Actin filaments and microtubules in dendritic spines

Dendritic spines are small protrusions emerging from their parent dendrites, and their morphological changes are involved in synaptic plasticity. These tiny structures are composed of thousands of different proteins belonging to several subfamilies such as membrane receptors, scaffold proteins, signal transduction proteins, and cytoskeletal proteins. Actin filaments in dendritic spines consist of double helix of actin protomers decorated with drebrin and ADF/cofilin, and the balance of the two is closely related to the actin dynamics, which may govern morphological and functional synaptic plasticity. During development, the accumulation of drebrin‐binding type actin filaments is one of the initial events occurring at the nascent excitatory postsynaptic site, and plays a pivotal role in spine formation as well as small GTPases. It has been recently reported that microtubules transiently appear in dendritic spines in correlation with synaptic activity. Interestingly, it is suggested that microtubule dynamics might couple with actin dynamics. In this review, we will summarize the contribution of both actin filaments and microtubules to the formation and regulation of dendritic spines, and further discuss the role of cytoskeletal deregulation in neurological disorders.     

Adducin Is an In Vivo Substrate for Protein Kinase C: Phosphorylation in the MARCKS-related Domain Inhibits Activity in Promoting Spectrin–Actin Complexes and Occurs in Many Cells,Including Dendritic Spines of Neurons

Adducin is a heteromeric protein with subunits containing a COOH-terminal myristoylated alanine-rich C kinase substrate (MARCKS)-related domain that caps and preferentially recruits spectrin to the fast-growing ends of actin filaments. The basic MARCKS-related domain, present in α, β, and γ adducin subunits, binds calmodulin and contains the major phosphorylation site for protein kinase C (PKC). This report presents the first evidence that phosphorylation of the MARCKS-related domain modifies in vitro and in vivo activities of adducin involving actin and spectrin, and we demonstrate that adducin is a prominent in vivo substrate for PKC or other phorbol 12-myristate 13-acetate (PMA)-activated kinases in multiple cell types, including neurons. PKC phosphorylation of native and recombinant adducin inhibited actin capping measured using pyrene-actin polymerization and abolished activity of adducin in recruiting spectrin to ends and sides of actin filaments. A polyclonal antibody specific to the phosphorylated state of the RTPS-serine, which is the major PKC phosphorylation site in the MARCKS-related domain, was used to evaluate phosphorylation of adducin in cells. Reactivity with phosphoadducin antibody in immunoblots increased twofold in rat hippocampal slices, eight- to ninefold in human embryonal kidney (HEK 293) cells, threefold in MDCK cells, and greater than 10-fold in human erythrocytes after treatments with PMA, but not with forskolin. Thus, the RTPS-serine of adducin is an in vivo phosphorylation site for PKC or other PMA-activated kinases but not for cAMP-dependent protein kinase in a variety of cell types. Physiological consequences of the two PKC phosphorylation sites in the MARCKS-related domain were investigated by stably transfecting MDCK cells with either wild-type or PKC-unphosphorylatable S716A/S726A mutant α adducin. The mutant α adducin was no longer concentrated at the cell membrane at sites of cell–cell contact, and instead it was distributed as a cytoplasmic punctate pattern. Moreover, the cells expressing the mutant α adducin exhibited increased levels of cytoplasmic spectrin, which was colocalized with the mutant α adducin in a punctate pattern. Immunofluorescence with the phosphoadducin-specific antibody revealed the RTPS-serine phosphorylation of adducin in postsynaptic areas in the developing rat hippocampus. High levels of the phosphoadducin were detected in the dendritic spines of cultured hippocampal neurons. Spectrin also was a component of dendritic spines, although at distinct sites from the ones containing phosphoadducin. These data demonstrate that adducin is a significant in vivo substrate for PKC or other PMA-activated kinases in a variety of cells, and that phosphorylation of adducin occurs in dendritic spines that are believed to respond to external signals by changes in morphology and reorganization of cytoskeletal structures.     

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca²⁺-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.     

Actin Out: Regulation of the Synaptic Cytoskeleton

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes that encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.     

Homer Tetramer Promotes Actin Bundling Activity of Drebrin

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Synaptopodin, a molecule involved in the formation of the dendritic spine apparatus, is a dual actin/alpha-actinin binding protein

Synaptopodin (SYNPO) is a cytoskeletal protein that is preferentially located in mature dendritic spines, where it accumulates in the spine neck and closely associates with the spine apparatus. Formation of the spine apparatus critically depends on SYNPO. To further determine its molecular action, we screened for cellular binding partners. Using the yeast two-hybrid system and biochemical assays, SYNPO was found to associate with both F-actin and alpha-actinin. Ectopic expression of SYNPO in neuronal and non-neuronal cells induced actin aggregates, thus confirming a cytoplasmic interaction with the actin cytoskeleton. Whereas F-actin association is mediated by a central SYNPO motif, binding to alpha-actinin requires the C-terminal domain. Notably, the alpha-actinin binding domain is also essential for dendritic targeting and postsynaptic accumulation of SYNPO in primary neurons. Taken together, our data suggest that dendritic spine accumulation of SYNPO critically depends on its interaction with postsynaptic alpha-actinin and that SYNPO may regulate spine morphology, motility and function via its distinct modes of association with the actin cytoskeleton.     

