Regulation Of Spine Morphology Research Articles

Exosomes from WT Mice Enhances Memory Recall-induced IEG Expression and Rescue Dendritic Spines Deficit in Angelman Syndrome Mice

Angelman syndrome (AS) is a rare neurogenetic disorder caused by deletion or mutations in the UBE3A gene, which lead to the deficiency of the UBE3A protein in neurons. UBE3A is crucial for normal neuronal communication, as it regulates the turnover of synaptic proteins and synaptic plasticity. Exosomes, small extracellular vesicles released by various cells, including neurons, play important roles in intercellular communication. Exosomes contain a variety of molecules, including proteins and nucleic acids, and can transfer these components between cells. Emerging evidence indicates that exosomes are critical for normal brain functions, and in recent years many studies have investigated the therapeutic potential of exosomes as vehicles for delivering therapeutic cargoes in various neurological disorders. In this study, we investigated the potential role of exosomes in regulating spine morphology in AS mice. Our results showed that activation of the lysosomal calcium channel TRPML1 stimulated exosome release from synaptosomes prepared from adult WT mice, but not from synaptosomes prepared from adult AS mice. Intriguingly, UBE3A was present in exosomes secreted from WT mice. It has been previously shown that hippocampal neurons of AS mice exhibit fewer dendritic spines, as compared to those from WT mice. Incubation of cultured hippocampal neurons from AS mice with conditioned medium from cultured hippocampal neurons from WT mice rescued the number of dendritic spines. Our data suggest that this effect is most likely due to exosomes in the medium, since purified exosomes from conditioned medium of WT neurons produced a similar rescue effect in cultured neurons from AS mice. Moreover, intravenous injection of exosomes prepared from synaptosomes of adult WT mice seemed to enhance contextual memory recall and associated immediate early gene (IEG) expression, and te number of dendritic spines in CA1 pyramidal neurons in adult AS mice. Whether and to what degree UBE3A present in WT exosomes participates in the rescue of AS neuronal morphology remains to be determined. Together, these results reveal both altered exosomal secretion and composition in AS mice. Treatment of AS neurons or adult AS mice with exosomes rescued dendritic spine deficits in vitro and in vivo. These results open new therapeutical approaches for using exosomes as a potential treatment for this disease.

Kainate receptors GluK1 and GluK2 differentially regulate synapse morphology.

The regulation of dendritic spine morphology is a critical aspect of neuronal network refinement during development and modulation of neurotransmission. Previous studies revealed that glutamatergic transmission plays a central role in synapse development. AMPA receptors and NMDA receptors regulate spine morphology in an activity dependent manner. However, whether and how Kainate receptors (KARs) regulate synapse development remains poorly understood. In this study, we found that GluK1 and GluK2 may play distinct roles in synapse development. In primary cultured hippocampal neurons, we found overexpression of the calcium-permeable GluK2(Q) receptor variant increased spine length and spine head area compared to overexpression of the calcium-impermeable GluK2(R) variant or EGFP transfected, control neurons, indicating that Q/R editing may play a role in GluK2 regulation of synapse development. Intriguingly, neurons transfected with GluK1(Q) showed decreased spine length and spine head area, while the density of dendritic spines was increased, suggesting that GluK1(Q) and GluK2(Q) have different effects on synaptic development. Swapping the critical domains between GluK2 and GluK1 demonstrated the N-terminal domain (NTD) is responsible for the different effects of GluK1 and GluK2. In conclusion, Kainate receptors GluK1 and GluK2 have distinct roles in regulating spine morphology and development, a process likely relying on the NTD.

KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization.

