Postsynaptic Compartments Research Articles

TDP-43 proteinopathy impairs mRNP granule mediated postsynaptic translation and mRNA metabolism.

Background: Local protein synthesis and mRNA metabolism mediated by mRNP granules in the dendrites and the postsynaptic compartment is essential for synaptic remodeling and plasticity in neuronal cells. Dysregulation of these processes caused by TDP-43 proteinopathy leads to neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis.Methods: Using biochemical analysis and imaging techniques, including super-resolution microscopy, we provide evidence, for the first time, for the postsynaptic localization of TDP-43 in mammalian synapses and we show that TDP-43 is a component of neuronal mRNP granules.Results: With activity stimulation and various molecular approaches, we further demonstrate activity-dependent mRNP granule dynamics involving disassembly of mRNP granules, release of mRNAs, activation of local protein translation, and the impairment of granule disassembly in cellular, animal and human models of TDP-43 proteinopathy.Conclusion: Our study elucidates the interplay between TDP-43 and neuronal mRNP granules in normal physiology and TDP-43 proteinopathy in the regulation of local protein translation and mRNA metabolism in the postsynaptic compartment.

The glomerular network of the zebrafish olfactory bulb.

Each zebrafish olfactory bulb contains ~ 140 glomeruli that are distinguishable based on size, location, neurochemistry and function. Here we examine the mitral cell innervation of differently sized glomeruli in adult zebrafish. Type 1 glomeruli had diameters of 80.9 ± 8.1μm and were innervated by 5.9 ± 0.9 mitral cells. The Type 1 mediodorsal glomeruli (mdG) were innervated by both uniglomerular (innervating only single glomeruli) and multiglomerular mitral cells (innervating two or more glomeruli). In contrast, the Type 1 ventroposterior (vpG) and lateral glomeruli (lG) were only innervated by uniglomerular mitral cells. Type 2 ventral glomeruli were 46 ± 5.1μm in diameter and were innervated by 3.3 ± 0.2 mitral cells. Type 2 ventromedial glomeruli (vmG) were innervated exclusively by uniglomerular mitral cells. Type 3 glomeruli had diameters of 17 ± 2.5μm and were innervated by 1.1 ± 0.6 multiglomerular mitral cells each. Finally, Type 4 glomeruli were small, with average diameters of 4.8 ± 3.9μm and were restricted to the lateral plexus. These glomeruli were innervated mainly by multiglomerular mitral cells with extensively branching dendrites. This study provides the first specific associations between uni- and multiglomerular mitral cells with known zebrafish glomeruli. Our results suggest that glomeruli are distinguishable based on their postsynaptic compartment and that distinct input-output computations occur in different types of zebrafish glomeruli.

Глюкокортикоидная регуляция глутаматергического синапса: механизмы стресс-зависимой нейропластичности

Glucocorticoids released from the adrenal cortex under the influence of stress factors are the most important messengers in the integrative regulation of adaptive plasticity of the brain, executed by the neurohumoral hypothalamic-pituitary-adrenal system. Excitatory synapses are considered as key participants in synaptic plasticity and behavioral adaptation. The review presents the accumulated data on the mechanisms of glucocorticoid regulation of the glutamatergic synapse, primarily on the examples of the hippocampus and prefrontal cortex. Glucocorticoids, by triggering signal transduction through mineralocorticoid and glucocorticoid receptors located on synaptic membranes and in the cytosol of glutamatergic neurons, regulate synapse plasticity at the level of pre - and postsynaptic compartments. Glucocorticoids modulate synapse excitability through changes in vesicular transport and glutamate release, and mediate changes in the expression, composition, and properties of ionotropic NMDA and AMPA receptors as well as other glutamate receptors. A detailed scheme of multiple regulatory mechanisms implemented in the glutamatergic synapse during the binding of glucocorticoids to specific receptors is presented.

Synaptic activity and strength are reflected by changes in the post-synaptic secretory pathway

Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.

