Synaptic Actin Research Articles

Nonhomogeneous distribution of filamentous actin in the presynaptic terminals on the spinal motoneurons

Although actin is known to play important roles in synapses, immunocytochemical and structural studies on synaptic actin have resulted in conflicting data, and the presence and precise localization of filamentous actin (F-actin) in the synapses have still not been well clarified. We recently described a phalloidin-based antifluorescein isothiocyanate system, which has been successfully developed for ultrastructural investigations of F-actin in the ependymal microvilli. By this technique, the present study has demonstrated the presence of F-actin in both the pre- and the postsynaptic regions in the synapses of spinal motoneurons. In the presynaptic terminal, F-actin was localized predominantly in the active zones and the adjacent synaptic vesicle clusters, including the vesicles docked at the active zones and a population of recycling vesicles. By contrast, the proximally located vesicle pool was much less intensely stained. In the postsynaptic region, F-actin was concentrated at the postsynaptic densities and stretched some way into the surrounding cytoplasm. Insofar as the axonal terminals analyzed in this study touched on either the cell body or the dendritic shaft, where the postsynaptic regions did not form spine-like specializations, our results cannot shed any light on the distribution of F-actin within spines. However, the present study has provided a hitherto unreported ultrastructural view of the subcellular distribution of F-actin in the synapse, which is thought to be helpful for understanding the roles of the synaptic actin cytoskeleton.

Proteomic Profiling of the Epileptic Dentate Gyrus

The development of epilepsy is often associated with marked changes in central nervous system cell structure and function. Along these lines, reactive gliosis and granule cell axonal sprouting within the dentate gyrus of the hippocampus are commonly observed in individuals with temporal lobe epilepsy (TLE). Here we used the pilocarpine model of TLE in mice to screen the proteome and phosphoproteome of the dentate gyrus to identify molecular events that are altered as part of the pathogenic process. Using a two-dimensional gel electrophoresis-based approach, followed by liquid chromatography-tandem mass spectrometry, 24 differentially expressed proteins, including 9 phosphoproteins, were identified. Functionally, these proteins were organized into several classes, including synaptic physiology, cell structure, cell stress, metabolism and energetics. The altered expression of three proteins involved in synaptic physiology, actin, profilin 1 and α-synuclein was validated by secondary methods. Interestingly, marked changes in protein expression were detected in the supragranular cell region, an area where robust mossy fibers sprouting occurs. Together, these data provide new molecular insights into the altered protein profile of the epileptogenic dentate gyrus and point to potential pathophysiologic mechanisms underlying epileptogenesis.

Retrograde BMP Signaling Controls Synaptic Growth at the NMJ by Regulating Trio Expression in Motor Neurons

Retrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ.

Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

The role of the inositol polyphosphate 5-phosphatases in cellular function and human disease

Phosphoinositides are membrane-bound signalling molecules that regulate cell proliferation and survival, cytoskeletal reorganization and vesicular trafficking by recruiting effector proteins to cellular membranes. Growth factor or insulin stimulation induces a canonical cascade resulting in the transient phosphorylation of PtdIns(4,5)P(2) by PI3K (phosphoinositide 3-kinase) to form PtdIns(3,4,5)P(3), which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) back to PtdIns(4,5)P(2), or by the 5-ptases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). The 5-ptases also hydrolyse PtdIns(4,5)P(2), forming PtdIns4P. Ten mammalian 5-ptases have been identified, which share a catalytic mechanism similar to that of the apurinic/apyrimidinic endonucleases. Gene-targeted deletion of 5-ptases in mice has revealed that these enzymes regulate haemopoietic cell proliferation, synaptic vesicle recycling, insulin signalling, endocytosis, vesicular trafficking and actin polymerization. Several studies have revealed that the molecular basis of Lowe's syndrome is due to mutations in the 5-ptase OCRL (oculocerebrorenal syndrome of Lowe). Futhermore, the 5-ptases SHIP [SH2 (Src homology 2)-domain-containing inositol phosphatase] 2, SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) and 72-5ptase (72 kDa 5-ptase)/Type IV/Inpp5e (inositol polyphosphate 5-phosphatase E) are implicated in negatively regulating insulin signalling and glucose homoeostasis in specific tissues. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. Gene profiling studies have identified changes in the expression of various 5-ptases in specific cancers. In addition, 5-ptases such as SHIP1, SHIP2 and 72-5ptase/Type IV/Inpp5e regulate macrophage phagocytosis, and SHIP1 also controls haemopoietic cell proliferation. Therefore the 5-ptases are a significant family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Emerging studies have implicated their loss or gain of function in human disease.

