Dendritic Spine Morphogenesis Research Articles

PLD1 promotes dendritic spine morphogenesis via activating PKD1

Dendritic spines on the dendrites of pyramidal neurons are one of the most important components for excitatory synapses, where excitatory information exchanges and integrates. The defects of dendritic spine development have been closely connected with many nervous system diseases including autism, intellectual disability and so forth. Based on our previous studies, we here report a new functional signaling link between phospholipase D1 (PLD1) and protein kinase D1 (PKD1) in dendritic spine morphogenesis. Coimmunoprecipitation assays showed that PLD1 associates with PKD1. A series of knocking down and rescuing experiments demonstrated that PLD1 acts upstream of PKD1 in positively regulating dendritic spine morphogenesis. Using PLD1 inhibitor, we found that PLD1 activates PKD1 to promote dendritic spine morphogenesis. Thus, we further reveal the roles of the two different enzymes in neuronal development.

Altered Glutamate Receptor Ionotropic Delta Subunit 2 Expression in Stau2-Deficient Cerebellar Purkinje Cells in the Adult Brain.

Staufen2 (Stau2) is an RNA-binding protein that is involved in dendritic spine morphogenesis and function. Several studies have recently investigated the role of Stau2 in the regulation of its neuronal target mRNAs, with particular focus on the hippocampus. Here, we provide evidence for Stau2 expression and function in cerebellar Purkinje cells. We show that Stau2 downregulation (Stau2GT) led to an increase of glutamate receptor ionotropic delta subunit 2 (GluD2) in Purkinje cells when animals performed physical activity by voluntary wheel running compared with the age-matched wildtype (WT) mice (C57Bl/6J). Furthermore, Stau2GT mice showed lower performance in motor coordination assays but enhanced motor learning abilities than did WT mice, concomitantly with an increase in dendritic GluD2 expression. Together, our results suggest the novel role of Stau2 in Purkinje cell synaptogenesis in the mouse cerebellum.

Pituitary Adenylate Cyclase-Activating Polypeptide Modulates Dendritic Spine Maturation and Morphogenesis via MicroRNA-132 Upregulation.

Alterations in pituitary adenylate cyclase-activating polypeptide (PACAP), a multifunctional neuropeptide, and its receptors have been identified as risk factors for certain psychiatric disorders, including schizophrenia. Increasing evidence from human genetic and animal model studies suggest an association between various psychiatric disorders and altered dendritic spine morphology. In the present study, we investigated the role of exogenous and endogenous PACAP in spine formation and maturation. PACAP modified the density and morphology of PSD-95-positive spines in primary cultured hippocampal neurons. Notably, PACAP increased the levels of microRNA (miR)-132 and decreased expression of corresponding miR-132 target genes and protein expression of p250GAP, a miR-132 effector known to be involved in spine morphology regulation. In corroboration, PSD-95-positive spines were reduced in PACAP-deficient (PACAP-/-) mice versus WT mice. Golgi staining of hippocampal CA1 neurons revealed a reduced spine densities and atypical morphologies in the male PACAP-/- mice. Furthermore, viral miR-132 overexpression reversed the reduction in hippocampal spinal density in the male PACAP-/- mice. These results indicate that PACAP signaling plays a critical role in spine morphogenesis possibly via miR-132. We suggest that dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through its effects on spine formation.SIGNIFICANCE STATEMENT Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling dysfunction and dendritic spine morphology alterations have recently been suggested as important pathophysiological mechanisms underlying several psychiatric and neurological disorders. In this study, we investigated whether PACAP regulates dendritic spine morphogenesis. In a combination of pharmacological and viral gain- and loss-of-function approaches in vitro and in vivo experiments, we found PACAP to increase the size and density of dendritic spines via miR-132 upregulation. Together, our data suggest that a dysfunction of PACAP signaling may contribute to the pathogenesis of neuropsychiatric disorders, at least partly through abnormal spine formation.