Novel Features of the Light Chain of Microtubule-associated Protein MAP1B: Microtubule Stabilization,Self Interaction,Actin Filament Binding,and Regulation by the Heavy Chain

Previous studies on the role of microtubule-associated protein 1B (MAP1B) in adapting microtubules for nerve cell-specific functions have examined the activity of the entire MAP1B protein complex consisting of heavy and light chains and revealed moderate effects on microtubule stability. Here we have analyzed the effects of the MAP1B light chain in the absence or presence of the heavy chain by immunofluorescence microscopy of transiently transfected cells. Distinct from all other MAPs, the MAP1B light chain–induced formation of stable but apparently flexible microtubules resistant to the effects of nocodazole and taxol. Light chain activity was inhibited by the heavy chain. In addition, the light chain was found to harbor an actin filament binding domain in its COOH terminus. By coimmunoprecipitation experiments using epitope-tagged fragments of MAP1B we showed that light chains can dimerize or oligomerize. Furthermore, we localized the domains for heavy chain–light chain interaction to regions containing sequences homologous to MAP1A. Our findings assign several crucial activities to the MAP1B light chain and suggest a new model for the mechanism of action of MAP1B in which the heavy chain might act as the regulatory subunit of the MAP1B complex to control light chain activity.     

The Phosphodiesterase 9 Inhibitor PF‐04449613 Promotes Dendritic Spine Formation and Performance Improvement after Motor Learning

The cyclic nucleotide cGMP is an intracellular second messenger with important roles in neuronal functions and animals' behaviors. The phosphodiesterases (PDEs) are a family of enzymes that hydrolyze the second messengers cGMP and cAMP. Inhibition of phosphodiesterase 9 (PDE9), a main isoform of PDEs hydrolyzing cGMP, has been shown to improve learning and memory as well as cognitive function in rodents. However, the role of PDE9 in regulating neuronal structure and function in vivo remains unclear. Here we used in vivo two‐photon microscopy to investigate the effect of a selective PDE9 inhibitor PF‐04449613 on the activity and plasticity of dendritic spines of layer V pyramidal neurons in the mouse primary motor cortex. We found that administration of PF‐04449613 increased calcium activity of dendrites and dendritic spines of layer V pyramidal neurons in mice under resting and running conditions. Chronic treatment of PF‐04449613 over weeks increased dendritic spine formation and elimination under basal conditions. Furthermore, PF‐04449613 treatment over 1–7 days increased the formation and survival of new spines as well as performance improvement after rotarod motor training. Taken together, our studies suggest that elevating the level of cGMP with the PDE9 inhibitor PF‐04449613 increases synaptic calcium activity and learning‐dependent synaptic plasticity, thereby contributing to performance improvement after learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000–000, 2018     

Matrix metalloproteinase-7 disrupts dendritic spines in hippocampal neurons through NMDA receptor activation

Dendritic spines are protrusions from the dendritic shaft that host most excitatory synapses in the brain. Although they first emerge during neuronal maturation, dendritic spines remain plastic through adulthood, and recent advances in the molecular mechanisms governing spine morphology have shown them to be exquisitely sensitive to changes in the micro-environment. Among the many factors affecting spine morphology are components and regulators of the extracellular matrix (ECM). Modification of the ECM is critical to the repair of injuries throughout the body, including the CNS. Matrix metalloproteinase (MMP)-7/matrilysin is a key regulator of the ECM during pathogen infection, after nerve crush and in encephalitogenic disorders. We have investigated the effects of MMP-7 on dendritic spines in hippocampal neuron cultures and found that it induces the transformation of mature, short mushroom-shaped spines into long, thin filopodia reminiscent of immature spines. These changes were accompanied by a dramatic redistribution of F-actin from spine heads into thick, rope-like structures in the dendritic shaft. Strikingly, MMP-7 effects on dendritic spines were similar to those of NMDA treatment, and both could be blocked by channel-specific antagonists. These findings are the first direct evidence that MMPs can influence the morphology of mature dendritic spines, and hence synaptic stability.     

PICK1 inhibition of the Arp2/3 complex controls dendritic spine size and synaptic plasticity

Activity-dependent remodelling of dendritic spines is essential for neural circuit development and synaptic plasticity, but the precise molecular mechanisms that regulate this process are unclear. Activators of Arp2/3-mediated actin polymerisation are required for spine enlargement; however, during long-term depression (LTD), spines shrink via actin depolymerisation and Arp2/3 inhibitors in this process have not yet been identified. Here, we show that PICK1 regulates spine size in hippocampal neurons via inhibition of the Arp2/3 complex. PICK1 knockdown increases spine size, whereas PICK1 overexpression reduces spine size. NMDA receptor activation results in spine shrinkage, which is blocked by PICK1 knockdown or overexpression of a PICK1 mutant that cannot bind Arp2/3. Furthermore, we show that PICK1-Arp2/3 interactions are required for functional hippocampal LTD. This work demonstrates that PICK1 is a novel regulator of spine dynamics. Via Arp2/3 inhibition, PICK1 has complementary yet distinct roles during LTD to regulate AMPA receptor trafficking and spine size, and therefore functions as a crucial factor in both structural and functional plasticity.     

Activity-Dependent Nucleation of Dynamic Microtubules at Presynaptic Boutons Controls Neurotransmission

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Expression and Electrophysiological Function of Actin in Chick Cerebellar Neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a ¹²⁵I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.     

Co-polymers of Actin and Tropomyosin Account for a Major Fraction of the Human Actin Cytoskeleton

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Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis ¹. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.     

Homeostatic Plasticity in the Hippocampus Facilitates Memory Extinction

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Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.