Formin-Dependent Synaptic Growth: Evidence That Dlar Signals via Diaphanous to Modulate Synaptic Actin and Dynamic Pioneer Microtubules

The diaphanous gene is the founding member of a family of Diaphanous-related formin proteins (DRFs). We identified diaphanous in a screen for genes that are necessary for the normal growth and stabilization of the Drosophila neuromuscular junction (NMJ). Here, we demonstrate that diaphanous mutations perturb synaptic growth at the NMJ. Diaphanous protein is present both presynaptically and postsynaptically. However, genetic rescue experiments in combination with additional genetic interaction experiments support the conclusion that dia is necessary presynaptically for normal NMJ growth. We then document defects in both the actin and microtubule cytoskeletons in dia mutant nerve terminals. In so doing, we define and characterize a population of dynamic pioneer microtubules within the NMJ that are distinct from the bundled core of microtubules identified by the MAP1b-like protein Futsch. Defects in both synaptic actin and dynamic pioneer microtubules are correlated with impaired synaptic growth in dia mutants. Finally, we present genetic evidence that Dia functions downstream of the presynaptic receptor tyrosine phosphatase Dlar and the Rho-type GEF (guanine nucleotide exchange factor) trio to control NMJ growth. Based on the established function of DRFs as Rho-GTPase-dependent regulators of the cell cytoskeleton, we propose a model in which Diaphanous links receptor tyrosine phosphatase signaling at the plasma membrane to growth-dependent modulation of the synaptic actin and microtubule cytoskeletons.

ROCK, PAK, and Toll of synapses in Alzheimer’s disease

Alzheimer’s disease is a neurodegenerative disorder where the cognitive deficit is the hallmark symptom reflecting the progression of the disease. Synaptic dysfunction is a sensitive parameter of the AD pathology. Rho GTPases and the Rho kinases, ROCK1/2, and PAK1-3, are important regulators of synaptic plasticity, especially in maintaining the actin cytoskeleton of dendritic spines. Recent studies have revealed that β-amyloid oligomers can inhibit PAK and stimulate ROCK-mediated signaling. Both of these effects enhance the disassembly of synaptic actin filaments and ultimately evoke synaptic loss. Brain tissue in AD recognizes the β-amyloid peptide oligomers as foreign protein particles and mounts an inflammatory defense via Toll-like receptor (TLR) signaling which causes synaptic impairment. We will review here the dysfunction of ROCK, PAK, and Toll signaling associated with AD pathology. The protection of synapses in AD may provide new therapeutic approaches to combatting the cognitive impairment in AD.

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.

Postsynaptic enrichment of Eps8 at dendritic shaft synapses of unipolar brush cells in rat cerebellum

