Loss of the actin regulator cyclase-associated protein 1 (CAP1) modestly affects dendritic spine remodeling during synaptic plasticity

Dendritic spines form the postsynaptic compartment of most excitatory synapses in the vertebrate brain. Morphological changes of dendritic spines contribute to major forms of synaptic plasticity such as long-term potentiation (LTP) or depression (LTD). Synaptic plasticity underlies learning and memory, and defects in synaptic plasticity contribute to the pathogeneses of human brain disorders. Hence, deciphering the molecules that drive spine remodeling during synaptic plasticity is critical for understanding the neuronal basis of physiological and pathological brain function. Since actin filaments (F-actin) define dendritic spine morphology, actin-binding proteins (ABP) that accelerate dis-/assembly of F-actin moved into the focus as critical regulators of synaptic plasticity. We recently identified cyclase-associated protein 1 (CAP1) as a novel actin regulator in neurons that cooperates with cofilin1, an ABP relevant for synaptic plasticity. We therefore hypothesized a crucial role for CAP1 in structural synaptic plasticity. By exploiting mouse hippocampal neurons, we tested this hypothesis in the present study. We found that induction of both forms of synaptic plasticity oppositely altered concentration of exogenous, myc-tagged CAP1 in dendritic spines, with chemical LTP (cLTP) decreasing and chemical LTD (cLTD) increasing it. cLTP induced spine enlargement in CAP1-deficient neurons. However, it did not increase the density of large spines, different from control neurons. cLTD induced spine retraction and spine size reduction in control neurons, but not in CAP1-KO neurons. Together, we report that postsynaptic myc-CAP1 concentration oppositely changed during cLTP and cTLD and that CAP1 inactivation modestly affected structural plasticity.     

Preserved morphology and physiology of excitatory synapses in profilin1-deficient mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity.     

Targeting of the Arpc3 actin nucleation factor by miR-29a/b regulates dendritic spine morphology

Previous studies have demonstrated that microribonucleic acids (miRs) are key regulators of protein expression in the brain and modulate dendritic spine morphology and synaptic activity. To identify novel miRs involved in neuronal plasticity, we exposed adult mice to chronic treatments with nicotine, cocaine, or amphetamine, which are psychoactive drugs that induce well-documented neuroadaptations. We observed brain region- and drug-specific changes in miR expression levels and identified miR-29a/b as regulators of synaptic morphology. In vitro imaging experiments indicated that miR-29a/b reduce mushroom-shaped dendritic spines on hippocampal neurons with a concomitant increase in filopodial-like outgrowths, suggesting an effect on synapse formation via actin cytoskeleton remodeling. We identified Arpc3, a component of the ARP2/3 actin nucleation complex, as a bona fide target for down-regulation by miR-29a/b. This work provides evidence that targeting of Arpc3 by miR-29a/b fine tunes structural plasticity by regulating actin network branching in mature and developing spines.     

Protein Tyrosine Phosphatase PTP1B Is Involved in Hippocampal Synapse Formation and Learning

ER-bound PTP1B is expressed in hippocampal neurons, and accumulates among neurite contacts. PTP1B dephosphorylates ß-catenin in N-cadherin complexes ensuring cell-cell adhesion. Here we show that endogenous PTP1B, as well as expressed GFP-PTP1B, are present in dendritic spines of hippocampal neurons in culture. GFP-PTP1B overexpression does not affect filopodial density or length. In contrast, impairment of PTP1B function or genetic PTP1B-deficiency leads to increased filopodia-like dendritic spines and a reduction in mushroom-like spines, while spine density is unaffected. These morphological alterations are accompanied by a disorganization of pre- and post-synapses, as judged by decreased clustering of synapsin-1 and PSD-95, and suggest a dynamic synaptic phenotype. Notably, levels of ß-catenin-Tyr-654 phosphorylation increased ∼5-fold in the hippocampus of adult PTP1B^−/− (KO) mice compared to wild type (WT) mice and this was accompanied by a reduction in the amount of ß-catenin associated with N-cadherin. To determine whether PTP1B-deficiency alters learning and memory, we generated mice lacking PTP1B in the hippocampus and cortex (PTP1B^fl/fl–Emx1-Cre). PTP1B^fl/fl–Emx1-Cre mice displayed improved performance in the Barnes maze (decreased time to find and enter target hole), utilized a more efficient strategy (cued), and had better recall compared to WT controls. Our results implicate PTP1B in structural plasticity within the hippocampus, likely through modulation of N-cadherin function by ensuring dephosphorylation of ß-catenin on Tyr-654. Disruption of hippocampal PTP1B function or expression leads to elongation of dendritic filopodia and improved learning and memory, demonstrating an exciting novel role for this phosphatase.     