Mapping Phosphodiesterase 4D (PDE4D) in Macaque Dorsolateral Prefrontal Cortex: Postsynaptic Compartmentalization in Layer III Pyramidal Cell Circuits.

cAMP signaling has powerful, negative effects on cognitive functions of the primate dorsolateral prefrontal cortex (dlPFC), opening potassium channels to reduce firing and impair working memory, and increasing tau phosphorylation in aging neurons. This contrasts with cAMP actions in classic circuits, where it enhances plasticity and transmitter release. PDE4 isozymes regulate cAMP actions, and thus have been a focus of research and drug discovery. Previous work has focused on the localization of PDE4A and PDE4B in dlPFC, but PDE4D is also of great interest, as it is the predominant PDE4 isoform in primate association cortex, and PDE4D expression decreases with aging in human dlPFC. Here we used laser-capture microdissection transcriptomics and found that PDE4D message is enriched in pyramidal cells compared to GABAergic PV-interneurons in layer III of the human dlPFC. A parallel study in rhesus macaques using high-spatial resolution immunoelectron microscopy revealed the ultrastructural locations of PDE4D in primate dlPFC with clarity not possible in human post-mortem tissue. PDE4D was especially prominent in dendrites associated with microtubules, mitochondria, and likely smooth endoplasmic reticulum (SER). There was substantial postsynaptic labeling in dendritic spines, associated with the SER spine-apparatus near glutamatergic-like axospinous synapses, but sparse labeling in axon terminals. We also observed dense PDE4D labeling perisynaptically in astroglial leaflets ensheathing glutamatergic connections. These data suggest that PDE4D is strategically positioned to regulate cAMP signaling in dlPFC glutamatergic synapses and circuits, especially in postsynaptic compartments where it is localized to influence cAMP actions on intracellular trafficking, mitochondrial physiology, and internal calcium release.

Activity-Dependent Remodeling of Synaptic Protein Organization Revealed by High Throughput Analysis of STED Nanoscopy Images.

The organization of proteins in the apposed nanodomains of pre- and postsynaptic compartments is thought to play a pivotal role in synaptic strength and plasticity. As such, the alignment between pre- and postsynaptic proteins may regulate, for example, the rate of presynaptic release or the strength of postsynaptic signaling. However, the analysis of these structures has mainly been restricted to subsets of synapses, providing a limited view of the diversity of synaptic protein cluster remodeling during synaptic plasticity. To characterize changes in the organization of synaptic nanodomains during synaptic plasticity over a large population of synapses, we combined STimulated Emission Depletion (STED) nanoscopy with a Python-based statistical object distance analysis (pySODA), in dissociated cultured hippocampal circuits exposed to treatments driving different forms of synaptic plasticity. The nanoscale organization, characterized in terms of coupling properties, of presynaptic (Bassoon, RIM1/2) and postsynaptic (PSD95, Homer1c) scaffold proteins was differently altered in response to plasticity-inducing stimuli. For the Bassoon - PSD95 pair, treatments driving synaptic potentiation caused an increase in their coupling probability, whereas a stimulus driving synaptic depression had an opposite effect. To enrich the characterization of the synaptic cluster remodeling at the population level, we applied unsupervised machine learning approaches to include selected morphological features into a multidimensional analysis. This combined analysis revealed a large diversity of synaptic protein cluster subtypes exhibiting differential activity-dependent remodeling, yet with common features depending on the expected direction of plasticity. The expanded palette of synaptic features revealed by our unbiased approach should provide a basis to further explore the widely diverse molecular mechanisms of synaptic plasticity.

Alternative splicing of neurexins 1–3 is modulated by neuroinflammation in the prefrontal cortex of a murine model of multiple sclerosis

Mounting evidence points to immune-mediated synaptopathy and impaired plasticity as early pathogenic events underlying cognitive decline (CD) in Multiple sclerosis (MS) and in the experimental autoimmune encephalomyelitis (EAE) mouse model of the disease. However, knowledge of the neurobiology of synaptic dysfunction is still incomplete. Splicing regulation represents a flexible and powerful mechanism involved in dynamic remodeling of the synapse, which allows the expression of synaptic protein variants that dynamically control the specificity of contacts between neurons. The pre-synaptic adhesion molecules neurexins (NRXNs) 1–3 play a relevant role in cognition and are alternatively spliced to yield variants that differentially cluster specific ligands in the postsynaptic compartment and modulate functional properties of the synaptic contact. Notably, mutations in these genes or disruption of their splicing program are associated with neuropsychiatric disorders. Herein, we have investigated how inflammatory changes imposed by EAE impact on alternative splicing of the Nrxn 1–3 mouse genes in the acute phase of disease. Due to its relevance in cognition, we focused on the prefrontal cortex (PFC) of SJL/J mice, in which EAE-induced inflammatory lesions extend to the rostral forebrain. We found that inclusion of the Nrxn 1–3 AS4 exon is significantly increased in the PFC of EAE mice and that splicing changes are correlated with local Il1β-expression levels. This correlation is sustained by the concomitant downregulation of SLM2, the main splicing factor involved in skipping of the AS4 exon, in EAE mice displaying high levels of Il1β- expression. We also observed that Il1β-expression levels correlate with changes in parvalbumin (PV)-positive interneuron connectivity. Moreover, exposure to environmental enrichment (EE), a condition known to stimulate neuronal connectivity and to improve cognitive functions in mice and humans, modified PFC phenotypes of EAE mice with respect to Il1β-, Slm2-expression, Nrxn AS4 splicing and PV-expression, by limiting changes associated with high levels of inflammation. Our results reveal that local inflammation results in early splicing modulation of key synaptic proteins and in remodeling of GABAergic circuitry in the PFC of SJL/J mice. We also suggest EE as a tool to counteract these inflammation-associated events, thus highlighting potential therapeutic targets for limiting the progressive CD occurring in MS.

Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and Neuromuscular Junction Development

Neuromuscular junctions (NMJs) govern efficient neuronal communication with muscle cells, relying on proper architecture of specialized postsynaptic compartments. However, the intrinsic mechanism in muscle cells contributing to NMJ development remains unclear. In this study, we reveal that dynamin-2 (Dyn2) is involved in postsynaptic development of NMJs. Mutations of Dyn2 have been linked to human muscular disorder and centronuclear myopathy (CNM), as well as featured with muscle atrophy and defective NMJs, yet the function of Dyn2 at the postsynaptic membrane is largely unknown. We demonstrate that Dyn2 is enriched at the postsynaptic membrane and regulates NMJ development via actin remodeling. Dyn2 functions as an actin-bundling GTPase to regulate podosome turnover and cytoskeletal organization of the postsynaptic apparatus, and CNM-Dyn2 mutations display abnormal actin remodeling and electrophysiological activity of fly NMJs. Altogether, Dyn2 primarily regulates actin cytoskeleton remodeling and NMJ morphogenesis at the postsynaptic membrane, which is distinct from its endocytosis regulatory role at the presynaptic membrane.

Systematic Quantification of Synapses in Primary Neuronal Culture.

SummaryMost neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

The TOR Pathway at the Neuromuscular Junction: More Than a Metabolic Player?

The neuromuscular junction (NMJ) is the chemical synapse connecting motor neurons and skeletal muscle fibers. NMJs allow all voluntary movements, and ensure vital functions like breathing. Changes in the structure and function of NMJs are hallmarks of numerous pathological conditions that affect muscle function including sarcopenia, the age-related loss of muscle mass and function. However, the molecular mechanisms leading to the morphological and functional perturbations in the pre- and post-synaptic compartments of the NMJ remain poorly understood. Here, we discuss the role of the metabolic pathway associated to the kinase TOR (Target of Rapamycin) in the development, maintenance and alterations of the NMJ. This is of particular interest as the TOR pathway has been implicated in aging, but its role at the NMJ is still ill-defined. We highlight the respective functions of the two TOR-associated complexes, TORC1 and TORC2, and discuss the role of localized protein synthesis and autophagy regulation in motor neuron terminals and sub-synaptic regions of muscle fibers and their possible effects on NMJ maintenance.

Neurogranin, Encoded by the Schizophrenia Risk Gene NRGN, Bidirectionally Modulates Synaptic Plasticity via Calmodulin-Dependent Regulation of the Neuronal Phosphoproteome