Epidermal growth factor receptor pathway substrate 8 (Eps8) is a widely expressed multidomain signaling protein that coordinates two disparate GTPase-dependent mechanisms: actin reorganization via Ras/Rac pathways and receptor trafficking via Rab5. Expression of Eps8, the gene encoding the founding member of the Eps8 family of proteins, was found in cerebellum by virtual Northern analysis and in situ hybridization. Because the cerebellum has a well-known cellular architecture and is a favored model to study synaptic plasticity and actin dynamics, we sought to analyze Eps8 localization in rat cerebellar neurons and synapses by light and electron microscopy.Specificity of Eps8-antibody was demonstrated by immunoblots and in brain sections. In cerebellum, unipolar brush cells (UBCs) were densely Eps8 immunopositive and granule cells were moderately immunostained. In both types of neuron immunoreaction product was localized to the somatodendritic and axonal compartments. Postsynaptic immunostained foci were demonstrated in the glomeruli in correspondence of the synapses formed by mossy fiber terminals with granule cell and UBC dendrites. These foci appeared especially evident in the UBC brush, which contains an extraordinary postsynaptic apparatus of actin microfilaments facing synaptic junctions of the long and segmented varieties. Eps8 immunoreactivity was conspicuously absent in Purkinje cells and their actin-rich dendritic spines, in all types of inhibitory interneurons of the cerebellum, cerebellar nuclei neurons, and astrocytes. In conclusion, Eps8 protein in cerebellum is expressed exclusively by excitatory cortical interneurons and is intracellularly compartmentalized in a cell-class specific manner. This is the first demonstration of the presence of a member of the Eps8 protein family in UBCs and its enrichment at postsynaptic sites.

Akt1 deficiency affects neuronal morphology and predisposes to abnormalities in prefrontal cortex functioning.

There is accumulating evidence that AKT signaling plays a role in the pathogenesis of schizophrenia. We asked whether Akt1 deficiency in mice results in structural and functional abnormalities in prefrontal cortex (PFC). Exploratory transcriptional profiling revealed concerted alterations in the expression of PFC genes controlling synaptic function, neuronal development, myelination, and actin polymerization, and follow-up ultrastructural analysis identified consistent changes in the dendritic architecture of pyramidal neurons. Behavioral analysis indicated that Akt1-mutant mice have normal acquisition of a PFC-dependent cognitive task but abnormal working memory retention under neurochemical challenge of three distinct neurotransmitter systems. Thus, Akt1 deficiency creates a context permissive for gene-gene and gene-environment interactions that modulate PFC functioning and contribute to the disease risk associated with this locus, the severity of the clinical syndrome, or both.

Phosphorylation of Microtubule-associated Protein STOP by Calmodulin Kinase II

STOP proteins are microtubule-associated, calmodulin-regulated proteins responsible for the high degree of stabilization displayed by neuronal microtubules. STOP suppression in mice induces synaptic defects affecting both short and long term synaptic plasticity in hippocampal neurons. Interestingly, STOP has been identified as a component of synaptic structures in neurons, despite the absence of microtubules in nerve terminals, indicating the existence of mechanisms able to induce a translocation of STOP from microtubules to synaptic compartments. Here we have tested STOP phosphorylation as a candidate mechanism for STOP relocalization. We show that, both in vitro and in vivo, STOP is phosphorylated by the multifunctional enzyme calcium/calmodulin-dependent protein kinase II (CaMKII), which is a key enzyme for synaptic plasticity. This phosphorylation occurs on at least two independent sites. Phosphorylated forms of STOP do not bind microtubules in vitro and do not co-localize with microtubules in cultured differentiating neurons. Instead, phosphorylated STOP co-localizes with actin assemblies along neurites or at branching points. Correlatively, we find that STOP binds to actin in vitro. Finally, in differentiated neurons, phosphorylated STOP co-localizes with clusters of synaptic proteins, whereas unphosphorylated STOP does not. Thus, STOP phosphorylation by CaMKII may promote STOP translocation from microtubules to synaptic compartments where it may interact with actin, which could be important for STOP function in synaptic plasticity.

Activity-Induced Rapid Synaptic Maturation Mediated by Presynaptic Cdc42 Signaling

Maturation of presynaptic transmitter secretion machinery is a critical step in synaptogenesis. Here we report that a brief train of presynaptic action potentials rapidly converts early nonfunctional contacts between cultured hippocampal neurons into functional synapses by enhancing presynaptic glutamate release. The enhanced release was confirmed by a marked increase in the number of depolarization-induced FM4-64 puncta in the presynaptic axon. This rapid presynaptic maturation can be abolished by treatments that interfered with presynaptic BDNF and Cdc42 signaling or actin polymerization. Activation of Cdc42 by applying BDNF or bradykinin mimicked the effect of electrical activity in promoting synaptic maturation. Furthermore, activity-induced increase in presynaptic actin polymerization, as revealed by increased concentration of actin-YFP at axon boutons, was abolished by inhibiting BDNF and Cdc42 signaling. Thus, rapid presynaptic maturation induced by neuronal activity is mediated by presynaptic activation of the Cdc42 signaling pathway.

