Small GTPase Rab17 regulates dendritic morphogenesis and postsynaptic development of hippocampal neurons

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.     

Influence of Olfactory Epithelium on Mitral/Tufted Cell Dendritic Outgrowth

Stereotypical connections between olfactory sensory neuron axons and mitral cell dendrites in the olfactory bulb establish the first synaptic relay for olfactory perception. While mechanisms of olfactory sensory axon targeting are reported, molecular regulation of mitral cell dendritic growth and refinement are unclear. During embryonic development, mitral cell dendritic distribution overlaps with olfactory sensory axon terminals in the olfactory bulb. In this study, we investigate whether olfactory sensory neurons in the olfactory epithelium influence mitral cell dendritic outgrowth in vitro. We report a soluble trophic activity in the olfactory epithelium conditioned medium which promotes mitral/tufted cell neurite outgrowth. While the trophic activity is present in both embryonic and postnatal olfactory epithelia, only embryonic but not postnatal mitral/tufted cells respond to this activity. We show that BMP2, 5 and 7 promote mitral/tufted cells neurite outgrowth. However, the BMP antagonist, Noggin, fails to neutralize the olfactory epithelium derived neurite growth promoting activity. We provide evidence that olfactory epithelium derived activity is a protein factor with molecular weight between 50–100 kD. We also observed that Follistatin can effectively neutralize the olfactory epithelium derived activity, suggesting that TGF-beta family proteins are involved to promote mitral/tufted dendritic elaboration.     

Combinatorial assembly of neurons: from chromatin to dendrites

The generation and refinement of dendrites is essential for normal brain development and function. However, the molecular mechanisms that govern dendritic morphogenesis are poorly understood. Recent studies from the Crabtree laboratory have uncovered a requirement for the neuron-specific chromatin-remodeling enzyme nBAF in dendritic growth and branching in response to neuronal activity. These findings highlight the significance of epigenetic mechanisms in activity-dependent dendritic morphogenesis, with important implications in brain development and plasticity.     

Differentiated neurons retain the capacity to generate axons from dendrites

Cutting the axon of a morphologically polarized neuron (stage 3) close to the cell body causes another neurite to grow as an axon [1-3]. Stage 3 neurons still lack molecular segregation of axonal and dendritic proteins, however. Axonal and dendritic compartments acquire their distinct composition at stage 4 (4-5days in culture), when proteins such as the microtubule-associated protein 2 (MAP-2) and the glutamate receptor subunit GluR1 localize to the dendrites and disappear from the axon [4,5]. We investigated whether cultured hippocampal neurons retained axon/dendrite plasticity after axons and dendrites have created their distinct cytoskeletal architecture and acquired their specific membrane composition. We found that axotomy of stage 4 neurons transformed a dendrite into an axon. Using axonal and dendritic markers, we tested whether cytoskeletal changes could cause similar transformations, and found that actin depolymerization induced multiple axons in unpolarized neurons. Moreover, depletion of actin filaments from both morphologically and molecularly polarized cells also resulted in the growth of multiple axons from pre-existing dendrites. These results imply that dendrites retain the potential to become axons even after molecular segregation has occurred and that the dendritic fate depends on the integrity of the actin cytoskeleton.     

The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines

NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. We report that the Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with RNAi and dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines.     

Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.     

Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis

To study the roles of intracellular factors in neuronal morphogenesis, we used the mosaic analysis with a repressible cell marker (MARCM) technique to visualize identifiable single multiple dendritic (MD) neurons in living Drosophila larvae. We found that individual neurons in the peripheral nervous system (PNS) developed clear morphological polarity and diverse dendritic branching patterns in larval stages. Each MD neuron in the same dorsal cluster developed a unique dendritic field, suggesting that they have specific physiological functions. Single-neuron analysis revealed that Flamingo did not affect the general dendritic branching patterns in postmitotic neurons. Instead, Flamingo limited the extension of one or more dorsal dendrites without grossly affecting lateral branches. The dendritic overextension phenotype was partially conferred by the precocious initiation of dorsal dendrites in flamingo mutant embryos. In addition, Flamingo is required cell autonomously to promote axonal growth and to prevent premature axonal branching of PNS neurons. Our molecular analysis also indicated that the amino acid sequence near the first EGF motif is important for the proper localization and function of Flamingo. These results demonstrate that Flamingo plays a role in early neuronal differentiation and exerts specific effects on dendrites and axons.     

Shikonin induces immunogenic cell death in tumor cells and enhances dendritic cell-based cancer vaccine

Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.     

RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport

The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.     

