Plexin-B family members demonstrate non-redundant expression patterns in the developing mouse nervous system: an anatomical basis for morphogenetic effects of Sema4D during development

Semaphorins and their receptors play important roles in patterning the connectivity of the developing nervous system and recent data suggest that members of the plexin-B family of semaphorin receptors may be involved in axonal guidance. Here we show that the mRNAs of the three plexin-B genes, plxnb1, plxnb2 and plxnb3 (plexin-B1, plexin-B2 and plexin-B3), respectively, are expressed in highly specific and non-redundant patterns in peripheral and central components of the nervous system over defined periods during murine development. Whereas plexin-B1 and plexin-B2 are strongly expressed in the neuroepithelium and developing neurons, plexin-B3 mRNA is selectively localized to the white matter. Moreover, plexin-B1 and its ligand Sema4D are expressed in complementary patterns in several regions such as the developing neopallial cortex, the dorsal root ganglia and the spinal cord over embryonic stages. The Sema4d gene demonstrates a dramatic switch from prenatal expression in neuronal populations to a postnatal expression in oligodendrocytes. In contrast to its collapsing activity on growth cones of embryonic retinal ganglion cells and hippocampal neurons, soluble Sema4D enhances axonal outgrowth in embryonic cortical explants cultured in collagen matrices. Thus, plexin-B family members and Sema4D are likely to play complex and non-redundant roles during the development of the nervous system.     

Semaphorin 4D/Plexin-B1-mediated M-Ras GAP activity regulates actin-based dendrite remodeling through Lamellipodin

Semaphorins have been identified as repulsive guidance molecules in the developing nervous system. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 induces repulsion in axon and dendrites by functioning as a GTPase-activating protein (GAP) for R-Ras and M-Ras, respectively. In axons, Sema4D stimulation induces growth cone collapse, and downregulation of R-Ras activity by Plexin-B1-mediated GAP activity is required for the action. Axonal R-Ras GAP activity downregulates phosphatidylinositol 3-kinase signaling pathway, and thereby induces inactivation of a microtubule assembly promoter protein, CRMP-2. However, in contrast to the well studied roles of semaphorins and plexins in axonal guidance, signaling molecules linking M-Ras GAP to dendritic cytoskeleton remain obscure. Here we identified an Ena/VASP ligand, Lamellipodin (Lpd), as a novel effector of M-Ras in dendrites. Lpd was expressed in F-actin-rich distal dendritic processes and was required for both basal and M-Ras-mediated dendrite development. Subcellular fractionation showed M-Ras-dependent membrane translocation of Lpd, which was suppressed by Sema4D. Furthermore, the Ena/VASP-binding region within Lpd was required for dendrite development, and its membrane targeting was sufficient to overcome the Sema4D-mediated reduction of dendritic outgrowth and disappearance of F-actin from distal dendrites. Furthermore, in utero electroporation experiments also indicated that regulation of the M-Ras-Lpd system by the GAP activity of Plexin is involved in the normal development of cortical dendrites in vivo. Overall, our study sheds light on how repulsive guidance molecules regulate actin cytoskeleton in dendrites, revealing a novel mechanism that the M-Ras-Lpd system regulates actin-based dendrite remodeling by Sema/Plexin in rats or mice of either sex.     

Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development

Semaphorins and Plexins are cognate ligand-receptor families that regulate important steps during nervous system development. The Plexin-B2 receptor is critically involved in neural tube closure and cerebellar granule cell development, however, its specific ligands have only been suggested by in vitro studies. Here, we show by in vivo and in vitro analyses that the two Semaphorin-4 family members Sema4C and Sema4G are likely to be in vivo ligands of Plexin-B2. The Sema4C and Sema4G genes are expressed in the developing cerebellar cortex, and Sema4C and Sema4G proteins specifically bind to Plexin-B2 expressing cerebellar granule cells. To further elucidate their in vivo function, we have generated and analyzed Sema4C and Sema4G knockout mouse mutants. Like Plexin-B2-/- mutants, Sema4C-/- mutants reveal exencephaly and subsequent neonatal lethality with partial penetrance. Sema4C-/- mutants that bypass exencephaly are viable and fertile, but display distinctive defects of the cerebellar granule cell layer, including gaps in rostral lobules, fusions of caudal lobules, and ectopic granule cells in the molecular layer. In addition to neuronal defects, we observed in Sema4C-/- mutants also ventral skin pigmentation defects that are similar to those found in Plexin-B2-/- mutants. The Sema4G gene deletion causes no overt phenotype by itself, but combined deletion of Sema4C and Sema4G revealed an enhanced cerebellar phenotype. However, Sema4C/Sema4G double mutants showed overall less severe cerebellar phenotypes than Plexin-B2-/- mutants, indicating that further ligands of Plexin-B2 exist. In explant cultures of the developing cerebellar cortex, Sema4C promoted migration of cerebellar granule cell precursors in a Plexin-B2-dependent manner, supporting the model that a reduced migration rate of granule cell precursors is the basis for the cerebellar defects of Sema4C-/- and Sema4C/Sema4G mutants.     