The nuclear matrix protein Matr3 regulates processing of the synaptic microRNA-138-5p.

microRNA-dependent post-transcriptional control represents an important gene-regulatory layer in post-mitotic neuronal development and synaptic plasticity. We recently identified the brain-enriched miR-138 as a negative regulator of dendritic spine morphogenesis in rat hippocampal neurons. A potential involvement of miR-138 in cognition is further supported by a recent GWAS study on memory performance in a cohort of aged (>60 years) individuals. The expression of miR-138, which is encoded in two independent genomic loci (miR-138-1 and -2), is subject to both cell-type and developmental stage-specific regulation, the underlying molecular mechanisms however are poorly understood. Here, we show that miR-138-2 is the primary source of mature miR-138 in developing rat hippocampal neurons. Furthermore, we obtained evidence for the regulation of miR-138-2 biogenesis at the level of primary miRNA processing. Using biochemical pull-down assays, we identified the nuclear matrix protein Matrin-3 as pri/pre-miR-138 interacting protein and mapped the interaction to the pri/pre-miR-138-2 loop region. Matrin-3 loss-of-function experiments in HEK293 cells and primary neurons together with protein localization studies suggest an inhibitory function of Matrin-3 in nuclear pri-miR-138-2 processing. Together, our experiments unravel a new mechanism of miR-138 regulation in neurons, with important implications for miR-138 regulation during neuronal development, synaptic plasticity and memory-related processes.

Kalirin-7 prevents dendritic spine dysgenesis induced by amyloid beta-derived oligomers.

Synapse degeneration and dendritic spine dysgenesis are believed to be crucial early steps in Alzheimer's disease (AD), and correlate with cognitive deficits in AD patients. Soluble amyloid beta (Aβ)-derived oligomers, also termed Aβ-derived diffusible ligands (ADDLs), accumulate in the brain of AD patients and play a crucial role in AD pathogenesis. ADDLs bind to mature hippocampal neurons, induce structural changes in dendritic spines and contribute to neuronal death. However, mechanisms underlying structural and toxic effects are not fully understood. Here, we report that ADDLs bind to cultured mature cortical pyramidal neurons and induce spine dysgenesis. ADDL treatment induced the rapid depletion of kalirin-7, a brain-specific guanine-nucleotide exchange factor for the small GTPase Rac1, from spines. Kalirin-7 is a key regulator of dendritic spine morphogenesis and maintenance in forebrain pyramidal neurons and here we show that overexpression of kalirin-7 prevents ADDL-induced spine degeneration.Taken together, our results suggest that kalirin-7 may play a role in the early events leading to synapse degeneration, and its pharmacological activation may prevent or delay synapse pathology in AD.

Src-mediated phosphorylation of βPix-b regulates dendritic spine morphogenesis.

PAK-interacting guanine nucleotide exchange factor (βPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which βPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced βPix isoforms in the brain. Here, we report a novel finding that the b isoform of βPix (βPix-b) mediates the regulation of spine and synapse formation. We found that βPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of βPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of βPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of βPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of βPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which βPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation.

Wnt-7a Stimulates Dendritic Spine Morphogenesis and PSD-95 Expression Through Canonical Signaling.

Wnt signaling regulates brain development and synapse maturation; however, the precise molecular mechanism remains elusive. Here, we report that Wnt-7a stimulates dendritic spine morphogenesis in the hippocampus via glycogen synthase kinase-3 β (GSK-3β) inhibition, triggering β-catenin/T cell factor/lymphoid enhancer factor (TCF/LEF)-dependent gene transcription and promoting postsynaptic density-95 (PSD-95) protein expression. In addition, wild-type mice treated with an inhibitor of β-catenin/TCF/LEF-mediated transcription showed a reduction in spatial memory acquisition accompanied by a reduction in PSD-95 and decreases in spine density measured by Golgi staining, suggesting that PSD-95 is a novel Wnt target gene. Together, our data strongly demonstrate that Wnt-dependent target gene transcription is essential to hippocampal synaptic plasticity.

