical structure using different organic solvents, including a greener alternative to traditional toxic solvents such as dimethylacetamide, and tested their properties for potential use in solar sailing. Thin polyimide membranes with thickness below 3 μm were fabricated and their chemical and physical properties investigated using several experimental techniques, from infrared spectroscopy to calorimetry and water contact angle analysis. Results were used to assess the potential use of the in-house-made polyimide membranes for the Helianthus mission, a test study case of the research program on Solar Photonic Propulsion, which is under development jointly between Sapienza University of Rome and the Italian Space Agency (ASI)

Structural design of booms for the solar sail of Helianthus sailcraft

Solar sail is a promising propulsion concept that exploits solar pressure to navigate in space without the use of propellants, therefore enabling missions otherwise not attainable by traditional propulsion (i.e. electric or chemical propulsion). For instance, a synchronous solar sail with the Earth-Moon barycenter to be used as a long warning time of solar storms caused by Coronal Mass Ejections is the main objective of the Helianthus project, funded by the Italian Space Agency. This paper is aimed specifically at the presentation and description of the design of the structural subsystem for the solar sail of the Helianthus project. This subsystem is composed of four deployable ultralight booms, which deploy and keep the sail-membrane in tension. The booms have to withstand the axial load, generated by the tensioned membrane, which must be smaller than the critical load at buckling. At the same time, the booms need to have sufficient stiffness to prevent a large out-of-plane displacement of the membrane leading to reduction of the thrust. First, the geometry and the dimensions of the boom cross-section to optimize the stiffness is determined. Then, a structural numerical analysis on a full-scale model of a square solar sail (40 m x 40 m) with four supporting booms is performed. For such configuration, the sail tension is simulated in order to determine the axial load acting on the tip of each boom and the displacements due to the solar radiation pressure are evaluated. Simulations are carried out by finite element method using the software ABAQUS. Results are presented at both system and individual components level

The space weather x-ray spectrometer for the Helianthus sub-L1 mission with solar photonic propulsion

Helianthus is a phase A study of a space weather station with solar photonic propulsion. The scientific payload will be made of: an X-ray spectrometer to detect solar flares; SailCor, a coronagraph with a wide field of view; a plasma analyzer; a magnetometer. The maximum allowed mass for the entire scientific payload shall not exceed 5 kg. The two imaging devices (coronagraph and X-ray spectrometer) are of fundamental importance for the sake of remotely and timely mapping the status of the Sun and provide Earth stations with early warning of potentially disruptive events. An extensive research on available X-Ray detectors was performed and the Amptek FAST-SDD spectrometer was selected. It is a very light, compact and vacuum compatible instrument. In order to prove the device readiness for flight, a measurement campaign was organized to investigate its performance in terms of spectral range, spectral resolution, dynamic range and response speed. The campaign was run at the INAF XACT facility in Palermo (Italy). This paper describes the facility, the measurement campaign and the results