Dynamic microtubules play a critical role in cell structure and function. In nervous system, microtubules are the major route for cargo protein trafficking and they specially extend into and out of synapses to regulate synaptic development and plasticity. However, the detailed depolymerization mechanism that regulates dynamic microtubules in synapses and dendrites is still unclear. In this study, we find that KIF2C, a dynamic microtubule depolymerization protein without known function in the nervous system, plays a pivotal role in the structural and functional plasticity of synapses and regulates cognitive function in mice. Through its microtubule depolymerization capability, KIF2C regulates microtubule dynamics in dendrites, and regulates microtubule invasion of spines in neurons in a neuronal activity-dependent manner. Using RNAi knockdown and conditional knockout approaches, we showed that KIF2C regulates spine morphology and synaptic membrane expression of AMPA receptors. Moreover, KIF2C deficiency leads to impaired excitatory transmission, long-term potentiation, and altered cognitive behaviors in mice. Collectively, our study explores a novel function of KIF2C in the nervous system and provides an important regulatory mechanism on how activity-dependent microtubule dynamic regulates synaptic plasticity and cognition behaviors.

Alzheimer's disease risk factor CD2AP functions at synapses via spinal F-actin.

In late-onset Alzheimer's disease (LOAD), the causal mechanisms of synaptic dysfunction underlying memory deficits that lead to dementia remain unclear. Genetic studies of LOAD patients identified gene variants associated with AD. Now it is necessary to translate the genotype into the mechanism of synaptic dysfunction. Here we investigated the impact of the putative LOAD risk factor CD2AP loss of function. We hypothesized that CD2AP might regulate spinal actin since CD2AP is a regulator of actin dynamics in non-neuronal cells, and the actin cytoskeleton dynamics is essential for spine plasticity. We have used wild-type mouse primary cortical neurons after differentiation for 15-21 DIV. To knockdown CD2AP, we infected primary neurons with lentivirus expressing GFP and shCD2AP or non-targeting shRNA as control. We used immunofluorescence microscopy, using epifluorescence and confocal microscopy, and quantitative morphologic and subcellular analysis using ICY and Imaris software to analyze CD2AP synaptic localization, synapses and spines density, as well as spinal F-actin. We discovered that CD2AP is present in spines. CD2AP knockdown reduced spines density. In contrast, CD2AP overexpression increased spine density. Morphologically, CD2AP overexpression enlarged spine heads. Synapses density was, like spines, reduced by CD2AP knockdown. Mechanistically, we found that spinal F-actin decreased by CD2AP knockdown and increased upon CD2AP overexpression. These results indicate that CD2AP regulates spine morphology and thus synapses, likely by regulating actin dynamics at spines. Overall CD2AP variants may contribute to LOAD development by directly interfering with synapses.

CAMP Signaling–Mediated Phosphorylation of Diacylglycerol Lipase α Regulates Interaction With Ankyrin-G and Dendritic Spine Morphology

BackgroundDiacylglycerol lipase α (DAGLα), a major biosynthetic enzyme for endogenous cannabinoid signaling, has emerged as a risk gene in multiple psychiatric disorders. However, its role in the regulation of dendritic spine plasticity is unclear. MethodsDAGLα wild-type or point mutants were overexpressed in primary cortical neurons or human embryonic kidney 293T cells. The effects of mutated variants on interaction, dendritic spine morphology, and dynamics were examined by proximity ligation assay or fluorescence recovery after photobleaching. Behavioral tests and immunohistochemistry were performed with ankyrin-G conditional knockout and wild-type male mice. ResultsDAGLα modulated dendritic spine size and density, but the effects of changes in its protein level versus enzymatic activity were different, implicating either a 2-arachidonoylglycerol (2-AG)–dependent or –independent mechanism. The 2-AG–independent effects were mediated by the interaction of DAGLα with ankyrin-G, a multifunctional scaffold protein implicated in psychiatric disorders. Using superresolution microscopy, we observed that they colocalized in distinct nanodomains, which correlated with spine size. In situ proximity ligation assay combined with structured illumination microscopy revealed that DAGLα phosphorylation upon forskolin treatment enhanced the interaction with ankyrin-G in spines, leading to increased spine size and decreased DAGLα surface diffusion. Ankyrin-G conditional knockout mice showed significantly decreased DAGLα-positive neurons in the forebrain. In mice, ankyrin-G was required for forskolin-dependent reversal of depression-related behavior. ConclusionsTaken together, ANK3 and DAGLA, both neuropsychiatric disorder genes, interact in a complex to regulate spine morphology. These data reveal novel synaptic signaling mechanisms and potential therapeutic avenues.