CaMKIIβ is localized in dendritic spines as both drebrin-dependent and drebrin-independent pools.

Drebrin is a major F‐actin binding protein in dendritic spines that is critically involved in the regulation of dendritic spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin‐binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca2+/calmodulin‐dependent protein kinase II (CaMKIIβ) as a drebrin‐binding protein using a yeast two‐hybrid system, and investigated the drebrin–CaMKIIβ relationship in dendritic spines using rat hippocampal neurons. Drebrin knockdown resulted in diffuse localization of CaMKIIβ in dendrites during the resting state, suggesting that drebrin is involved in the accumulation of CaMKIIβ in dendritic spines. Fluorescence recovery after photobleaching analysis showed that drebrin knockdown increased the stable fraction of CaMKIIβ, indicating the presence of drebrin‐independent, more stable CaMKIIβ. NMDA receptor activation also increased the stable fraction in parallel with drebrin exodus from dendritic spines. These findings suggest that CaMKIIβ can be classified into distinct pools: CaMKIIβ associated with drebrin, CaMKIIβ associated with post‐synaptic density (PSD), and CaMKIIβ free from PSD and drebrin. CaMKIIβ appears to be anchored to a protein complex composed of drebrin‐binding F‐actin during the resting state. NMDA receptor activation releases CaMKIIβ from drebrin resulting in CaMKIIβ association with PSD.

NMDAR‐dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.

Differential effects of transient and sustained activation of BDNF-TrkB signaling.

Brain-derived neurotrophic factor (BDNF) serves a pleiotropic role in the central nervous system, ranging from promoting neuronal survival and differentiation during development and synaptic modulation in the adult. An important, yet unanswered question is how BDNF could serve such diverse functions, sometimes in the same cell. At least two modes of BDNF actions have been elucidated so far based on BDNF signaling kinetics and/or the activity status of the responding neurons. Acute and gradual increases in extracellular BDNF concentrations elicit, respectively, transient and sustained activation of TrkB receptor and its downstream signaling, leading to differential molecular and cellular functions. In cultured neurons, sustained TrkB activation promotes neuronal dendritic arborization and spinogenesis, whereas transient TrkB activation facilitates dendritic growth and spine morphogenesis. In hippocampal slices, slow delivery of BDNF facilitates LTP, whereas fast application of BDNF enhances basal synaptic transmission in schaffer collateral synapses. High-frequency stimulation of neurons converts BDNF-induced TrkB signaling from a transient to a sustained mode. These initial insights lay the foundation for future investigation of the BDNF-TrkB pathway, and analogous signaling pathways to gain a comprehensive understanding to enable translational research. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 647-659, 2018.

Short-term mastication after weaning upregulates GABAergic signalling and reduces dendritic spine in thalamus

Mastication enhances brain function and mental health, but little is known about the molecular mechanisms underlying the effects of mastication on neural development in early childhood. Therefore, we analysed the gene expression in juvenile neural circuits in rats fed with a soft or chow diet immediately after weaning. We observed that the gene expression patterns in the thalamus varied depending on the diet. Furthermore, gene ontology analysis revealed that two terms were significantly enhanced: chemical synaptic transmission and positive regulation of dendritic spine morphogenesis. With respect to chemical synaptic transmission, glutamate decarboxylase and GABA receptors were upregulated in the chow diet group. The related genes, including vesicular GABA transporter, were also upregulated, suggesting that mastication activates GABAergic signalling. With respect to dendritic spine morphogenesis, Ingenuity Pathway Analysis predicted fewer extension of neurites and neurons and fewer number of branches in the chow diet group. The numbers of spines in the ventral posterolateral and posteromedial regions were significantly decreased. These results suggest that mastication in the early developing period upregulates GABAergic signalling genes, with a decrease of spines in the thalamus.