The role of calcium signaling in early axonal and dendritic morphogenesis of rat cerebral cortex neurons under non-stimulated growth conditions

The effects of depolarizing stimuli on neurite outgrowth have been shown to depend on an influx of extracellular calcium. However, the role of calcium under non-stimulated growth conditions is less well established. Here we investigated the contribution of calcium signaling to early neuronal morphogenesis of rat cerebral cortex neurons at three levels by blocking L-type voltage sensitive calcium channels, by depleting intracellular calcium or by blocking myosin light chain kinase. Detailed quantitative morphological analysis of neurons treated for 1 day revealed that depletion of intracellular calcium strongly decreased the density of filopodia, arrested axonal outgrowth and strongly decreased dendritic branching. Preventing calcium influx through L-type voltage sensitive calcium channels and blocking of myosin light chain kinase activity selectively decreased dendritic branching. Our observations support an essential role for basal intracellular calcium levels in axonal elongation. Furthermore, under non-stimulated conditions calcium entry through L-type voltage sensitive calcium channels and myosin light chain kinase play an important role in dendritic branching.     

Erythropoietin promotes axonal growth in a model of neuronal polarization

Erythropoietin (EPO) enhances neurogenesis, neuroprotection and regeneration. Here, we examined the effects of EPO on axonal and dendritic growth in a model of neuronal polarization. EPO did not effect survival or the polarized morphology of hippocampal neurons but its effect on neurite outgrowth depended upon the stage of polarization. When added to neurons in the process of establishing polarity (0–2 days in vitro (DIV)), it enhanced axonal and dendritic growth, while EPO added to early polarized cultures at 3–4 DIV promoted the growth of axons but not dendrites. EPO stimulated the phosphorylation of Akt at serine-473 and co-incubation of the Akt/PI-3 kinase pathway inhibitor LY294002 with EPO abolished its effects on Akt phosphorylation and axonal growth. However, while Leukemia Inhibitory Factor (LIF) similarly stimulated phosphorylation of Akt, it had no effect on axonal or dendritic growth, indicating that AKT phosphorylation is necessary but not sufficient for neurite outgrowth in hippocampal neurons.     

CRP1, a protein localized in filopodia of growth cones, is involved in dendritic growth

The cysteine-rich protein (CRP) family is a subgroup of LIM domain proteins. CRP1, which cross-links actin filaments to make actin bundles, is the only CRP family member expressed in the CNS with little known about its function in nerve cells. Here, we report that CRP1 colocalizes with actin in the filopodia of growth cones in cultured rat hippocampal neurons. Knockdown of CRP1 expression by short hairpin RNA interference results in inhibition of filopodia formation and dendritic growth in neurons. Overexpression of CRP1 increases filopodia formation and neurite branching, which require its actin-bundling activity. Expression of CRP1 with a constitutively active form of Cdc42, a GTPase involved in filopodia formation, increases filopodia formation in COS-7 cells, suggesting cooperation between the two proteins. Moreover, we demonstrate that neuronal activity upregulates CRP1 expression in hippocampal neurons via Ca2? influx after depolarization. Ca2?/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein mediate the Ca2?-induced upregulation of CRP1 expression. Furthermore, CRP1 is required for the dendritic growth induced by Ca2? influx or CaMKIV. Together, these data are the first to demonstrate a role for CRP1 in dendritic growth.     

