ABSTRACT
Volume reconstruction from electron microscopy datasets is a tool increasingly used to study the ultrastructure of the synapse in the broader context of neuronal network and brain organization. Fine modifications of synapse structure, such as activity-dependent dendritic spine enlargement and changes in the size and shape of the postsynaptic density, occur upon maturation and plasticity. The lack of structural plasticity or the inability to stabilize potentiated synapses are associated with synaptic and neuronal functional impairment. Mapping these rearrangements with the high resolution of electron microscopy proved to be essential in order to establish precise correlations between the geometry of synapses and their functional states. In this review we discuss recent discoveries on the substructure of the postsynaptic compartment of central excitatory synapses and how those are correlated with functional states of the neuronal network. The added value of volume electron microscopy analyses with respect to conventional transmission electron microscopy studies is highlighted considering that some limitations of volume-based methods imposed several adjustments to describe the geometry of this synaptic compartment and new parameters-that are good indicators of synapses strength and activity-have been introduced.
ABSTRACT
Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.
ABSTRACT
BACKGROUND: The radio- and chemo-resistance of glioblastoma stem-like cells (GSCs), together with their innate tumor-initiating aptitude, make this cell population a crucial target for effective therapies. However, targeting GSCs is hardly difficult and complex, due to the presence of the blood-brain barrier (BBB) and the infiltrative nature of GSCs arousing their dispersion within the brain parenchyma. METHODS: Liposomes (LIPs), surface-decorated with an Apolipoprotein E-modified peptide (mApoE) to enable BBB crossing, were loaded with doxorubicin (DOXO), as paradigm of cytotoxic drug triggering immunogenic cell death (ICD). Patient-derived xenografts (PDXs) obtained by GSC intracranial injection were treated with mApoE-DOXO-LIPs alone or concomitantly with radiation. RESULTS: Our results indicated that mApoE, through the engagement of the low-density lipoprotein receptor (LDLR), promotes mApoE-DOXO-LIPs transcytosis across the BBB and confers target specificity towards GSCs. Irradiation enhanced LDLR expression on both BBB and GSCs, thus further promoting LIP diffusion and specificity. When administered in combination with radiations, mApoE-DOXO-LIPs caused a significant reduction of in vivo tumor growth due to GSC apoptosis. GSC apoptosis prompted microglia/macrophage phagocytic activity, together with the activation of the antigen-presenting machinery crucially required for anti-tumor adaptive immune response. CONCLUSIONS: Our results advocate for radiotherapy and adjuvant administration of drug-loaded, mApoE-targeted nanovectors as an effective strategy to deliver cytotoxic molecules to GSCs at the surgical tumor margins, the forefront of glioblastoma (GBM) recurrence, circumventing BBB hurdles. DOXO encapsulation proved in situ immune response activation within GBM microenvironment.
ABSTRACT
Rho GTPases are a class of G-proteins involved in several aspects of cellular biology, including the regulation of actin cytoskeleton. The most studied members of this family are RHOA and RAC1 that act in concert to regulate actin dynamics. Recently, Rho GTPases gained much attention as synaptic regulators in the mammalian central nervous system (CNS). In this context, ARHGAP22 protein has been previously shown to specifically inhibit RAC1 activity thus standing as critical cytoskeleton regulator in cancer cell models; however, whether this function is maintained in neurons in the CNS is unknown. Here, we generated a knockout animal model for arhgap22 and provided evidence of its role in the hippocampus. Specifically, we found that ARHGAP22 absence leads to RAC1 hyperactivity and to an increase in dendritic spine density with defects in synaptic structure, molecular composition, and plasticity. Furthermore, arhgap22 silencing causes impairment in cognition and a reduction in anxiety-like behavior in mice. We also found that inhibiting RAC1 restored synaptic plasticity in ARHGAP22 KO mice. All together, these results shed light on the specific role of ARHGAP22 in hippocampal excitatory synapse formation and function as well as in learning and memory behaviors.
Subject(s)
Cognition/physiology , GTPase-Activating Proteins/metabolism , Glutamic Acid/metabolism , Hippocampus/metabolism , Neurons/metabolism , Neuropeptides/metabolism , Synapses/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Anxiety/genetics , Anxiety/metabolism , Behavior, Animal/physiology , Dendritic Spines/metabolism , GTPase-Activating Proteins/genetics , Maze Learning/physiology , Mice , Mice, Knockout , Motor Activity/physiology , Neuronal Plasticity/genetics , Neuropeptides/genetics , Synapses/genetics , Synaptosomes/metabolism , rac1 GTP-Binding Protein/geneticsABSTRACT
Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.