BackgroundNeurogranin (Ng), encoded by the schizophrenia risk gene NRGN, is a calmodulin-binding protein enriched in the postsynaptic compartments, and its expression is reduced in the postmortem brains of patients with schizophrenia. Experience-dependent translation of Ng is critical for encoding contextual memory, and Ng regulates developmental plasticity in the primary visual cortex during the critical period. However, the overall impact of Ng on the neuronal signaling that regulates synaptic plasticity is unknown. MethodsAltered Ng expression was achieved via virus-mediated gene manipulation in mice. The effect on long-term potentiation (LTP) was accessed using spike timing–dependent plasticity protocols. Quantitative phosphoproteomics analyses led to discoveries in significant phosphorylated targets. An identified candidate was examined with high-throughput planar patch clamp and was validated with pharmacological manipulation. ResultsNg bidirectionally modulated LTP in the hippocampus. Decreasing Ng levels significantly affected the phosphorylation pattern of postsynaptic density proteins, including glutamate receptors, GTPases, kinases, RNA binding proteins, selective ion channels, and ionic transporters, some of which highlighted clusters of schizophrenia- and autism-related genes. Hypophosphorylation of NMDA receptor subunit Grin2A, one significant phosphorylated target, resulted in accelerated decay of NMDA receptor currents. Blocking protein phosphatase PP2B activity rescued the accelerated NMDA receptor current decay and the impairment of LTP mediated by Ng knockdown, implicating the requirement of synaptic PP2B activity for the deficits. ConclusionsAltered Ng levels affect the phosphorylation landscape of neuronal proteins. PP2B activity is required for mediating the deficit in synaptic plasticity caused by decreasing Ng levels, revealing a novel mechanistic link of a schizophrenia risk gene to cognitive deficits.

Cytoskeletal regulation of synaptogenesis in a model of human fetal brain development.

Excitatory synapse formation begins in mid-fetal gestation. However, due to our inability to image fetal synaptogenesis, the initial formation of synapses remains understudied. The recent development of human fetal brain spheroids provides access to this critical period of synapse formation. Using human neurons and brain spheroids, we address how altered actin regulation impacts the formation of excitatory synapses during fetal brain development. Prior to synapse formation, inhibition of RhoA kinase (ROCK) signaling promotes neurite elongation and branching. In addition to increasing neural complexity, ROCK inhibition increases the length of protrusions along the neurite, ultimately promoting excitatory synapse formation in human cortical brain spheroids. A corresponding increase in Rac1-driven actin polymerization drives this increase in excitatory synaptogenesis. Using STORM super-resolution microscopy, we demonstrate that actomyosin regulators, including the Rac1 regulator, α-PIX, and the RhoA regulator, p115-RhoGEF, localize to nascent excitatory synapses, where they preferentially localize to postsynaptic compartments. These results demonstrate that coordinated RhoGTPase activities underlie the initial formation of excitatory synapses and identify critical cytoskeletal regulators of early synaptogenic events.

Pyk2 overexpression in postsynaptic neurons blocks amyloid β1-42-induced synaptotoxicity in microfluidic co-cultures.

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer’s disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer’s disease, deciphering the impact of Alzheimer’s risk genes on synapse formation and maintenance is of great interest. In this article, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid β peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid β1–42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid β suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta—an Alzheimer’s disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer’s disease brains at gene expression and protein levels—selectively in postsynaptic neurons is protective against amyloid β1–42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically relevant model of Alzheimer’s disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments.

Neurons must establish and stabilize connections made with diverse targets, each with distinct demands and functional characteristics. At Drosophila neuromuscular junctions (NMJs), synaptic strength remains stable in a manipulation that simultaneously induces hypo-innervation on one target and hyper-innervation on the other. However, the expression mechanisms that achieve this exquisite target-specific homeostatic control remain enigmatic. Here, we identify the distinct target-specific homeostatic expression mechanisms. On the hypo-innervated target, an increase in postsynaptic glutamate receptor (GluR) abundance is sufficient to compensate for reduced innervation, without any apparent presynaptic adaptations. In contrast, a target-specific reduction in presynaptic neurotransmitter release probability is reflected by a decrease in active zone components restricted to terminals of hyper-innervated targets. Finally, loss of postsynaptic GluRs on one target induces a compartmentalized, homeostatic enhancement of presynaptic neurotransmitter release called presynaptic homeostatic potentiation (PHP) that can be precisely balanced with the adaptations required for both hypo- and hyper-innervation to maintain stable synaptic strength. Thus, distinct anterograde and retrograde signaling systems operate at pre- and post-synaptic compartments to enable target-specific, homeostatic control of neurotransmission.

Presynaptic APP levels and synaptic homeostasis are regulated by Akt phosphorylation of huntingtin.

Studies have suggested that amyloid precursor protein (APP) regulates synaptic homeostasis, but the evidence has not been consistent. In particular, signaling pathways controlling APP transport to the synapse in axons and dendrites remain to be identified. Having previously shown that Huntingtin (HTT), the scaffolding protein involved in Huntington's disease, regulates neuritic transport of APP, we used a microfluidic corticocortical neuronal network-on-a-chip to examine APP transport and localization to the pre- and post-synaptic compartments. We found that HTT, upon phosphorylation by the Ser/Thr kinase Akt, regulates APP transport in axons but not dendrites. Expression of an unphosphorylatable HTT decreased axonal anterograde transport of APP, reduced presynaptic APP levels, and increased synaptic density. Ablating in vivo HTT phosphorylation in APPPS1 mice, which overexpress APP, reduced presynaptic APP levels, restored synapse number and improved learning and memory. The Akt-HTT pathway and axonal transport of APP thus regulate APP presynaptic levels and synapse homeostasis.

