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.

Gephyrin regulates GABAergic and glutamatergic synaptic transmission in hippocampal cell cultures.

Gephyrin is a scaffold protein essential for stabilizing glycine and GABA(A) receptors at inhibitory synapses. Here, recombinant intrabodies against gephyrin (scFv-gephyrin) were used to assess whether this protein exerts a transynaptic action on GABA and glutamate release. Pair recordings from interconnected hippocampal cells in culture revealed a reduced probability of GABA release in scFv-gephyrin-transfected neurons compared with controls. This effect was associated with a significant decrease in VGAT, the vesicular GABA transporter, and in neuroligin 2 (NLG2), a protein that, interacting with neurexins, ensures the cross-talk between the post- and presynaptic sites. Interestingly, hampering gephyrin function also produced a significant reduction in VGLUT, the vesicular glutamate transporter, an effect accompanied by a significant decrease in frequency of miniature excitatory postsynaptic currents. Overexpressing NLG2 in gephyrin-deprived neurons rescued GABAergic but not glutamatergic innervation, suggesting that the observed changes in the latter were not due to a homeostatic compensatory mechanism. Pulldown experiments demonstrated that gephyrin interacts not only with NLG2 but also with NLG1, the isoform enriched at excitatory synapses. These results suggest a key role of gephyrin in regulating transynaptic signaling at both inhibitory and excitatory synapses.

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.

Pre-synaptic BK channels selectively control glutamate versus GABA release from cortical and hippocampal nerve terminals.

In the present study, by means of genetic, biochemical, morphological, and electrophysiological approaches, the role of large-conductance voltage- and Ca(2+)-dependent K(+) channels (BK channels) in the release of excitatory and non-excitatory neurotransmitters at hippocampal and non-hippocampal sites has been investigated. The results obtained show that the pharmacological modulation of pre-synaptic BK channels selectively regulates [(3)H]D-aspartate release from cortical and hippocampal rat synaptosomes, but it fails to influence the release of excitatory neurotransmitters from cerebellar nerve endings or that of [(3)H]GABA, [(3)H]Noradrenaline, or [(3)H]Dopamine from any of the brain regions investigated. Confocal immunofluorescence experiments in hippocampal or cerebrocortical nerve terminals revealed that the main pore-forming BK α subunit was more abundantly expressed in glutamatergic (vGLUT1(+)) versus GABAergic (GAD(65-67)(+)) nerve terminals. Double patch recordings in monosynaptically connected hippocampal neurons in culture confirmed a preferential control exerted by BK channels on glutamate over GABA release. Altogether, the present results highlight a high degree of specificity in the regulation of the release of various neurotransmitters from distinct brain regions by BK channels, supporting the concept that BK channel modulators can be used to selectively limit excessive excitatory amino acid release, a major pathogenetic mechanism in several neuropsychiatric disorders.

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.

Dopamine, Alpha-Synuclein, and Mitochondrial Dysfunctions in Parkinsonian Eyes.

Parkinson's disease (PD) is characterized by motor dysfunctions including bradykinesia, tremor at rest and motor instability. These symptoms are associated with the progressive degeneration of dopaminergic neurons originating in the substantia nigra pars compacta and projecting to the corpus striatum, and by accumulation of cytoplasmic inclusions mainly consisting of aggregated alpha-synuclein, called Lewy bodies. PD is a complex, multifactorial disorder and its pathogenesis involves multiple pathways and mechanisms such as α-synuclein proteostasis, mitochondrial function, oxidative stress, calcium homeostasis, axonal transport, and neuroinflammation. Motor symptoms manifest when there is already an extensive dopamine denervation. There is therefore an urgent need for early biomarkers to apply disease-modifying therapeutic strategies. Visual defects and retinal abnormalities, including decreased visual acuity, abnormal spatial contrast sensitivity, color vision defects, or deficits in more complex visual tasks are present in the majority of PD patients. They are being considered for early diagnosis together with retinal imaging techniques are being considered as non-invasive biomarkers for PD. Dopaminergic cells can be found in the retina in a subpopulation of amacrine cells; however, the molecular mechanisms leading to visual deficits observed in PD patients are still largely unknown. This review provides a comprehensive analysis of the retinal abnormalities observed in PD patients and animal models and of the molecular mechanisms underlying neurodegeneration in parkinsonian eyes. We will review the role of α-synuclein aggregates in the retina pathology and/or in the onset of visual symptoms in PD suggesting that α-synuclein aggregates are harmful for the retina as well as for the brain. Moreover, we will summarize experimental evidence suggesting that the optic nerve pathology observed in PD resembles that seen in mitochondrial optic neuropathies highlighting the possible involvement of mitochondrial abnormalities in the development of PD visual defects. We finally propose that the eye may be considered as a complementary experimental model to identify possible novel disease' pathways or to test novel therapeutic approaches for PD.

Tuning GABAergic Inhibition: Gephyrin Molecular Organization and Functions.