The late‐onset Alzheimer's disease genetic risk factor CD2AP impacts synapses via actin dynamics

AbstractBackgroundIn late‐onset Alzheimer’s disease (LOAD) the causal mechanisms of synaptic dysfunction that underlie the memory deficits that lead to dementia are not established. The search for genetic risk factors in LOAD led to the identification of more than 20 genes and three main mechanisms: endocytic trafficking, lipid metabolism and immune response. Here we aim to establish causality between a mutation in CD2AP, one of the endocytic trafficking risk factors, and synaptic dysfunction. Since CD2AP is a regulator of the actin cytoskeleton dynamics which is essential for spine morphology, we hypothesize that a LOAD mutation CD2AP may impact spines by deregulating actin.MethodWe have analyzed synapses and spines density as well as spinal actin in cortical mouse primary neurons either transiently overexpressing wild‐type and mutant CD2AP‐GFP as well as GFP as control or infected with lentivirus expressing GFP and shCD2AP or non‐targeting shRNA as control. Moreover, we investigated CD2AP synaptic localization by immunofluorescence microscopy. We used quantitative single‐cell analysis using ICY and Imaris software.ResultWe discovered that a pool of CD2AP is present in spines; and that both CD2AP wild‐type and mutant are significantly enriched in spines. Spines length decreased with larger spine heads, by CD2AP wild‐type and more pronouncedly by mutant CD2AP overexpression. Indicating that CD2AP might function at synapses, by regulating spine morphology. Indeed, synapses density was reduced by CD2AP knockdown. Mechanistically, we found that spinal F‐actin increased by CD2AP wild‐type and more pronouncedly by mutant CD2AP overexpression compared to control neurons. We are currently investigating whether spinal F‐actin is altered by CD2AP knockdown.ConclusionThese results indicate that CD2AP regulates spine morphology and thus synapses. A LOAD mutant CD2AP may interfere with its function by dysregulating actin dynamics at spines. Overall CD2AP variants may contribute to LOAD development by directly interfering with synapses.

Impact of JNK and Its Substrates on Dendritic Spine Morphology.

The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates.

Vasodilator-stimulated phosphoprotein (VASP) is recruited into dendritic spines via G-actin-dependent mechanism and contributes to spine enlargement and stabilization.

Actin organization and dynamics are modulated by diverse actin regulators during dendritic spine development. To understand the molecular network that regulates actin organization and spine morphology, it is important to investigate dynamic redistribution of actin regulators during spine development. One of the actin regulators, vasodilator-stimulated phosphoprotein (VASP), has multiple functions in actin regulation and is known to regulate spine morphology. However, dynamics and temporal regulation of VASP during spine development have not been clarified. In this study, we performed time-lapse imaging of mouse hippocampal dissociated neurons to analyse the change in localization of VASP during spine development. We found that accumulation of VASP within spines precedes the start of persistent F-actin increase, which are temporally coupled with spine enlargement. Using domain deletion or mutation constructs of VASP, we revealed that the interaction with G-actin is important for the preceding accumulation of VASP. Furthermore, we showed that accumulation of VASP contributes to actin enrichment within spines and stabilization of spine morphology by dominant negative experiments. These data suggest that G-actin-dependent VASP recruitment has dual functions in spine development, enlargement and stabilization, through the interaction with actin and other cytoskeletal regulators.