Subject(s)
Cognitive Dysfunction/genetics , Cognitive Dysfunction/physiopathology , Fragile X Mental Retardation Protein/metabolism , Mutation, Missense/physiology , Receptors, Glutamate/metabolism , Animals , Biotinylation , Brain/metabolism , Brain/physiopathology , Cells, Cultured , Cognitive Dysfunction/metabolism , Female , Fragile X Mental Retardation Protein/genetics , Hippocampus/metabolism , Hippocampus/physiopathology , Humans , Immunoblotting , Long-Term Potentiation/genetics , Long-Term Potentiation/physiology , Male , Mice , Mutation, Missense/genetics , Patch-Clamp Techniques , Receptors, Glutamate/geneticsABSTRACT
Three-dimensional (3D) reconstruction from electron microscopy (EM) datasets is a widely used tool that has improved our knowledge of synapse ultrastructure and organization in the brain. Rearrangements of synapse structure following maturation and in synaptic plasticity have been broadly described and, in many cases, the defective architecture of the synapse has been associated to functional impairments. It is therefore important, when studying brain connectivity, to map these rearrangements with the highest accuracy possible, considering the affordability of the different EM approaches to provide solid and reliable data about the structure of such a small complex. The aim of this work is to compare quantitative data from two dimensional (2D) and 3D EM of mouse hippocampal CA1 (apical dendrites), to define whether the results from the two approaches are consistent. We examined asymmetric excitatory synapses focusing on post synaptic density and dendritic spine area and volume as well as spine density, and we compared the results obtained with the two methods. The consistency between the 2D and 3D results questions the need-for many applications-of using volumetric datasets (costly and time consuming in terms of both acquisition and analysis), with respect to the more accessible measurements from 2D EM projections.
Subject(s)
CA1 Region, Hippocampal/ultrastructure , Dendritic Spines/ultrastructure , Pyramidal Cells/ultrastructure , Animals , Imaging, Three-Dimensional , Mice , Microscopy, Electron , Synapses/ultrastructureABSTRACT
Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1-Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.
Subject(s)
Autism Spectrum Disorder , Proto-Oncogene Proteins c-akt , Animals , Autism Spectrum Disorder/genetics , Humans , Mice , Mice, Knockout , Microfilament Proteins , Nerve Tissue Proteins/genetics , SynapsesABSTRACT
Although acetylcholine is the major neurotransmitter operating at the skeletal neuromuscular junction of many invertebrates and of vertebrates, glutamate participates in modulating cholinergic transmission and plastic changes in the last. Presynaptic terminals of neuromuscular junctions contain and release glutamate that contribute to the regulation of synaptic neurotransmission through its interaction with pre- and post-synaptic receptors activating downstream signaling pathways that tune synaptic efficacy and plasticity. During vertebrate development, the chemical nature of the neurotransmitter at the vertebrate neuromuscular junction can be experimentally shifted from acetylcholine to other mediators (including glutamate) through the modulation of calcium dynamics in motoneurons and, when the neurotransmitter changes, the muscle fiber expresses and assembles new receptors to match the nature of the new mediator. Finally, in adult rodents, by diverting descending spinal glutamatergic axons to a denervated muscle, a functional reinnervation can be achieved with the formation of new neuromuscular junctions that use glutamate as neurotransmitter and express ionotropic glutamate receptors and other markers of central glutamatergic synapses. Here, we summarize the past and recent experimental evidences in support of a role of glutamate as a mediator at the synapse between the motor nerve ending and the skeletal muscle fiber, focusing on the molecules and signaling pathways that are present and activated by glutamate at the vertebrate neuromuscular junction.
Subject(s)
Acetylcholine/metabolism , Glutamic Acid/physiology , Membrane Transport Proteins/metabolism , Neuromuscular Junction/physiology , Synaptic Transmission/physiology , Animals , Calcium/metabolism , Humans , Mice , Motor Neurons/cytology , Motor Neurons/metabolism , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Presynaptic Terminals/metabolism , Rats , Receptors, Neurotransmitter/metabolismABSTRACT
In ungulates the stability of the fetlock joint is dependent on several muscles, which are exposed to high stress and strain. Among those muscles, the proximal sesamoidean ligament or PSL (also known as the suspensory ligament or Ruini's elasto-tendinous organ) is organized at birth in layers of muscle fibres alternated with abundant tendinous tissue that, during the postnatal development, becomes the predominant tissue. In this study we analysed the PSL of the sheep at the age of 1, 30 and 180â¯days and determined the expression of several genes which either (a) are markers of muscle fibre growth and maturation, or (b) play a role as signal molecules. We observed an accelerated maturation, as indicated by the transition of MyHC isoform expression towards the slow isoforms and a reduced regenerative potential indicated by the low Pax7 expression and the altered Wnt signalling. We also found a specific myogenic expression pattern of MyoD, Myf5 and Myogenin in the developing PSL and high mRNA levels of specific fibrogenic factors, as TGF-ß1, that, undoubtedly, stimulate the growth of connective tissue. Our observations confirmed, at molecular level, the peculiarity of the fast involution observed in PSL a muscle that undergoes a very specific active differentiation process during early development, which implies myofibres involution and their replacement with connective tissue.