CNF1 improves astrocytic ability to support neuronal growth and differentiation in vitro

Modulation of cerebral Rho GTPases activity in mice brain by intracerebral administration of Cytotoxic Necrotizing Factor 1 (CNF1) leads to enhanced neurotransmission and synaptic plasticity and improves learning and memory. To gain more insight into the interactions between CNF1 and neuronal cells, we used primary neuronal and astrocytic cultures from rat embryonic brain to study CNF1 effects on neuronal differentiation, focusing on dendritic tree growth and synapse formation, which are strictly modulated by Rho GTPases. CNF1 profoundly remodeled the cytoskeleton of hippocampal and cortical neurons, which showed philopodia-like, actin-positive projections, thickened and poorly branched dendrites, and a decrease in synapse number. CNF1 removal, however, restored dendritic tree development and synapse formation, suggesting that the toxin can reversibly block neuronal differentiation. On differentiated neurons, CNF1 had a similar effacing effect on synapses. Therefore, a direct interaction with CNF1 is apparently deleterious for neurons. Since astrocytes play a pivotal role in neuronal differentiation and synaptic regulation, we wondered if the beneficial in vivo effect could be mediated by astrocytes. Primary astrocytes from embryonic cortex were treated with CNF1 for 48 hours and used as a substrate for growing hippocampal neurons. Such neurons showed an increased development of neurites, in respect to age-matched controls, with a wider dendritic tree and a richer content in synapses. In CNF1-exposed astrocytes, the production of interleukin 1β, known to reduce dendrite development and complexity in neuronal cultures, was decreased. These results demonstrate that astrocytes, under the influence of CNF1, increase their supporting activity on neuronal growth and differentiation, possibly related to the diminished levels of interleukin 1β. These observations suggest that the enhanced synaptic plasticity and improved learning and memory described in CNF1-injected mice are probably mediated by astrocytes.     

MAP1B‐dependent Rac activation is required for AMPA receptor endocytosis during long‐term depression

The microtubule‐associated protein 1B (MAP1B) plays critical roles in neurite growth and synapse maturation during brain development. This protein is well expressed in the adult brain. However, its function in mature neurons remains unknown. We have used a genetically modified mouse model and shRNA techniques to assess the role of MAP1B at established synapses, bypassing MAP1B functions during neuronal development. Under these conditions, we found that MAP1B deficiency alters synaptic plasticity by specifically impairing long‐term depression (LTD) expression. Interestingly, this is due to a failure to trigger AMPA receptor endocytosis and spine shrinkage during LTD. These defects are accompanied by an impaired targeting of the Rac1 activator Tiam1 at synaptic compartments. Accordingly, LTD and AMPA receptor endocytosis are restored in MAP1B‐deficient neurons by providing additional Rac1. Therefore, these results indicate that the MAP1B‐Tiam1‐Rac1 relay is essential for spine structural plasticity and removal of AMPA receptors from synapses during LTD. This work highlights the importance of MAPs as signalling hubs controlling the actin cytoskeleton and receptor trafficking during plasticity in mature neurons.     

Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 - A quantitative Golgi cox study

Gangliosides are characteristic plasma membrane constituents of vertebrate brain used as milestones of neuronal development. As neuronal morphology is a good indicator of neuronal differentiation, we analyzed how lack of the ganglioside biosynthetic gene Galgt1 whose product is critical for production of four major adult mammalian brain complex gangliosides (GM1, GD1a, GD1b and GT1b) affects neuronal maturation in vivo. To define maturation of cortical neurons in mice lacking B4galnt1 we performed a morphological analysis of Golgi-Cox impregnated pyramidal neurons in primary motor cortex and granular cells of dentate gyrus in 3, 21 and 150 days old B4galnt1-null and wild type mice. Quantitative analysis of basal dendritic tree on layer III pyramidal neurons in the motor cortex showed very immature dendritic picture in both mice at postnatal day 3. At postnatal day 21 both mice reached adult values in dendritic length, complexity and spine density. No quantitative differences were found between B4galnt1-null and wild type mice in pyramidal cells of motor cortex or granular cells of dentate gyrus at any examined age. In addition, the general structural and neuronal organization of all brain structures, qualitatively observed on Nissl and Golgi-Cox, were similar Our results demonstrate that neurons can develop normal dendritic complexity and length without presence of complex gangliosides in vivo. Therefore, behavioral differences observed in B4galnt1-null mice may be attributed to functional rather than morphological level of dendrites and spines of cortical pyramidal neurons.     

REELIN and schizophrenia: a disease at the interface of the genome and the epigenome

The downregulation of the Reelin gene (RELN) that occurs in schizophrenic brains, which are characterized by pyramidal neurons with shortened dendrites and by reduced expression densities of dendritic spines, may well result from hypermethylation of the RELN promoter. In the adult mammalian brain, gamma-aminoburytic acid-secreting (GABAergic) interneurons release RELN into the extracellular matrix, where it binds with high affinity to the integrin receptors present at dendritic spine postsynaptic densities and likely plays a role, elaborated in this article, in synaptic plasticity. In heterozygous reeler mice, which are haploinsufficient in RELN, inhibitors of histone deacetylase increase DNA demethylase activity and restore RELN expression. Such inhibitors could thus be of therapeutic value in mitigating vulnerability to schizophrenia among high-risk individuals.     

MAP1B Regulates Axonal Development by Modulating Rho-GTPase Rac1 Activity

Cultured neurons obtained from MAP1B-deficient mice have a delay in axon outgrowth and a reduced rate of axonal elongation compared with neurons from wild-type mice. Here we show that MAP1B deficiency results in a significant decrease in Rac1 and cdc42 activity and a significant increase in Rho activity. We found that MAP1B interacted with Tiam1, a guanosine nucleotide exchange factor for Rac1. The decrease in Rac1/cdc42 activity was paralleled by decreases in the phosphorylation of the downstream effectors of these proteins, such as LIMK-1 and cofilin. The expression of a constitutively active form of Rac1, cdc42, or Tiam1 rescued the axon growth defect of MAP1B-deficient neurons. Taken together, these observations define a new and crucial function of MAP1B that we show to be required for efficient cross-talk between microtubules and the actin cytoskeleton during neuronal polarization.     

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. We have identified an ephrinB-EphB receptor trans-synaptic signaling pathway which regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin, or dominant-negative Rac1, or inhibition of PAK eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity.     

GSK-3β and MMP-9 Cooperate in the Control of Dendritic Spine Morphology

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3β (GSK-3β) has been implicated. Despite much evidence of the involvement of GSK-3β in functional synaptic plasticity, it is unclear how GSK-3β controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3β in neurons to investigate how GSK-3β affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3β activity increased the number of thin spines, whereas lacking GSK-3β increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3β activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3β overexpressing mice. Administration of the nonspecific GSK-3β inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3β and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3β activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3β-induced synaptic alterations.     