EphA4 loss improves social memory performance and alters dendritic spine morphology without changes in amyloid pathology in a mouse model of Alzheimer\u2019s disease

BackgroundEphA4 is a receptor of the ephrin system regulating spine morphology and plasticity in the brain. These processes are pivotal in the pathophysiology of Alzheimer’s disease (AD), characterized by synapse dysfunction and loss, and the progressive loss of memory and other cognitive functions. Reduced EphA4 signaling has been shown to rescue beta-amyloid-induced dendritic spine loss and long-term potentiation (LTP) deficits in cultured hippocampal slices and primary hippocampal cultures. In this study, we investigated whether EphA4 ablation might preserve synapse function and ameliorate cognitive performance in the APPPS1 transgenic mouse model of AD.MethodsA postnatal genetic ablation of EphA4 in the forebrain was established in the APPPS1 mouse model of AD, followed by a battery of cognitive tests at 9 months of age to investigate cognitive function upon EphA4 loss. A Golgi-Cox staining was used to explore alterations in dendritic spine density and morphology in the CA1 region of the hippocampus.ResultsUpon EphA4 loss in APPPS1 mice, we observed improved social memory in the preference for social novelty test without affecting other cognitive functions. Dendritic spine analysis revealed altered synapse morphology as characterized by increased dendritic spine length and head width. These modifications were independent of hippocampal plaque load and beta-amyloid peptide levels since these were similar in mice with normal versus reduced levels of EphA4.ConclusionLoss of EphA4 improved social memory in a mouse model of Alzheimer’s disease in association with alterations in spine morphology.

Dendritic spine morphology and memory formation depend on postsynaptic Caskin proteins

CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.

The role of CaMKII-Tiam1 complex on learning and memory.

A stimulation inducing long-term potentiation (LTP) of synaptic transmission induces a persistent expansion of dendritic spines, a phenomenon known as structural LTP (sLTP). We previously proposed that the formation of a reciprocally activating kinase-effector complex (RAKEC) between CaMKII and Tiam1, an activator of the small G-protein Rac1, locks CaMKII into an active conformation, which in turn maintains the phosphorylation status of Tiam1. This makes Rac1 persistently active, specifically in the stimulated spine. To understand the significance of the CaMKII-Tiam1 RAKEC in vivo, we generated a Tiam1 mutant knock-in mouse line in which critical residues for CaMKII binding were mutated into alanines. We confirmed the central role of this interaction on sLTP by observing that KI mice showed reduced Rac1 activity, had smaller spines and a diminished sLTP as compared to their wild-type littermates. Moreover, behavioral tests showed that the novel object recognition memory of these animals was impaired. We thus propose that the CaMKII-Tiam1 interaction regulates spine morphology in vivo and is required for memory storage.

Drebrin E regulates neuroblast proliferation and chain migration in the adult brain.

F-actin-binding protein drebrin has two major isoforms: drebrin A and drebrin E. Drebrin A is the major isoform in the adult brain and is highly concentrated in dendritic spines, regulating spine morphology and synaptic plasticity. Conversely, drebrin E is the major isoform in the embryonic brain and regulates neuronal morphological differentiation, but it is also expressed in neurogenic regions of the adult brain. The subventricular zone (SVZ) is one of the brain regions where adult neurogenesis occurs. Neuroblasts migrate to the olfactory bulb (OB) and integrate into existing neuronal networks, after which drebrin expression changes from E to A, suggesting that drebrin E plays a specific role in neuroblasts in the adult brain. Therefore, to understand the role of drebrin E in the adult brain, we immunohistochemically analyzed adult neurogenesis using drebrin-null-mutant (DXKO) mice. In DXKO mice, the number of neuroblasts and cell proliferation decreased, although cell death remained unchanged. These results suggest that drebrin E regulates cell proliferation in the adult SVZ. Surprisingly, the decreased number of neuroblasts in the SVZ did not result in less neurons in the OB. This was because the survival rate of newly generated neurons in the OB increased in DXKO mice. Additionally, when neuroblasts reached the OB, the change in the migratory pathway from tangential to radial was partly disturbed in DXKO mice. These results suggest that drebrin E is involved in a chain migration of neuroblasts.