Examining UBE3A's Possible Role in Dendritic Spine Morphogenesis

Examining UBE3A's Possible Role in Dendritic Spine Morphogenesis

FRMPD4 mutations cause X-linked intellectual disability and disrupt dendritic spine morphogenesis.

FRMPD4 (FERM and PDZ Domain Containing 4) is a neural scaffolding protein that interacts with PSD-95 to positively regulate dendritic spine morphogenesis, and with mGluR1/5 and Homer to regulate mGluR1/5 signaling. We report the genetic and functional characterization of 4 FRMPD4 deleterious mutations that cause a new X-linked intellectual disability (ID) syndrome. These mutations were found to be associated with ID in ten affected male patients from four unrelated families, following an apparent X-linked mode of inheritance. Mutations include deletion of an entire coding exon, a nonsense mutation, a frame-shift mutation resulting in premature termination of translation, and a missense mutation involving a highly conserved amino acid residue neighboring FRMPD4-FERM domain. Clinical features of these patients consisted of moderate to severe ID, language delay and seizures alongside with behavioral and/or psychiatric disturbances. In-depth functional studies showed that a frame-shift mutation, FRMPD4p.Cys618ValfsX8, results in a disruption of FRMPD4 binding with PSD-95 and HOMER1, and a failure to increase spine density in transfected hippocampal neurons. Behavioral studies of frmpd4-KO mice identified hippocampus-dependent spatial learning and memory deficits in Morris Water Maze test. These findings point to an important role of FRMPD4 in normal cognitive development and function in humans and mice, and support the hypothesis that FRMPD4 mutations cause ID by disrupting dendritic spine morphogenesis in glutamatergic neurons.

The E3 ubiquitin ligase IDOL regulates synaptic ApoER2 levels and is important for plasticity and learning.

Neuronal ApoE receptors are linked to learning and memory, but the pathways governing their abundance, and the mechanisms by which they affect the function of neural circuits are incompletely understood. Here we demonstrate that the E3 ubiquitin ligase IDOL determines synaptic ApoER2 protein levels in response to neuronal activation and regulates dendritic spine morphogenesis and plasticity. IDOL-dependent changes in ApoER2 abundance modulate dendritic filopodia initiation and synapse maturation. Loss of IDOL in neurons results in constitutive overexpression of ApoER2 and is associated with impaired activity-dependent structural remodeling of spines and defective LTP in primary neuron cultures and hippocampal slices. IDOL-deficient mice show profound impairment in experience-dependent reorganization of synaptic circuits in the barrel cortex, as well as diminished spatial and associative learning. These results identify control of lipoprotein receptor abundance by IDOL as a post-transcriptional mechanism underlying the structural and functional plasticity of synapses and neural circuits.

Role of G Protein-Coupled Receptors in the Regulation of Structural Plasticity and Cognitive Function.

Cognition and other higher brain functions are known to be intricately associated with the capacity of neural circuits to undergo structural reorganization. Structural remodelling of neural circuits, or structural plasticity, in the hippocampus plays a major role in learning and memory. Dynamic modifications of neuronal connectivity in the form of dendritic spine morphology alteration, as well as synapse formation and elimination, often result in the strengthening or weakening of specific neural circuits that determine synaptic plasticity. Changes in dendritic complexity and synapse number are mediated by cellular processes that are regulated by extracellular signals such as neurotransmitters and neurotrophic factors. As many neurotransmitters act on G protein-coupled receptors (GPCRs), it has become increasingly apparent that GPCRs can regulate structural plasticity through a myriad of G protein-dependent pathways and non-canonical signals. A thorough understanding of how GPCRs exert their regulatory influence on dendritic spine morphogenesis may provide new insights for treating cognitive impairment and decline in various age-related diseases. In this article, we review the evidence of GPCR-mediated regulation of structural plasticity, with a special emphasis on the involvement of common as well as distinct signalling pathways that are regulated by major neurotransmitters.