Scaffold Protein X11α Interacts with Kalirin-7 in Dendrites and Recruits It to Golgi Outposts

Pyramidal neurons in the mammalian forebrain receive their synaptic inputs through their dendritic trees, and dendritic spines are the sites of most excitatory synapses. Dendritic spine structure is important for brain development and plasticity. Kalirin-7 is a guanine nucleotide-exchange factor for the small GTPase Rac1 and is a critical regulator of dendritic spine remodeling. The subcellular localization of kalirin-7 is thought to be important for regulating its function in neurons. A yeast two-hybrid screen has identified the adaptor protein X11α as an interacting partner of kalirin-7. Here, we show that kalirin-7 and X11α form a complex in the brain, and this interaction is mediated by the C terminus of kalirin-7. Kalirin-7 and X11α co-localize at excitatory synapses in cultured cortical neurons. Using time-lapse imaging of fluorescence recovery after photobleaching, we show that X11α is present in a mobile fraction of the postsynaptic density. X11α also localizes to Golgi outposts in dendrites, and its overexpression induces the removal of kalirin-7 from spines and accumulation of kalirin-7 in Golgi outposts. In addition, neurons overexpressing X11α displayed thinner spines. These data support a novel mechanism of regulation of kalirin-7 localization and function in dendrites, providing insight into signaling pathways underlying neuronal plasticity. Dissecting the molecular mechanisms of synaptic structural plasticity will improve our understanding of neuropsychiatric and neurodegenerative disorders, as kalirin-7 has been associated with schizophrenia and Alzheimer disease.     

Dicer and eIF2c are enriched at postsynaptic densities in adult mouse brain and are modified by neuronal activity in a calpain-dependent manner

We have hypothesized that small RNAs may participate in learning and memory mechanisms. Because dendritic spines are important in synaptic plasticity and learning, we asked whether dicer, the rate-limiting enzyme in the formation of small RNAs, is enriched within dendritic spines. In adult mouse brain, dicer and the RNA-induced silencing complex (RISC) component eIF2c were expressed in the somatodendritic compartment of principal neurons and some interneurons in many regions, and dicer was enriched in dendritic spines and postsynaptic densities (PSDs). A portion of dicer and eIF2c were associated with each other and with fragile X mental retardation protein (FMRP), as assessed by co-immunoprecipitation. Calpain I treatment of recombinant dicer or immunopurified brain dicer caused a marked increase in RNAse III activity. Purified PSDs did not exhibit RNAse III activity, but calpain caused release of dicer from PSDs in an enzymatically active form, together with eIF2c. NMDA stimulation of hippocampal slices, or calcium treatment of synaptoneurosomes, caused a 75 kDa dicer fragment to appear in a calpain-dependent manner. The findings support a model whereby acute neuronal stimulation at excitatory synapses increases intracellular calcium, which activates calpain, which liberates dicer and eIF2c bound to PSDs. This supports the hypothesis that dicer could be involved in synaptic plasticity.     

Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons.     

β-adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the α- and γ-adducin subunits

Adducins are a family of proteins found in cytoskeleton junctional complexes, which bind and regulate actin filaments and actin-spectrin complexes. In brain, adducin is expressed at high levels and is identified as a constituent of synaptic structures, such as dendritic spines and growth cones of neurons. Adducin-induced changes in dendritic spines are involved in activity-dependent synaptic plasticity processes associated with learning and memory, but the mechanisms underlying these functions remain to be elucidated. Here, β-adducin knockout (KO) mice were used to obtain a deeper insight into the role of adducin in these processes. We showed that β-adducin KO mice showed behavioral, motor coordination and learning deficits together with an altered expression and/or phosphorylation levels of α-adducin and γ-adducin. We found that β-adducin KO mice exhibited deficits in learning and motor performances associated with an impairment of long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. These effects were accompanied by a decrease in phosphorylation of adducin, a reduction in α-adducin expression levels and upregulation of γ-adducin in hippocampus, cerebellum and neocortex of mutant mice. In addition, we found that the mRNA encoding β-adducin is also located in dendrites, where it may participate in the fine modulation of LTP and LTD. These results strongly suggest coordinated expression and phosphorylation of adducin subunits as a key mechanism underlying synaptic plasticity, motor coordination performance and learning behaviors.     

