Postsynaptic protein organization revealed by electron microscopy.

Neuronal synapses are key devices for transmitting and processing information in the nervous system. Synaptic plasticity, generally regarded as the cellular basis of learning and memory, involves changes of subcellular structures that take place at the nanoscale. High-resolution imaging methods, especially electron microscopy (EM), have allowed for quantitative analysis of such nanoscale structures in different types of synapses. In particular, the semi-ordered organization of neurotransmitter receptors and their interacting scaffolds in the postsynaptic density have been characterized for both excitatory and inhibitory synapses by studies using various EM techniques such as immuno-EM, electron tomography of high-pressure freezing and freeze-substituted samples, and cryo electron tomography. These techniques, in combination with new correlative approaches, will further facilitate our understanding of the molecular organization underlying diverse functions of neuronal synapses

The CaMKII/NMDA receptor complex controls hippocampal synaptic transmission by kinase-dependent and independent mechanisms.

CaMKII is one of the most studied synaptic proteins, but many critical issues regarding its role in synaptic function remain unresolved. Using a CRISPR-based system to delete CaMKII and replace it with mutated forms in single neurons, we have rigorously addressed its various synaptic roles. In brief, basal AMPAR and NMDAR synaptic transmission both require CaMKIIα, but not CaMKIIβ, indicating that, even in the adult, synaptic transmission is determined by the ongoing action of CaMKIIα. While AMPAR transmission requires kinase activity, NMDAR transmission does not, implying a scaffolding role for the CaMKII protein instead. LTP is abolished in the absence of CaMKIIα and/or CaMKIIβ and with an autophosphorylation impaired CaMKIIα (T286A). With the exception of NMDAR synaptic currents, all aspects of CaMKIIα signaling examined require binding to the NMDAR, emphasizing the essential role of this receptor as a master synaptic signaling hub

Neuroadaptations in the Cellular and Postsynaptic Group 1 Metabotropic Glutamate Receptor mGluR5 and Homer Proteins Following Extinction of Cocaine Self-administration

This study examined the role of group1 metabotropic glutamate receptor mGluR5 and associated postsynaptic scaffolding protein Homer1b/c in behavioral plasticity after three withdrawal treatments from cocaine self-administration. Rats self-administered cocaine or saline for 14 days followed by a withdrawal period during which rats underwent extinction training, remained in their home cages, orwere placed in the self-administration chambers in the absence of extinction. Subsequently, the tissue level and distribution of proteins in the synaptosomal fraction associated with the postsynaptic densitywere examined. Cocaine self-administration followed by home cage exposure reduced the mGluR5 protein in nucleus accumbens (NA) shell and dorsolateral striatum. While extinction training reduced mGluR5 protein in NAshell, NAcore and dorsolateral striatum did not display any change. The scaffolding protein PSD95 increased in NAcore of the extinguished animals. Extinction of drug seeking was associated with a significant decrease in the synaptosomal mGluR5 protein in NAshell and an increase in dorsolateral striatum, while that of NAcore was not modified. Interestingly, both Homer1b/c and PSD95 scaffolding proteins were decreased in the synaptosomal fraction after extinction training in NAshell but not NAcore. Extinguished drug-seeking behavior was also associated with an increase in the synaptosomal actin proteins in dorsolateral striatum. Therefore, extinction of cocaine seeking is associated with neuroadaptations in mGluR5 expression and distribution that are region-specific and consist of extinction-induced reversal of cocaine-induced adaptations aswell as emergent extinction-induced alterations. Concurrent plasticity in the scaffolding proteins further suggests that mGluR5 receptor neuroadaptations may have implications for synaptic function

Evolutionary insights into premetazoan functions of the neuronal protein homer.

Reconstructing the evolution and ancestral functions of synaptic proteins promises to shed light on how neurons first evolved. The postsynaptic density (PSD) protein Homer scaffolds membrane receptors and regulates Ca(2+) signaling in diverse metazoan cell types (including neurons and muscle cells), yet its ancestry and core functions are poorly understood. We find that the protein domain organization and essential biochemical properties of metazoan Homer proteins, including their ability to tetramerize, are conserved in the choanoflagellate Salpingoeca rosetta, one of the closest living relatives of metazoans. Unlike in neurons, Homer localizes to the nucleoplasm in S. rosetta and interacts directly with Flotillin, a protein more commonly associated with cell membranes. Surprisingly, we found that the Homer/Flotillin interaction and its localization to the nucleus are conserved in metazoan astrocytes. These findings suggest that Homer originally interacted with Flotillin in the nucleus of the last common ancestor of metazoans and choanoflagellates and was later co-opted to function as a membrane receptor scaffold in the PSD

Common Ribs of Inhibitory Synaptic Dysfunction in the Umbrella of Neurodevelopmental Disorders