Subject(s)
Ligaments/growth & development , Muscle Development/genetics , Myosins/genetics , Sheep, Domestic/genetics , Age Factors , Animals , Cell Differentiation , Myogenic Regulatory Factors , Myosins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sesamoid Bones , Sheep, Domestic/growth & developmentABSTRACT
PCDH19 is considered one of the most clinically relevant genes in epilepsy, second only to SCN1A. To date about 150 mutations have been identified as causative for PCDH19-female epilepsy (also known as early infantile epileptic encephalopathy-9, EIEE9), which is characterized by early onset epilepsy, intellectual disabilities, and behavioral disturbances. Although little is known about the physiological role of PCDH19 and the pathogenic mechanisms that lead to EIEE9, in this review, we will present latest researches focused on these aspects, underlining protein expression, its known functions and the mechanisms by which the protein acts, with particular interest in PCDH19 extracellular and intracellular roles in neurons.
Subject(s)
Cadherins/genetics , Mutation/genetics , Neurodevelopmental Disorders/genetics , Spasms, Infantile/genetics , Animals , Humans , Intellectual Disability/etiology , Intellectual Disability/genetics , Neurodevelopmental Disorders/complications , Protocadherins , Spasms, Infantile/complicationsABSTRACT
The flavivirus capsid protein (C) is separated from the downstream premembrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the endoplasmic reticulum (ER). To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of Zika virus was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression in cis with respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of heterologous, but not homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.IMPORTANCE The capsid anchor (Ca) is a single-pass transmembrane domain at the C terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further examine the role of Ca in Zika virus life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.
Subject(s)
Capsid Proteins/metabolism , Capsid/physiology , Morphogenesis , Protein Precursors/metabolism , Viral Envelope Proteins/metabolism , Zika Virus Infection/virology , Zika Virus/physiology , Amino Acid Sequence , Animals , Capsid Proteins/genetics , Chlorocebus aethiops , Cytosol/metabolism , Cytosol/virology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/virology , HEK293 Cells , Humans , Protein Precursors/genetics , Sequence Homology , Vero Cells , Viral Envelope Proteins/genetics , Virus Assembly , Zika Virus Infection/metabolismABSTRACT
KEY POINTS: The present study demonstrates, by in vitro and in vivo analyses, the novel concept that signal transmission between sympathetic neurons and the heart, underlying the physiological regulation of cardiac function, operates in a quasi-synaptic fashion. This is a result of the direct coupling between neurotransmitter releasing sites and effector cardiomyocyte membranes. ABSTRACT: Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer-based imaging of cAMP in co-cultures, as a readout of cardiac ß-adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino-atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20-50 mg kg-1 ), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site.
Subject(s)
Cardiac Output , Myocytes, Cardiac/physiology , Neurons/physiology , Sympathetic Nervous System/physiology , Synapses/physiology , Synaptic Transmission , Animals , Cell Communication , Cells, Cultured , Coculture Techniques , Heart Rate , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Cardiac/cytology , Neurons/cytology , Norepinephrine/metabolism , Optogenetics , Rats , Rats, Sprague-DawleyABSTRACT
Neuromuscular junction assembly and plasticity during embryonic, postnatal, and adult life are tightly regulated by the continuous cross-talk among motor nerve endings, muscle fibers, and glial cells. Altered communications among these components is thought to be responsible for the physiological age-related changes at this synapse and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS). ALS is characterized by the degeneration and death of motor neurons leading to skeletal muscle denervation, atrophy and, most often, death of the patient within five years from diagnosis. ALS is a non-cell autonomous disease as, besides motor neuron degeneration, glial cells, and possibly muscle fibers, play a role in its onset and progression. Here, we will review the recent literature regarding the mechanisms leading to neuromuscular junction disassembly and muscle denervation focusing on the role of the three players of this peripheral tripartite synapse.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Neuromuscular Junction/pathology , Aging , Amyotrophic Lateral Sclerosis/physiopathology , Animals , Humans , Motor Neurons/pathology , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Neuroglia/pathology , Neuromuscular Junction/physiopathology , Schwann Cells/pathology , Synapses/pathologyABSTRACT
Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.