To be highly reliable, synaptic transmission needs postsynaptic receptors (Rs) in precise apposition to the presynaptic release sites. At inhibitory synapses, the postsynaptic protein gephyrin self-assembles to form a scaffold that anchors glycine and GABAARs 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 interaction and downstream signaling pathways. In addition, through the constant exchange of scaffolding elements and receptors 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 autism spectrum disorders, schizophrenia, temporal lobe epilepsy and Alzheimer's disease, all associated with severe deficits of GABAergic signaling. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Computational Modeling of Inhibitory Transsynaptic Signaling in Hippocampal and Cortical Neurons Expressing Intrabodies Against Gephyrin.

GABAergic transmission regulates neuronal excitability, dendritic integration of synaptic signals and oscillatory activity, thought to be involved in high cognitive functions. By anchoring synaptic receptors just opposite to release sites, the scaffold protein gephyrin plays a key role in these tasks. In addition, by regulating GABAA receptor trafficking, gephyrin contributes to maintain, at the network level, an appropriate balance between Excitation (E) and Inhibition (I), crucial for information processing. An E/I imbalance leads to neuropsychiatric disorders such as epilepsy, schizophrenia and autism. In this article, we exploit a previously published computational method to fit spontaneous synaptic events, using a simplified model of the subcellular pathways involving gephyrin at inhibitory synapses. The model was used to analyze experimental data recorded under different conditions, with the main goal to gain insights on the possible consequences of gephyrin block on IPSCs. The same approach can be useful, in general, to analyze experiments designed to block a single protein. The results suggested possible ways to correlate the changes observed in the amplitude and time course of individual events recorded after different experimental protocols with the changes that may occur in the main subcellular pathways involved in gephyrin-dependent transsynaptic signaling.

Neuroinflammatory Processes, A1 Astrocyte Activation and Protein Aggregation in the Retina of Alzheimer's Disease Patients, Possible Biomarkers for Early Diagnosis.

Alzheimer's disease (AD), a primary cause of dementia in the aging population, is characterized by extracellular amyloid-beta peptides aggregation, intracellular deposits of hyperphosphorylated tau, neurodegeneration and glial activation in the brain. It is commonly thought that the lack of early diagnostic criteria is among the main causes of pharmacological therapy and clinical trials failure; therefore, the actual challenge is to define new biomarkers and non-invasive technologies to measure neuropathological changes in vivo at pre-symptomatic stages. Recent evidences obtained from human samples and mouse models indicate the possibility to detect protein aggregates and other pathological features in the retina, paving the road for non-invasive rapid detection of AD biomarkers. Here, we report the presence of amyloid beta plaques, tau tangles, neurodegeneration and detrimental astrocyte and microglia activation according to a disease associated microglia phenotype (DAM). Thus, we propose the human retina as a useful site for the detection of cellular and molecular changes associated with Alzheimer's disease.

Microglia control the glycinergic but not the GABAergic synapses via prostaglandin E2 in the spinal cord.

Microglia control excitatory synapses, but their role in inhibitory neurotransmission has been less well characterized. Herein, we show that microglia control the strength of glycinergic but not GABAergic synapses via modulation of the diffusion dynamics and synaptic trapping of glycine (GlyR) but not GABAA receptors. We further demonstrate that microglia regulate the activity-dependent plasticity of glycinergic synapses by tuning the GlyR diffusion trap. This microglia-synapse cross talk requires production of prostaglandin E2 by microglia, leading to the activation of neuronal EP2 receptors and cyclic adenosine monophosphate-dependent protein kinase. Thus, we now provide a link between microglial activation and synaptic dysfunctions, which are common early features of many brain diseases.

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.

Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction.

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2's ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1-/-) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction.

Developmental regulation of GABAergic signalling in the hippocampus of neuroligin 3 R451C knock-in mice: an animal model of Autism.

Autism Spectrum Disorders (ASDs) comprise an heterogeneous group of neuro-developmental abnormalities, mainly of genetic origin, characterized by impaired social interactions, communications deficits, and stereotyped behaviors. In a small percentage of cases, ASDs have been found to be associated with single mutations in genes involved in synaptic function. One of these involves the postsynaptic cell adhesion molecule neuroligin (NL) 3. NLs interact with presynaptic neurexins (Nrxs) to ensure a correct cross talk between post and presynaptic specializations. Here, transgenic mice carrying the human R451C mutation of Nlgn3, were used to study GABAergic signaling in the hippocampus early in postnatal life. Whole cell recordings from CA3 pyramidal neurons in slices from NL3(R451C) knock-in mice revealed an enhanced frequency of Giant Depolarizing Potentials (GDPs), 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 GABAA-mediated (GPSCs), but not AMPA-mediated postsynaptic currents (EPSCs). Changes in frequency of mGPSCs were associated with an acceleration of their decay kinetics, in the absence of any change in unitary synaptic conductance or in the number of GABAA receptor channels, as assessed by peak scaled non-stationary fluctuation analysis. The enhanced GABAergic but not glutamatergic transmission early in postnatal life may change the excitatory/inhibitory balance known to play a key role in the construction and refinement of neuronal circuits during postnatal development. This may lead to behavioral deficits reminiscent of those observed in ASDs patients.