Autaptic Cultures: Methods and Applications.

Neurons typically form daisy chains of synaptic connections with other neurons, but they can also form synapses with themselves. Although such self-synapses, or autapses, are comparatively rare in vivo, they are surprisingly common in dissociated neuronal cultures. At first glance, autapses in culture seem like a mere curiosity. However, by providing a simple model system in which a single recording electrode gives simultaneous access to the pre- and postsynaptic compartments, autaptic cultures have proven to be invaluable in facilitating important and elegant experiments in the area of synaptic neuroscience. Here, I provide detailed protocols for preparing and recording from autaptic cultures (also called micro-island or microdot cultures). Variations on the basic procedure are presented, as well as practical tips for optimizing the outcomes. I also illustrate the utility of autaptic cultures by reviewing the types of experiments that have used them over the past three decades. These examples serve to highlight the power and elegance of this simple model system, and will hopefully inspire new experiments for the interrogation of synaptic function.

Density of GABAB Receptors Is Reduced in Granule Cells of the Hippocampus in a Mouse Model of Alzheimer's Disease.

Metabotropic γ-aminobutyric acid (GABAB) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABAB receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABAB1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABAB receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABAB receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABAB1 at cytoplasmic sites. GABAB receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABAB receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABAB receptors in the DG suggest the alteration of the GABAB-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD.

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30–40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100–120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60–70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60–75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68–77 nm), than in control (59–63 nm) and stimulated samples (49–53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and the Maturation of Neuromuscular Junction

The neuromuscular junction (NMJ) governs rapid and efficient neuronal communication with muscle cells, which relies on the proper activity of postsynaptic compartment specialized with unique membrane and actin-dependent cytoskeleton organization. However, the intrinsic mechanism in muscle cells that contributes to the development and maintenance of the elaborate NMJ morphology is unclear. In this study, we discover that the large GTPase, dynamin, best-known for catalyzing synaptic vesicle endocytosis at the presynaptic membrane, is also involved in postsynaptic morphogenesis. By using cultured mouse myotube, Drosophila, and in vitro reconstitution assay, we demonstrate that dynamin-2 (Dyn2) is enriched in the postsynaptic membrane of muscle cells and is involved in the maturation of neurotransmitter receptor clusters and the functional integrity of NMJ. During the early stage of NMJ development, Dyn2 functions as a molecular girdle to regulate synaptic podosome turnover and promote the maturation of postsynaptic apparatus. After the maturation, Dyn2 remains enriched at NMJ to maintain proper actin cytoskeleton organization and its electrophysiological function. The self-assembly, GTP hydrolysis ability and Y597 phosphorylation of Dyn2 determine its localization at podosome and the actin bundling activity. Together, our results uncover a new function of Dyn2 on cytoskeleton remodeling and organization at the postsynaptic membrane of NMJ.

Functional interactions of ion channels with the actin cytoskeleton: does coupling to dynamic actin regulate NMDA receptors?

Synapses are enriched in the cytoskeletal protein actin, which determines the shape of the pre- and postsynaptic compartments, organizes the neurotransmitter release machinery, and provides a framework for trafficking of components. In the postsynaptic compartment, interactions with actin or its associated proteins are also critical for the localization and activity of synaptic neurotransmitter receptors and ion channels. Actin binding proteins, including spectrin and α-actinin, serve as molecular linkages between the actin cytoskeleton and a diverse collection of receptors, including the NMDA receptor (NMDAR) and voltage-gated Na+ channels. The actin cytoskeleton can regulate neurotransmitter receptors and ion channels by controlling their trafficking and localization at the synapse and by directly gating receptor channel opening. We highlight evidence that synaptic actin couples physically and functionally to the NMDAR and supports its activity. The molecular mechanisms by which actin regulates NMDARs are only just emerging, and recent advancements in light and electron microscopy-based imaging techniques should aide in elucidating these mechanisms.