A role for talin in presynaptic function.

Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Iγ, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]–synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin–PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits with wide necks, as previously observed after perturbing actin at these synapses. Thus, the interaction of PIP kinase with talin in presynaptic compartments provides a mechanism to coordinate PI(4,5)P2 synthesis, actin dynamics, and endocytosis, and further supports a functional link between actin and clathrin-mediated endocytosis.

Remodeling of Synaptic Actin Induced by Photoconductive Stimulation

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals.

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.

Bin2, a Functionally Nonredundant Member of the BAR Adaptor Gene Family

BAR family proteins are a unique class of adaptor proteins characterized by a common N-terminal fold of undetermined function termed the BAR domain. This set of adaptors, which includes the mammalian proteins amphiphysin and Bin1 and the yeast proteins Rvs167p and Rvs161p, has been implicated in diverse cellular processes, including synaptic vesicle endocytosis, actin regulation, differentiation, cell survival, and tumorigenesis. Here we report the identification and characterization of Bin2, a novel protein that contains a BAR domain but that is otherwise structurally dissimilar to other members of the BAR adaptor family. The Bin2 gene is located at chromosome 4q22.1 and is expressed predominantly in hematopoietic cells. Bin2 is upregulated during differentiation of granulocytes, suggesting that it functions in that lineage. Bin2 formed a stable complex in cells with Bin1, but not with amphiphysin, in a BAR domain-dependent manner. This finding indicates that BAR domains have specific preferences for interaction. However, Bin2 did not influence endocytosis in the same manner as brain-specific splice isoforms of Bin1, nor did it exhibit the tumor suppressor properties inherent to ubiquitous splice isoforms of Bin1. Thus, Bin2 appears to encode a nonredundant function in the BAR adaptor gene family.

The actin cytoskeleton and neurotransmitter release: An overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.

Synaptojanin family members are implicated in endocytic membrane traffic in yeast.

The synaptojanins represent a subfamily of inositol 5'-phosphatases that contain an NH2-terminal Sac1p homology domain. A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function. The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth. We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis. A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant. None of the mutants are impaired in invertase secretion. The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects. Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function. These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Changes in dynamin and actin mRNA expression in the dorsal column-medial lemniscal system following dorsal column lesion.

Transection of the third cervical hindlimb dorsal column nerve fibers in the spinal cord leads to a partial deafferentation atrophy of the neurons of the ascending dorsal column-medial lemniscal neural network (DC-ML) up to the cortex. We now examine the alteration of the steady-state level mRNA coding for the synaptic vesicle protein, dynamin I and the cytoskeletal protein beta-actin as early indicators of direct and trans-synaptic changes in the relay nuclei of the DC-ML. Rats were sacrificed at 6, 24, 72, and 240 hr after C3 hindlimb dorsal column or sham lesion. By 24 hr, there are changes in the steady-state levels of mRNA coding for both dynamin I and beta-actin in regions of the brain containing the first (nucleus gracilis of medulla) and third synaptic relays (cortex). Beta-actin mRNA is increased at both 6 and 24 hr in the nucleus gracilis. The changes in dynamin I mRNA in the nucleus gracilis are early and biphasic, elevated at 6 hr but decreased compared to sham by 24 hr. In both regions, the initial fluctuations of dynamin I and beta-actin mRNA levels are transient. By 72 hr, the levels are no different from those of sham-lesioned animals. In the somatomotor cortex, there is an additional increase in beta-actin mRNA levels at 240 hr. The increased steady-state levels of dynamin and actin mRNA following a hindlimb dorsal column lesion suggest that increased synaptic vesicle recycling and actin cytoskeleton rearrangement are some of the early responses to deafferentation made by the neurons of the DC-ML synaptic relays.