A macromolecular complex involving the amyloid precursor protein (APP) and the cytosolic adapter FE65 is a negative regulator of axon branching

Several studies suggest a role for the amyloid precursor protein (APP) in neurite outgrowth and synaptogenesis, but the downstream interactions that mediate the function of APP during neuron development are unknown. By introducing interaction-deficient FE65 into cultured hippocampal neurons using adenovirus, we show that a complex including APP, FE65 and an additional protein is involved in neurite outgrowth at early stages of neuronal development. Both FE65 that is unable to interact with APP (PID2 mutants) or a WW mutant increased axon branching. Although the FE65 mutants did not affect total neurite output, both mutants decreased axon segment length, consistent with an overall slowing of axonal growth cones. FE65 mutants did not alter the localization of either APP or FE65 in axonal growth cones, suggesting that the effects on neurite outgrowth are achieved by alterations in local complex formation within the axonal growth cone.     

Exogenous BDNF, NT-3 and NT-4 differentially regulate neurite outgrowth in cultured hippocampal neurons

Multiple growth factors contribute to the differentiation of dendritic and axonal processes by a neuron. Cultured hippocampal cells elaborate dendritic and axonal processes following well-defined steps. We used this culture system to determine the specific effects of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on dendritic and axonal differentiation in hippocampal pyramidal neurons. We demonstrated that each of these neurotrophins exert distinct effects on neurite outgrowth. Both BDNF and NT-3 had positive effects on the outgrowth of undifferentiated neurites, called minor neurites, and on the axonal process of hippocampal pyramidal neurons. However, the effect of NT-3 was more important than that of BDNF. On the other hand, NT-4 did not enhance axonal outgrowth but had only an effect on the outgrowth of minor neurites. Since cytoskeletal proteins play crucial roles in promoting neurite outgrowth, we examined the protein levels of some of these proteins that are associated with neurite outgrowth: beta-actin, gamma-actin, alpha-tubulin, MAP2 and tau. Surprisingly, we did not detect any change in their protein levels. Taken together, our results show that BDNF, NT-3 and NT-4 exert distinct effects on the neuritic compartments of hippocampal neurons.     

Increased proximal bifurcation of CA1 pyramidal apical dendrites in sema3A mutant mice

Semaphorin‐3A (Sema3A) is an attractive guidance molecule for cortical apical dendrites. To elucidate the role of Sema3A in hippocampal dendritic formation, we examined the Sema3A expression pattern in the perinatal hippocampal formation and analyzed hippocampal dendrites of the brains from young adult sema3A mutant mice. Sema3A protein was predominantly expressed in the hippocampal plate and the inner marginal zone at the initial period of apical dendritic growth. Neuropilin‐1 and plexin‐A, the receptor components for Sema3A, were also localized in the same regions. The Golgi impregnation method revealed that in wildtype mice more than 90% of hippocampal CA1 pyramidal neurons extended a single trunk or apical trunks bifurcated in stratum radiatum. Seven percent of the pyramidal neurons showed proximal bifurcation of apical trunks in stratum pyramidale or at the border of the stratum pyramidale and stratum radiatum. In sema3A mutant mice, proximally bifurcated apical dendrites were increased to 32%, while the single apical dendritic pyramidal neurons were decreased. We designate this phenotype in sema3A mutant mice as “proximal bifurcation.” In the dissociated culture system, approximately half of the hippocampal neurons from wildtype mice resembled pyramidal neurons, which possess a long, thick, and tapered dendrite. In contrast, only 30% of the neurons from sema3A mutants exhibited pyramidal‐like morphology. Proximal bifurcation of CA1 pyramidal neurons was also increased in the mutant mice of p35, an activator of cyclin‐dependent kinase 5 (Cdk5). Thus, Sema3A may facilitate the initial growth of CA1 apical dendrites via the activation of p35/Cdk5, which may in turn signal hippocampal development. J. Comp. Neurol. 516:360–375, 2009. © 2009 Wiley‐Liss, Inc.     

Lipid raft‐targeted Akt promotes axonal branching and growth cone expansion via mTOR and Rac1, respectively

The molecular mechanisms by which extracellular guidance cues regulate axonal morphology are not fully understood. Recent findings suggest that increased activity of the protein kinase Akt promotes dendritic branching and elongation in hippocampal neurons. We tested whether expression of constitutively active Akt (CA‐Akt) in primary sensory neurons would promote axonal branching and whether targeting CA‐Akt to lipid rafts, common sites of Akt function, would differentially regulate axonal morphology. Biolistic transduction of sensory neurons induced a rapid expression of CA‐Akt, resulting in increased axonal branching, cell hypertrophy, and growth cone expansion. Additionally, we found that targeting of CA‐Akt to lipid rafts significantly potentiated growth cone expansion compared with expression of CA‐Akt throughout the neuron. Because lipid rafts are concentrated within the growth cone, this finding suggests that signaling of expansion is likely regulated locally. We found that CA‐Akt‐mediated growth cone expansion, but not axonal branching, was attenuated by coexpression of dominant‐negative Rac1. In contrast, blockade of mammalian target of rapamycin (mTOR) prevented axonal branching and hypertrophy in response to CA‐Akt, but not growth cone expansion. These data indicate that Akt activity can regulate growth cone expansion via localized Rac1 signaling and regulate axonal branching and soma size via activation of mTOR. © 2009 Wiley‐Liss, Inc.     