Formation of dendritic spines with GABAergic synapses induced by whisker stimulation in adult mice

During development, alterations in sensory experience modify the structure of cortical neurons, particularly at the level of the dendritic spine. Are similar adaptations involved in plasticity of the adult cortex? Here we show that a 24 hr period of single whisker stimulation in freely moving adult mice increases, by 36%, the total synaptic density in the corresponding cortical barrel. This is due to an increase in both excitatory and inhibitory synapses found on spines. Four days after stimulation, the inhibitory inputs to the spines remain despite total synaptic density returning to pre-stimulation levels. Functional analysis of layer IV cells demonstrated altered response properties, immediately after stimulation, as well as four days later. These results indicate activity-dependent alterations in synaptic circuitry in adulthood, modifying the flow of sensory information into the cerebral cortex.     

The p21-activated Kinase PAK3 Forms Heterodimers with PAK1 in Brain Implementing Trans-regulation of PAK3 Activity

p21-activated kinase 1 (PAK1) and PAK3 belong to group I of the PAK family and control cell movement and division. They also regulate dendritic spine formation and maturation in the brain, and play a role in synaptic transmission and synaptic plasticity. PAK3, in particular, is known for its implication in X-linked intellectual disability. The pak3 gene is expressed in neurons as a GTPase-regulated PAK3a protein and also as three splice variants which display constitutive kinase activity. PAK1 regulation is based on its homodimerization, forming an inactive complex. Here, we analyze the PAK3 capacity to dimerize and show that although PAK3a is able to homodimerize, it is more likely to form heterodimeric complexes with PAK1. We further show that two intellectual disability mutations impair dimerization with PAK1. The b and c inserts present in the regulatory domain of PAK3 splice variants decrease the dimerization but retain the capacity to form heterodimers with PAK1. PAK1 and PAK3 are co-expressed in neurons, are colocalized within dendritic spines, co-purify with post-synaptic densities, and co-immunoprecipitate in brain lysates. Using kinase assays, we demonstrate that PAK1 inhibits the activity of PAK3a but not of the splice variant PAK3b in a trans-regulatory manner. Altogether, these results show that PAK3 and PAK1 signaling may be coordinated by heterodimerization.     

Comparison between basal and apical dendritic spines in estrogen-induced rapid spinogenesis of CA1 principal neurons in the adult hippocampus

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Here, we demonstrated the rapid effect of 17beta-estradiol on the density and morphology of spines in the stratum oriens (s.o., basal side) and in the stratum lacunosum-moleculare (s.l.m., apical side) by imaging Lucifer Yellow-injected CA1 neurons in adult male rat hippocampal slices, because spines in s.o. and s.l.m. have been poorly understood as compared with spines in the stratum radiatum. The application of 1nM estradiol-induced a rapid increase in the density of spines of pyramidal neurons within 2h. This increase by estradiol was blocked by Erk MAP kinase inhibitor and estrogen receptor inhibitor in both regions. Effect of blockade by agonists of AMPA receptors and NMDA receptors was different between s.o. and s.l.m. In both regions, ERalpha agonist PPT induced the same enhancing effect of spinogenesis as that induced by estradiol.     

Altered cortical synaptic morphology and impaired memory consolidation in forebrain- specific dominant-negative PAK transgenic mice

Molecular and cellular mechanisms for memory consolidation in the cortex are poorly known. To study the relationships between synaptic structure and function in the cortex and consolidation of long-term memory, we have generated transgenic mice in which catalytic activity of PAK, a critical regulator of actin remodeling, is inhibited in the postnatal forebrain. Cortical neurons in these mice displayed fewer dendritic spines and an increased proportion of larger synapses compared to wild-type controls. These alterations in basal synaptic morphology correlated with enhanced mean synaptic strength and impaired bidirectional synaptic modifiability (enhanced LTP and reduced LTD) in the cortex. By contrast, spine morphology and synaptic plasticity were normal in the hippocampus of these mice. Importantly, these mice exhibited specific deficits in the consolidation phase of hippocampus-dependent memory. Thus, our results provide evidence for critical relationships between synaptic morphology and bidirectional modifiability of synaptic strength in the cortex and consolidation of long-term memory.