The term neurodevelopmental disorder (NDD) is an umbrella term used to group together a heterogeneous class of disorders characterized by disruption in cognition, emotion, and behavior, early in the developmental timescale. These disorders are heterogeneous, yet they share common behavioral symptomatology as well as overlapping genetic contributors, including proteins involved in the formation, specialization, and function of synaptic connections. Advances may arise from bridging the current knowledge on synapse related factors indicated from both human studies in NDD populations, and in animal models. Mounting evidence has shown a link to inhibitory synapse formation, specialization, and function among Autism, Angelman, Rett and Dravet syndromes. Inhibitory signaling is diverse, with numerous subtypes of inhibitory interneurons, phasic and tonic modes of inhibition, and the molecular and subcellular diversity of GABAA receptors. We discuss common ribs of inhibitory synapse dysfunction in the umbrella of NDD, highlighting alterations in the developmental switch to inhibitory GABA, dysregulation of neuronal activity patterns by parvalbumin-positive interneurons, and impaired tonic inhibition. Increasing our basic understanding of inhibitory synapses, and their role in NDDs is likely to produce significant therapeutic advances in behavioral symptom alleviation for interrelated NDDs. Highlights • Human studies and animal models need to be bridged in neurodevelopmental disorders • Inhibitory signaling emerges as a common contributor to neurodevelopmental disorders • Inhibitory signaling is diverse in mode, source, and target • Systematic evaluation of inhibitory diversity is lacking in neurodevelopment • Understanding of inhibitory signaling diversity will advance therapeutic strategie

Activity of nAChRs containing α9 subunits modulates synapse stabilization via bidirectional signaling programs

Although the synaptogenic program for cholinergic synapses of the neuromuscular junction is well known, little is known of the identity or dynamic expression patterns of proteins involved in non-neuromuscular nicotinic synapse development. We have previously demonstrated abnormal presynaptic terminal morphology following loss of nicotinic acetylcholine receptor (nAChR) α9 subunit expression in adult cochleae. However, the molecular mechanisms underlying these changes have remained obscure. To better understand synapse formation and the role of cholinergic activity in the synaptogenesis of the inner ear, we exploit the nAChR α9 subunit null mouse. In this mouse, functional acetylcholine (ACh) neurotransmission to the hair cells is completely silenced. Results demonstrate a premature, effusive innervation to the synaptic pole of the outer hair cells in α9 null mice coinciding with delayed expression of cell adhesion proteins during the period of effusive contact. Collapse of the ectopic innervation coincides with an age-related hyperexpression pattern in the null mice. In addition, we document changes in expression of presynaptic vesicle recycling/trafficking machinery in the α9 null mice that suggests a bidirectional information flow between the target of the neural innervation (the hair cells) and the presynaptic terminal that is modified by hair cell nAChR activity. Loss of nAChR activity may alter transcriptional activity, as CREB binding protein expression is decreased coincident with the increased expression of N-Cadherin in the adult α9 null mice. Finally, by using mice expressing the nondesensitizing α9 L90T point mutant nAChR subunit, we show that increased nAChR activity drives synaptic hyperinnervation. © 2009 Wiley Periodicals, Inc.Fil: Murthy, Vidya. Tufts University School of Medicine; EslovaquiaFil: Taranda, Julian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Tufts University School of Medicine; EslovaquiaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; ArgentinaFil: Vetter, Douglas E.. Tufts University School of Medicine; Eslovaqui

Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and Bayesian inference: application to the multi-scale dynamics of glycine receptors in the neuronal membrane

Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). When applying these analytical tools to glycine neurotransmitter receptors (GlyRs) at inhibitory synapses, we find that gephyrin scaffolds act as shallow energy traps (~3 kBT) for GlyRs, with a depth modulated by the biochemical properties of the receptor-gephyrin interaction loop. In turn, the inferred maps can be used to simulate the dynamics of proteins in the membrane, from the level of individual receptors to that of the population, and thereby, to model the stochastic fluctuations of physiological parameters (such as the number of receptors at synapses). Overall, our approach provides a powerful and comprehensive framework with which to analyze biochemical interactions in living cells and to decipher the multi-scale dynamics of biomolecules in complex cellular environments.Comment: 23 pages, 4 figure

CaMKIIalpha interacts with multi-PDZ domain protein MUPP1 in spermatozoa and prevents spontaneous acrosomal exocytosis

The success of acrosomal exocytosis, a complex process with a variety of inter-related steps, relies on the coordinated interaction of participating signaling molecules. Since the acrosome reaction resembles Ca(2+)-regulated exocytosis in neurons, we investigated whether cognate neuronal binding partners of the multi-PDZ domain protein MUPP1, which recruits molecules that control the initial tethering and/or docking between the acrosomal vesicle and the plasma membrane, are also expressed in spermatozoa, and whether they contribute to the regulation of acrosomal secretion. We observed that CaMKIIalpha colocalizes with MUPP1 in the acrosomal region of epididymal spermatozoa where the kinase selectively binds to a region encompassing PDZ domains 10-11 of MUPP1. Furthermore, we found that pre-treating mouse spermatozoa with a CaMKII inhibitor that directly blocks the catalytic region of the kinase, as well as a competitive displacement of CaMKIIalpha from PDZ domains 10-11, led to a significant increase in spontaneous acrosomal exocytosis. Since Ca(2+)-calmodulin releases CaMKIIalpha from the PDZ scaffolding protein, MUPP1 represents a central signaling platform to dynamically regulate the assembly and disassembly of binding partners pertinent to acrosomal secretion, thereby precisely adjusting an increase in Ca(2+) to synchronized fusion pore formation