Subject(s)
Excitatory Amino Acid Agents/pharmacology , Intellectual Disability/drug therapy , Intellectual Disability/metabolism , Membrane Proteins/deficiency , Nerve Tissue Proteins/deficiency , Psychotropic Drugs/pharmacology , Receptors, AMPA/metabolism , Allosteric Regulation , Animals , Carrier Proteins/metabolism , Cell Cycle Proteins , Disease Models, Animal , Gene Expression/drug effects , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/ultrastructure , Intellectual Disability/pathology , Male , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding/drug effects , Synapses/drug effects , Synapses/metabolism , Synapses/ultrastructure , Synaptic Transmission/drug effects , Synaptic Transmission/physiology , Tissue Culture TechniquesABSTRACT
AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.
Subject(s)
Action Potentials , Caveolin 3/genetics , Caveolin 3/metabolism , Fibroblasts/metabolism , Mutation , Myocytes, Cardiac/metabolism , 3T3 Cells , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , Caveolae/metabolism , Caveolin 1/deficiency , Caveolin 1/genetics , Computer Simulation , Fibroblasts/ultrastructure , Heart Rate , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ion Channel Gating , Kinetics , Kv1.5 Potassium Channel/genetics , Kv1.5 Potassium Channel/metabolism , Mice , Mice, Knockout , Models, Cardiovascular , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocardial Contraction , Myocytes, Cardiac/ultrastructure , Potassium Channels/genetics , Potassium Channels/metabolism , Rats, Sprague-Dawley , TransfectionABSTRACT
The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).
Subject(s)
Membrane Potentials/physiology , Motor Neurons/physiology , Zebrafish/embryology , Animals , Cell Communication , Embryo, NonmammalianABSTRACT
Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy.
Subject(s)
Hippocampus/metabolism , Microfilament Proteins/genetics , Nerve Tissue Proteins/genetics , Neurons/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-B/genetics , Synaptic Transmission/genetics , Animals , Dentate Gyrus/metabolism , Dentate Gyrus/pathology , Dentate Gyrus/ultrastructure , Embryo, Mammalian , Epilepsy/genetics , Epilepsy/metabolism , Epilepsy/pathology , Gene Expression Regulation , HEK293 Cells , Hippocampus/pathology , Hippocampus/ultrastructure , Humans , Injections, Intraventricular , Intellectual Disability/genetics , Intellectual Disability/metabolism , Intellectual Disability/pathology , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neural Inhibition , Neurogenesis/genetics , Neurons/pathology , Neurons/ultrastructure , Primary Cell Culture , Rats , Rats, Wistar , Receptor Cross-Talk , Receptors, GABA-A/metabolism , Receptors, GABA-B/metabolism , Synapses/metabolism , Synapses/pathology , Synapses/ultrastructureABSTRACT
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
Subject(s)
Elongation Factor 2 Kinase/metabolism , Epilepsy/enzymology , Neurons/enzymology , Synaptic Transmission/physiology , Animals , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Conditioning, Psychological/physiology , Disease Models, Animal , Elongation Factor 2 Kinase/antagonists & inhibitors , Elongation Factor 2 Kinase/genetics , Epilepsy/pathology , Fear/physiology , Hippocampus/drug effects , Hippocampus/enzymology , Hippocampus/pathology , Mice, Inbred C57BL , Mice, Knockout , Neural Inhibition/drug effects , Neural Inhibition/physiology , Neurons/drug effects , Neurons/pathology , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Synapsins/genetics , Synapsins/metabolism , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/metabolismABSTRACT
The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies.
Subject(s)
Action Potentials/drug effects , Action Potentials/genetics , Amyotrophic Lateral Sclerosis/genetics , Motor Neurons/drug effects , Motor Neurons/physiology , Phenylglyoxal/analogs & derivatives , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/diagnosis , Animals , Animals, Genetically Modified , Disease Models, Animal , Gene Expression , Locomotion , Motor Activity/drug effects , Muscles/pathology , Mutation , Neuromuscular Junction/metabolism , Phenotype , Phenylglyoxal/pharmacology , Riluzole/pharmacology , Spinal Cord/pathology , ZebrafishABSTRACT
Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a(+/-) mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a(+/-) mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses.