Phosphorylation of CRMP2 is involved in proper bifurcation of the apical dendrite of hippocampal CA1 pyramidal neurons

The neural circuit in the hippocampus is important for higher brain functions. Dendrites of CA1 pyramidal neurons mainly receive input from the axons of CA3 pyramidal neurons in this neural circuit. A CA1 pyramidal neuron has a single apical dendrite and multiple basal dendrites. In wild‐type mice, most of CA1 pyramidal neurons extend a single trunk, or alternatively, the apical dendrite bifurcates into two daughter trunks at the stratum radiatum layer. We previously reported the proximal bifurcation phenotype in Sema3A?/?, p35?/?, and CRMP4?/? mice. Cdk5/p35 phosphorylates CRMP2 at Ser522, and inhibition of this phosphorylation suppressed Sema3A‐induced growth cone collapse. In this study, we analyzed the bifurcation points of the apical dendrites of hippocampal CA1 pyramidal neurons in CRMP2KI/KI mice in which the Cdk5/p35‐phosphorylation site Ser522 was mutated into an Ala residue. The proximal bifurcation phenotype was not observed in CRMP2KI/KI mice; however, severe proximal bifurcation of apical dendrites was found in CRMP2KI/KI;CRMP4?/? mice. Cultured hippocampal neurons from CRMP2KI/KI and CRMP2KI/KI;CRMP4?/? embryos showed an increased number of dendritic branching points compared to those from wild‐type embryos. Sema3A increased the number of branching points and the total length of dendrites in wild‐type hippocampal neurons, but these effects of Sema3A for dendrites were notobserved in CRMP2KI/KI and CRMP2KI/KI;CRMP4?/?hippocampal neurons. Binding of CRMP2 to tubulin increased in both CRMP2KI/KI and CRMP2KI/KI:CRMP4?/? brain lysates. These results suggest that CRMP2 and CRMP4 synergistically regulate dendritic development, and CRMP2 phosphorylation is critical for proper bifurcation of apical dendrite of CA1 pyramidal neurons. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus

Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that consist of 5 members (CRMP 1–5). CRMP2 and CRMP4 regulate neurite outgrowth by binding to tubulin heterodimers, resulting in the assembly of microtubules. CRMP2 also mediates the growth cone collapse response to the repulsive guidance molecule semaphorin‐3A (Sema3A). However, the role of CRMP4 in Sema3A signaling and its function in the developing mouse brain remain unclear. We generated CRMP4?/? mice in order to study the in vivo function of CRMP4 and identified a phenotype of proximal bifurcation of apical dendrites in the CA1 pyramidal neurons of CRMP4?/? mice. We also observed increased dendritic branching in cultured CRMP4?/? hippocampal neurons as well as in cultured cortical neurons treated with CRMP4 shRNA. Sema3A induces extension and branching of the dendrites of hippocampal neurons; however, these inductions were compromised in the CRMP4?/? hippocampal neurons. These results suggest that CRMP4 suppresses apical dendrite bifurcation of CA1 pyramidal neurons in the mouse hippocampus and that this is partly dependent on Sema3A signaling. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons

Development of appropriate dendritic arbors is crucial for neuronal information transfer. We show, using seizure-related gene 6 (sez-6) null mutant mice, that Sez-6 is required for normal dendritic arborization of cortical neurons. Deep-layer pyramidal neurons in the somatosensory cortex of sez-6 null mice exhibit an excess of short dendrites, and cultured cortical neurons lacking Sez-6 display excessive neurite branching. Overexpression of individual Sez-6 isoforms in knockout neurons reveals opposing actions of membrane-bound and secreted Sez-6 proteins, with membrane-bound Sez-6 exerting an antibranching effect under both basal and depolarizing conditions. Layer V pyramidal neurons in knockout brain slices show reduced excitatory postsynaptic responses and a reduced dendritic spine density, reflected by diminished punctate staining for postsynaptic density 95 (PSD-95). In behavioral tests, the sez-6 null mice display specific exploratory, motor, and cognitive deficits. In conclusion, cell-surface protein complexes involving Sez-6 help to sculpt the dendritic arbor, in turn enhancing synaptic connectivity.     

Elimination of Aberrant DRG Circuitries in Sema3A Mutant Mice Leads to Extensive Neuronal Deficits

Axon guidance molecules determine the pattern of neuronal circuits. Accuracy of the process is ensured by unknown mechanisms that correct early guidance errors. Since the time frame of error correction in Sema3A null mice partly overlaps with the period of naturally occurring cell death in dorsal root ganglia (DRG) development, we tested the hypothesis that apoptosis of misguided neurons enables error correction. We crossed BAX null mice, in which DRG apoptosis is blocked, with Sema3A null mice to induce errors. Analyses of these double-null mouse embryos showed that the elimination of abnormal projections is not blocked in the absence of BAX. Surprisingly however, there are fewer surviving neurons in Sema3A null or Sema3A/BAX double-null newborn mice than in wild-type mice. These results suggest that guidance errors are corrected by a BAX-independent cell death mechanism. Thus, aberrant axonal guidance may lead to reductions in neuronal numbers to suboptimal levels, perhaps increasing the likelihood of neuropathological consequences later in life.     

HGF regulates the development of cortical pyramidal dendrites

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.     

The GTPase Rem2 regulates synapse development and dendritic morphology

Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir subfamily of small Ras-like GTPases that was identified as an important mediator of synapse development. We performed a comprehensive, loss- of-function analysis of Rem2 function in cultured hippocampal neurons using RNAi to substantially decrease Rem2 protein levels. We found that knockdown of Rem2 decreases the density and maturity of dendritic spines, the primary site of excitatory synapses onto pyramidal neurons in the hippocampus. Knockdown of Rem2 also alters the gross morphology of dendritic arborizations, increasing the number of dendritic branches without altering total neurite length. Thus, Rem2 functions to inhibit dendritic branching and promote the development of dendritic spines and excitatory synapses. Interestingly, binding to the calcium-binding protein calmodulin is required for the Rem2 regulation of dendritic branching. However, this interaction is completely dispensable for synapse development. Overall, our results suggest that Rem2 regulates dendritic branching and synapse development via distinct and overlapping signal transduction pathways.     

The protein dendrite arborization and synapse maturation 1 (Dasm-1) is dispensable for dendrite arborization

The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization.     

The rodent Four-jointed ortholog Fjx1 regulates dendrite extension

The extrinsic and intrinsic factors that regulate the size and complexity of dendritic arborizations are still poorly understood. Here we identify Fjx1, the rodent ortholog of the Drososphila planar cell polarity (PCP) protein Four-jointed (Fj), as a new inhibitory factor that regulates dendrite extension. The Drosophila gene four-jointed (fj) has been suggested to provide directional information in wing discs, but the mechanism how it acts is only poorly understood and the function of its mammalian homolog Fjx1 remains to be investigated. We analyzed the phenotype of a null mutation for mouse Fjx1. Homozygous Fjx1 mutants show an abnormal morphology of dendritic arbors in the hippocampus. In cultured hippocampal neurons from Fjx1 mutant mice, loss of Fjx1 resulted in an increase in dendrite extension and branching. Addition of Fjx1 to cultures of dissociated hippocampal neurons had the opposite effect and reduced the length of dendrites and decreased dendritic branching. Rescue experiments with cultured neurons showed that Fjx1 can act both cell-autonomously and non-autonomously. Our results identify Fjx1 as a new inhibitory factor that regulates dendrite extension.     

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.     

Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung

Semaphorin III/collapsin-1 (Sema3A) guides a specific subset of neuronal growth cones as a repulsive molecule. In this study, we have investigated a possible role of non-neuronal Sema3A in lung morphogenesis. Expression of mRNAs of Sema3A and neuropilin-1 (NP-1), a Sema3A receptor, was detected in fetal and adult lungs. Sema3A-immunoreactive cells were found in airway and alveolar epithelial cells of the fetal and adult lungs. Immunoreactivity for NP-1 was seen in fetal and adult alveolar epithelial cells as well as endothelial cells. Immunoreactivity of collapsin response mediator protein CRMP (CRMP-2), an intracellular protein mediating Sema3A signaling, was localized in alveolar epithelial cells, nerve tissue and airway neuroendocrine cells. The expression of CRMP-2 increased during the fetal, neonate and adult periods, and this pattern paralleled that of NP-1. In a two-day culture of lung explants from fetal mouse lung (E11.5), with exogenous Sema3A at a dose comparable to that which induces growth cone collapse of dorsal root ganglia neurons, the number of terminal buds was reduced in a dose-dependent manner when compared with control or untreated lung explants. This decrease was not accompanied with any alteration of the bromodeoxyuridine-positive DNA-synthesizing fraction. A soluble NP-1 lacking the transmembrane and intracellular region, neutralized the inhibitory effect of Sema3A. The fetal lung explants from neuropilin-1 homozygous null mice grew normally in vitro regardless of Sema3A treatment. These results provide evidence that Sema3A inhibits branching morphogenesis in lung bud organ cultures via NP-1 as a receptor or a component of a possible multimeric Sema3A receptor complex.     

Plexin‐B1 is a GTPase activating protein for M‐Ras,remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.     

Induction of spine growth and synapse formation by regulation of the spine actin cytoskeleton

We explored the relationship between regulation of the spine actin cytoskeleton, spine morphogenesis, and synapse formation by manipulating expression of the actin binding protein NrbI and its deletion mutants. In pyramidal neurons of cultured rat hippocampal slices, NrbI is concentrated in dendritic spines by binding to the actin cytoskeleton. Expression of one NrbI deletion mutant, containing the actin binding domain, dramatically increased the density and length of dendritic spines with synapses. This hyperspinogenesis was accompanied by enhanced actin polymerization and spine motility. Synaptic strengths were reduced to compensate for extra synapses, keeping total synaptic input per neuron constant. Our data support a model in which synapse formation is promoted by actin-powered motility.     

Dendritic morphology of hippocampal and amygdalar neurons in adolescent mice is resilient to genetic differences in stress reactivity

Many studies have shown that chronic stress or corticosterone over-exposure in rodents leads to extensive dendritic remodeling, particularly of principal neurons in the CA3 hippocampal area and the basolateral amygdala. We here investigated to what extent genetic predisposition of mice to high versus low stress reactivity, achieved through selective breeding of CD-1 mice, is also associated with structural plasticity in Golgi-stained neurons. Earlier, it was shown that the highly stress reactive (HR) compared to the intermediate (IR) and low (LR) stress reactive mice line presents a phenotype, with respect to neuroendocrine parameters, sleep architecture, emotional behavior and cognition, that recapitulates some of the features observed in patients suffering from major depression. In late adolescent males of the HR, IR, and LR mouse lines, we observed no significant differences in total dendritic length, number of branch points and branch tips, summated tip order, number of primary dendrites or dendritic complexity of either CA3 pyramidal neurons (apical as well as basal dendrites) or principal neurons in the basolateral amygdala. Apical dendrites of CA1 pyramidal neurons were also unaffected by the differences in stress reactivity of the animals; marginally higher length and complexity of the basal dendrites were found in LR compared to IR but not HR mice. In the same CA1 pyramidal neurons, spine density of distal apical tertiary dendrites was significantly higher in LR compared to IR or HR animals. We tentatively conclude that the dendritic complexity of principal hippocampal and amygdala neurons is remarkably stable in the light of a genetic predisposition to high versus low stress reactivity, while spine density seems more plastic. The latter possibly contributes to the behavioral phenotype of LR versus HR animals.     

Glycinergic transmission regulates dendrite size in organotypic culture

We previously demonstrated that inhibitory synaptic transmission influences dendrite development in vivo. We now report an analogous finding in an organotypic culture of a glycinergic projection nucleus, the medial nucleus of the trapezoid body (MNTB), and its postsynaptic target, the lateral superior olive (LSO) of gerbils. Cultures were generated at 6–7 days postnatal and grown in serum containing medium with or without the glycine receptor antagonist, strychnine (SN), at 2 μM. LSO neurons were then labeled with biocytin, and the dendritic arbors were analyzed morphometrically. Compared to neurons from age-matched in vivo tissue, the neurons cultured in control media were somewhat atrophic, including decreases in dendritic branching and length. Incubation in strychnine led to a dramatic increase in dendritic branching and total dendritic length. Control neurons averaged 6.3 branches, compared to 18 branches/neuron in SN-treated cultures. There was a similar increase in primary dendrites and total dendritic length. The physical elimination of MNTB cells did not mimic SN treatment, presumably because glycinergic LSO neurons generated intrinsic connections. In fact, the LSO soma area was significantly greater following MNTB removal, suggesting that these afferents provide a second signal to postsynaptic neurons. These results suggest that spontaneous glycinergic transmission regulates the growth of postsynaptic processes. © 1996 John Wiley & Sons, Inc.     

Semaphorin3A regulates neuronal polarization by suppressing axon formation and promoting dendrite growth

Semaphorin 3A (Sema3A) is a secreted factor known to guide axon/dendrite growth and neuronal migration. We found that it also acts as a polarizing factor for axon/dendrite development in cultured hippocampal neurons. Exposure of the undifferentiated neurite to localized Sema3A suppressed its differentiation into axon and promoted dendrite formation, resulting in axon formation away from the Sema3A source, and bath application of Sema3A to polarized neurons promoted dendrite growth but suppressed axon growth. Fluorescence resonance energy transfer (FRET) imaging showed that Sema3A elevated the cGMP but reduced cAMP and protein kinase A (PKA) activity, and its axon suppression is attributed to the downregulation of PKA-dependent phosphorylation of axon determinants LKB1 and GSK-3β. Downregulating Sema3A signaling in rat embryonic cortical progenitors via in utero electroporation of siRNAs against the Sema3A receptor neuropilin-1 also resulted in polarization defects in?vivo. Thus, Sema3A regulates the earliest step of neuronal morphogenesis by polarizing axon/dendrite formation.     

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.     

Null and conditional semaphorin 3B alleles using a flexible puroDeltatk loxP/FRT vector

In neural development, Semaphorin 3B (SEMA3B) is thought to play a role in guiding axons by repulsion. In nonneuronal tissue, SEMA3B has been postulated to be a tumor suppressor gene of lung and breast cancer. Much of the understanding of the function of members of the SEMA3 family has come from targeted deletion of these genes in mice (Sema3A, Sema3C, and Sema3F). Thus, targeted deletion of Sema3B in mice would prove invaluable in dissecting out its functions. To allow for maximum gene-targeting flexibility, we developed a generic targeting vector, pFlexible, containing the positive/negative selectable marker puroDeltatk and loxP and FRT recombination sites, and used it to target Sema3B in ES cells. Flpe- and Cre-mediated recombination in vitro generated ES cell lines that contained a conditional or null Sema3B allele, respectively, which were established as homozygous alleles in mice. Analysis of Sema3B null mice showed they were viable, fertile, and displayed no overt pathological abnormalities, suggesting an inherent correction mechanism or level of redundancy between the class 3 semaphorins. This targeting vector system has broad applicability in any knockout experiment and provides a flexible approach for the generation of modified alleles in mice.     

A critical role for system A amino acid transport in the regulation of dendritic development by brain-derived neurotrophic factor (BDNF)

Dendritic development is essential for the establishment of a functional nervous system. Among factors that control dendritic development, brain-derived neurotrophic factor (BDNF) has been shown to regulate dendritic length and complexity of cortical neurons. However, the cellular and molecular mechanisms that underlie these effects remain poorly understood. In this study, we examined the role of amino acid transport in mediating the effects of BDNF on dendritic development. We show that BDNF increases System A amino acid transport in cortical neurons by selective up-regulation of the sodium-coupled neutral amino acid transporter (SNAT)1. Up-regulation of SNAT1 expression and System A activity is required for the effects of BDNF on dendritic growth and branching of cortical neurons. Further analysis revealed that induction of SNAT1 expression and System A activity by BDNF is necessary in particular to enhance synthesis of tissue-type plasminogen activator, a protein that we demonstrate to be essential for the effects of BDNF on cortical dendritic morphology. Together, these data reveal that stimulation of neuronal differentiation by BDNF requires the up-regulation of SNAT1 expression and System A amino acid transport to meet the increased metabolic demand associated with the enhancement of dendritic growth and branching.     

Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.