CRMP2在大鼠海马神经元中的表达分布及对神经元突起生长的影响

目的探讨脑衰反应调节蛋白2(CRMP2)在大鼠海马神经元中的表达分布及对神经元突起生长的影响。方法CRMP2及其模拟磷酸化质粒CRMP2-S522D、CRMP2-T555D,转入体外培养的SD大鼠海马神经元,转染72 h后,采用免疫印迹法和免疫荧光染色分析CRMP2、CRMP2-S522D、CRMP2-T555D在神经元中的分布差异以及对神经元突起分枝和突起长度的影响。结果 CRMP2及CRMP2-T555D在神经元胞体和突起均有分布,CRMP2-S522D则主要分布于胞体。CRMP2-T555D对神经元的突起长度和分枝数均没有明显影响;CRMP2-S522D对突起数目没有明显影响,但可以显著降低突起长度。结论 CRMP2的不同磷酸化形式在大鼠海马神经元中存在分布差异,并对神经元的突起长度产生影响。     

Neuregulin-1beta induces neurite extension and arborization in cultured hippocampal neurons

Neuregulin-1 (NRG-1) growth and differentiation factors and their erbB receptors are hypothesized to promote embryonic hippocampal neuron differentiation via as yet unknown mechanisms. We have found that NRG-1beta increases the outgrowth of primary neurites, neuronal area, total neurite length, and neuritic branching in E18 hippocampal neurons. NRG-1beta effects on neurite extension and arborization are similar to, but not additive with, those of brain-derived neurotrophic factor and reflect direct NRG-1 action on hippocampal neurons as these cells express the NRG-1 receptors erbB2 and erbB4, the erbB-specific inhibitor PD158780 decreases NRG-1beta induced neurite outgrowth, and NRG-1beta stimulation induces p42/44 ERK phosphorylation. Pharmacological inhibition of p42/44 ERK and protein kinase C (PKC), but not PI3K or p38 MAP kinase, inhibits NRG-1beta-induced neurite extension and elaboration. We conclude that NRG-1beta stimulates hippocampal neurite extension and arborization via a signaling pathway that involves erbB membrane tyrosine kinases (erbB2 and/or erbB4), p42/44 ERK, and PKC.     

Neuregulin-1β induces neurite extension and arborization in cultured hippocampal neurons

Neuregulin-1 (NRG-1) growth and differentiation factors and their erbB receptors are hypothesized to promote embryonic hippocampal neuron differentiation via as yet unknown mechanisms. We have found that NRG-1beta increases the outgrowth of primary neurites, neuronal area, total neurite length, and neuritic branching in E18 hippocampal neurons. NRG-1beta effects on neurite extension and arborization are similar to, but not additive with, those of brain-derived neurotrophic factor and reflect direct NRG-1 action on hippocampal neurons as these cells express the NRG-1 receptors erbB2 and erbB4, the erbB-specific inhibitor PD158780 decreases NRG-1beta induced neurite outgrowth, and NRG-1beta stimulation induces p42/44 ERK phosphorylation. Pharmacological inhibition of p42/44 ERK and protein kinase C (PKC), but not PI3K or p38 MAP kinase, inhibits NRG-1beta-induced neurite extension and elaboration. We conclude that NRG-1beta stimulates hippocampal neurite extension and arborization via a signaling pathway that involves erbB membrane tyrosine kinases (erbB2 and/or erbB4), p42/44 ERK, and PKC.     

Nanomolar concentrations of nicotine and cotinine alter the development of cultured hippocampal neurons via non-acetylcholine receptor-mediated mechanisms.

We investigated the effects of nicotine and its metabolic byproduct cotinine on survival, differentiation and intracellular Ca2+ levels of cultured E18 rat hippocampal neurons. We used a range of concentrations from 1 nM to 10 microM, most of which are within the likely range of human fetal exposure from maternal smoking. Nicotine did not influence neuron survival or neurite production. However, at all concentrations tested, nicotine significantly increased branching of both axons and dendrites, an effect which was not reversed by co-culturing with alpha-bungarotoxin, which blocks the nicotinic acetylcholine receptors that predominate in hippocampal cultures (Alkondon and Albuquerque, 1993; Barrantes et al., 1995b). Cotinine at 100 nM and 1 microM significantly reduced neuron survival and neurite production of surviving neurons, but did not significantly alter axon or dendrite branching. These membrane-permeable compounds may work synergistically in the developing embryo to impair the survival and differentiation of hippocampal neurons via intracellular mechanisms.     

The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitro

In the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB(1)Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB(1)R expression as well as effects of CB(1)R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB(1)Rs are strongly expressed in both reelin-expressing Cajal-Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB(1)R protein is localized first to somato-dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB(1)Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP-55940 results in the relocation of receptors from axons to somato-dendritic endosomes, indicating the functional competence of embryonic CB(1)Rs. The adult phenotype of CB(1)R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB(1)R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB(1)R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB(1)R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption.     

Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Both 17beta-estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age-related memory changes. There is growing evidence that a number of estrogen receptor-mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.Low levels of estradiol (10nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen-stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S-nitroso-N-acetylpenicillamine (SNAP: 119%; 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl-cGMP: 173% of control). Estradiol-stimulated dendritic branching was reversed by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.     

NETMORPH: A Framework for the Stochastic Generation of Large Scale Neuronal Networks With Realistic Neuron Morphologies

We present a simulation framework, called NETMORPH, for the developmental generation of 3D large-scale neuronal networks with realistic neuron morphologies. In NETMORPH, neuronal morphogenesis is simulated from the perspective of the individual growth cone. For each growth cone in a growing axonal or dendritic tree, its actions of elongation, branching and turning are described in a stochastic, phenomenological manner. In this way, neurons with realistic axonal and dendritic morphologies, including neurite curvature, can be generated. Synapses are formed as neurons grow out and axonal and dendritic branches come in close proximity of each other. NETMORPH is a flexible tool that can be applied to a wide variety of research questions regarding morphology and connectivity. Research applications include studying the complex relationship between neuronal morphology and global patterns of synaptic connectivity. Possible future developments of NETMORPH are discussed.     

Dock4 regulates dendritic development in hippocampal neurons

Dendrite development is required for establishing proper neuronal connectivity. Rho-family small GTPases have been reported to play important roles in the regulation of dendritic growth and morphology. However, the molecular mechanisms that control the activities of Rho GTPases in developing dendrites are not well understood. In the present study we found Dock4, an activator of the small GTPase Rac, to have a role in regulating dendritic growth and branching in rat hippocampal neurons. Dock4 is highly expressed in the developing rat brain, predominantly in hippocampal neurons. In dissociated cultured hippocampal neurons, the expression of Dock4 protein is up-regulated after between 3 and 8 days in culture, when dendrites begin to grow. Knockdown of endogenous Dock4 results in reduced dendritic growth and branching. Conversely, overexpression of Dock4 with its binding partner ELMO2 enhances the numbers of dendrites and dendritic branches. These morphological effects elicited by Dock4 and ELMO2 require Rac activation and the C-terminal Crk-binding region of Dock4. Indeed, Dock4 forms a complex with ELMO2 and CrkII in hippocampal neurons. These findings demonstrate a new function of the Rac activator Dock4 in dendritic morphogenesis in hippocampal neurons.     

Reelin and mDab1 regulate the development of hippocampal connections

We analyze in this study the participation of Reelin and mDab1 in the development of hippocampal connections. We show that mDab1 is present in growth cones and axonal tracts of developing hippocampal afferents. mdab1-deficiency produces severe alterations in the entorhino-hippocampal and commissural connections identical to those described in reeler mice, including innervation of ectopic areas, formation of abnormal patches of fiber termination and a delay in the refinement of projections. Organotypic slice cultures combining tissue from mdab1-mutant and control mice demonstrate that the abnormalities observed in the mutant entorhino-hippocampal projection are caused by mdab1-deficiency in both the projecting neurons and target hippocampal cells. Axonal afferents that innervate the hippocampus react to Reelin by reducing axonal growth, and increasing growth cone collapse and axonal branching. Altogether these results indicate that Reelin and mDab1 participate in the development and refinement of hippocampal connections by regulating axonal extension, targeting and branching.