Early growth response‐1‐mediated down‐regulation of drebrin correlates with loss of dendritic spines

Post-synaptic dendritic spines are structurally composed of actin cytoskeleton, which undergoes dynamic morphological changes to accommodate incoming synaptic activity. Drebrin is an actin-binding protein highly expressed in dendritic spines that serves an important role in regulating spine morphology. Functionally, loss of drebrin directly correlates with deficits in learning and memory, as is the case observed in Alzheimer's disease. Despite these findings, the regulatory factor responsible for drebrin loss remains unclear. Here, we show that early growth response-1 (Egr-1), an inducible zinc finger transcription factor, down-regulates drebrin expression. Chromatin immunoprecipitation analyses identified Egr-1 binding sites upstream of the drebrin start site in neuronal cells. Over-expression of Egr-1 invitro in primary hippocampal neurons or invivo in homogenates prepared from the hippocampi of an inducible mouse model of Egr-1 show reduced drebrin mRNA and protein levels. Conversely, increased drebrin was detected in hippocampal samples isolated from Egr-1-deficient brain. These data demonstrate that Egr-1 interacts with the drebrin promoter and negatively regulates drebrin expression. Furthermore, immunocytochemical and Golgi staining analyses revealed reduced drebrin protein and dendritic spine density as well as reduced expression of synaptic markers in invitro hippocampal neurons over-expressing Egr-1 and invivo inducible mouse model of Egr-1. In contrast, increased drebrin expression correlated with increased dendritic spine density was detected in samples from Egr-1-deficient mice. These data provide evidence that Egr-1 is a novel regulator of drebrin expression, which is linked to changes in dendritic spine density.

Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70.

The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.

Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

Organic Anion Transporter 1 Deficiency Accelerates Learning and Memory Impairment in tg2576 Mice by Damaging Dendritic Spine Morphology and Activity.

To investigate whether and how organic anion transporter 1 (OAT1) is involved in the process of Alzheimer's disease (AD), we crossbred OAT1 knockout mice with tg2576, the widely used AD model mice. Results here showed the heterozygous OAT1-deficient tg2576 mice developed a learning- and memory-related behavior deficiency and higher soluble Abeta amount in early stage (3 months old). Furthermore, the heterozygous mice brain slice also showed impaired long-term potentiation (LTP) and spontaneous excitatory postsynaptic currents (sEPSC). By crossbreeding heterozygous OAT1-deficient tg2576 mice with Thy-1 YFP mice, we got autofluoresced (layer 4/5 cortical neuron) heterozygous mice. By using two-photon microscope in the direct observation of mice brain in vivo or single photon confocal on slices, compared with control tg2576 mice, we found that the OAT1-deficient mice showed a higher spine numbers but with a much lesser maturity extent. Finally, by using glutamate uncaging method, we induced chemical LTP in brain slices and found that OAT1-deficient mice showed abnormal chemical-induced LTP, which meant that the deficient behavior may be caused by abnormal spine morphology and activity. Our results indicated OAT1 may be involved in AD process by regulating spine morphology and activity.

Differential Tiam1/Rac1 Activation in Hippocampal and Cortical Neurons Mediates Differential Spine Shrinkage in Response to Oxygen/Glucose Deprivation

Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia.

α-Actinin-2 mediates spine morphology and assembly of the post-synaptic density in hippocampal neurons.

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis.

Spikar, a novel drebrin‐binding protein, regulates the formation and stabilization of dendritic spines

Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.

The Fragile X Mental Retardation Protein Regulates Matrix Metalloproteinase 9 mRNA at Synapses

Activity-dependent protein synthesis at synapses is dysregulated in the Fragile X syndrome (FXS). This process contributes to dendritic spine dysmorphogenesis and synaptic dysfunction in FXS. Matrix Metalloproteinase 9 (MMP-9) is an enzyme involved in activity-dependent reorganization of dendritic spine architecture and was shown to regulate spine morphology in a mouse model of FXS, the Fmr1 knock-out mice. Here we show that MMP-9 mRNA is part of the FMRP complex and colocalizes in dendrites. In the absence of FMRP MMP-9 mRNA translation is increased at synapses, suggesting that this mechanism contributes to the increased metalloproteinase level at synapses of Fmr1 knock-out mice. We propose that such a local effect can contribute to the aberrant dendritic spine morphology observed in the Fmr1 knock-out mice and in patients with FXS.