Incorrect dosage of IQSEC2, a known intellectual disability and epilepsy gene, disrupts dendritic spine morphogenesis

There is considerable genetic and phenotypic heterogeneity associated with intellectual disability (ID), specific learning disabilities, attention-deficit hyperactivity disorder, autism and epilepsy. The intelligence quotient (IQ) motif and SEC7 domain containing protein 2 gene (IQSEC2) is located on the X-chromosome and harbors mutations that contribute to non-syndromic ID with and without early-onset seizure phenotypes in both sexes. Although IQ and Sec7 domain mutations lead to partial loss of IQSEC2 enzymatic activity, the in vivo pathogenesis resulting from these mutations is not known. Here we reveal that IQSEC2 has a key role in dendritic spine morphology. Partial loss-of-function mutations were modeled using a lentiviral short hairpin RNA (shRNA) approach, which achieved a 57% knockdown of Iqsec2 expression in primary hippocampal cell cultures from mice. Investigating gross morphological parameters after 8 days of in vitro culture (8DIV) identified a 32% reduction in primary axon length, in contrast to a 27% and 31% increase in the number and complexity of dendrites protruding from the cell body, respectively. This increase in dendritic complexity and spread was carried through dendritic spine development, with a 34% increase in the number of protrusions per dendritic segment compared with controls at 15DIV. Although the number of dendritic spines had normalized by 21DIV, a reduction was noted in the number of immature spines. In contrast, when modeling increased dosage, overexpression of wild-type IQSEC2 led to neurons with shorter axons that were more compact and displayed simpler dendritic branching. Disturbances to dendritic morphology due to knockdown of Iqsec2 were recapitulated in neurons from Iqsec2 knockout mice generated in our laboratory using CRISPR/Cas9 technology. These observations provide evidence of dosage sensitivity for IQSEC2, which normally escapes X-inactivation in females, and links these disturbances in expression to alterations in the morphology of developing neurons.

Assembly of Excitatory Synapses in the Absence of Glutamatergic Neurotransmission

Synaptic excitation mediates a broad spectrum of structural changes in neural circuits across the brain. Here, we examine the morphologies, wiring, and architectures of single synapses of projection neurons in the murine hippocampus that developed in virtually complete absence of vesicular glutamate release. While these neurons had smaller dendritic trees and/or formed fewer contacts in specific hippocampal subfields, their stereotyped connectivity was largely preserved. Furthermore, loss of release did not disrupt the morphogenesis of presynaptic terminals and dendritic spines, suggesting that glutamatergic neurotransmission is unnecessary for synapse assembly and maintenance. These results underscore the instructive role of intrinsic mechanisms in synapse formation.

Drebrin and Spine Formation.

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses in the central nervous system. Thus, spines are supposed to act as a fundamental unit for information processing of the brains. Previous studies have demonstrated the roles of drebrin in the formation of dendritic spines and in the recruitment of synaptic proteins to postsynaptic sites. Further, a live imaging study has revealed the unique dynamics of drebrin in dendritic spines, which help to understand how drebrin is involved in dendritic spine formation. This review will provide a basic knowledge about dendritic spine and overview recent progresses in understanding of the roles of drebrin in dendritic spine morphogenesis and synaptogenesis.

Regulation of Cortical Dendrite Morphology and Spine Organization by Secreted Semaphorins: A Primary Culture Approach.

Primary tissue culture is an invaluable technique in cell biology and has a long history in demonstrating its versatility in characterizing cellular morphology, function, and behavior. Here, we describe a modified, low density, long-term, primary neuron culture system to characterize dendritic morphology and synaptic spine organization in developing mouse cortical neurons. While this method can be applied to investigate the signaling pathways of a range of extracellular cues' effect on neuronal development, we focus on how distinct secreted semaphorins regulate dendritic elaboration and spine morphogenesis in deep layer cortical neurons.

CDK5 downregulation enhances synaptic plasticity.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity-such as long-term potentiation (LTP)-have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.