Hepatocyte growth factor-induced c-Src-phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway inhibits dendritic cell activation by blocking IκB kinase activity

Hepatocyte growth factor modulates activation and antigen-presenting cell function of dendritic cells. However, the molecular basis for immunoregulation of dendritic cells by hepatocyte growth factor is undefined. In the current study, we demonstrate that hepatocyte growth factor exhibits inhibitory effect on dendritic cell activation by blocking IκB kinase activity and subsequent nuclear factor-κB activation. Inhibition of IκB kinase is mediated by hepatocyte growth factor-induced activation of c-Src. Proximal signaling events induced in dendritic cells by hepatocyte growth factor include a physical association of c-Src with the hepatocyte growth factor receptor c-MET and concomitant activation of c-Src. Activation of c-Src in turn establishes a complex consisting of phosphatidylinositol 3-kinase and c-MET, and promotes downstream activation of the phosphatidylinositol 3-kinase/AKT pathway and mammalian target of rapamycin. Blocking activation of c-Src, phosphatidylinositol 3-kinase and mammalian target of rapamycin prevents hepatocyte growth factor-induced inhibition of IκB kinase, nuclear factor-κB and dendritic cell activation. Notably, hepatocyte growth factor-stimulated c-Src activation results in induction of phosphatidylinositol 3-kinase complexes p85α/p110α and p85α/p110δ, which is required for activation of mammalian target of rapamycin, and consequent inhibition of IκB kinase and nuclear factor-κB activation. Our findings, for the first time, have identified the c-Src-phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway that plays a pivotal role in mediating the inhibitory effects of hepatocyte growth factor on dendritic cell activation by blocking nuclear factor-κB signaling.     

Maturation of dendritic architecture: lessons from insect identified neurons

The highly complex geometry of dendritic trees is crucial for neural signal integration and the proper wiring of neuronal circuits. The morphogenesis of dendritic trees is regulated by innate genetic factors, neuronal activity, and external molecular cues. How each of these factors contributes to dendritic maturation has been addressed in studies of the developing nervous systems of animals ranging from insects to mammals. This article reviews our current knowledge and understanding of the role of afferent input in the establishment of the architecture of mature dendritic trees, using insect neurons as models. With these model systems and using quantitative morphometry, it is possible to define the contributions of intrinsic and extrinsic factors in dendritic morphogenesis of identified neurons and to evaluate the impact of dendritic maturation on the integration of identified neurons into functional circuits subserving identified behaviors. The commonly held view of dendritic morphogenesis is that general structural features result from genetic instructions, whereas fine connectivity details rely mostly on substrate interactions and functional activity. During early dendritic maturation, dendritic growth cone formation produces new branches at all dendritic roots. The second phase is growth cone independent and afferent input dependent, during which branching is limited to high order distal dendrites. During the third phase, activity-dependent synaptic maturation occurs with limited or subtle remodeling of branching.     

Short- and Long-Term Effects of LRRK2 on Axon and Dendrite Growth

Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson''s disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.     

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.     

Cellular and molecular control of dendritic growth and development of cerebellar Purkinje cells

Purkinje cells are the principal neurons of the cerebellar cortex and are characterized by a large and highly branched dendritic tree. For this reason, they have for a long time been an attractive model system to study the regulation of dendritic growth and differentiation. In this article, I will first review studies on different aspects of Purkinje cell dendritic development and then go on to present studies which have aimed at experimentally altering Purkinje cell dendritic development. Some of the cellular and molecular mechanisms which have been shown by these studies to be important determinants of Purkinje cell dendritic development will be discussed, in particular the role of the parallel fiber input, of hormones, and of neuronal growth factors. The organotypic slice culture method will be introduced as an important experimental tool to study Purkinje cell dendritic development under controlled conditions. Using cerebellar slice cultures, protein kinase C (PKC) has been identified as a major determinant of Purkinje cell dendritic development and the contribution of specific isoforms of PKC will be discussed. Finally, it will be shown that Purkinje cell dendritic development in slice cultures does not depend on the activation of glutamate receptors and appears to be independent of the presence of the neurotrophin BDNF. These studies indicate that the initial outgrowth of the Purkinje cell dendritic tree can occur in the absence of signals derived from afferent fibers, but is under control of PKC signaling.     

大鼠骨髓来源未成熟与成熟树突状细胞的对比研究

目的:对比培养大鼠骨髓来源的未成熟树突状细胞与成熟树突状细胞,并从形态学、表型及功能检测等多方面进行对比研究,为后续的实验做出基础研究。方法:大鼠脱臼法处死后取两侧胫骨、股骨,PBS冲洗骨髓腔收集骨髓细胞,经GM-CSF和IL-4刺激培养六天后,对比研究经LPS刺激组与未经LPS刺激培养组细胞状况。结果:①成熟树突状细胞悬浮生长,集落分散,扫描电镜下见其突起数目明显多于未成熟树突状细胞。②成熟树突状细胞高表达表面标记分子CD80、CD86、MHCⅡ,而未成熟树突状细胞均低表达。③成熟树突状细胞培养基上清中IL-12水平高,而未成熟树突状细胞培养基上清中IL-12水平低。④成熟树突状细胞具有强的刺激T细胞增殖能力,而未成熟树突状细胞基本不具有诱导T细胞增殖能力。结论:未成熟状态的树突状细胞具备致耐受原性,可抑制T细胞的应答,而成熟状态的树突状细胞由于获得了免疫刺激潜能从而会对炎性刺激做出反应。     

Emerging aspects of membrane traffic in neuronal dendrite growth

Polarized growth of the neuron would logically require some form of membrane traffic to the tip of the growth cone, regulated in conjunction with other trafficking processes that are common to both neuronal and non-neuronal cells. Unlike axons, dendrites are endowed with membranous organelles of the exocytic pathway extending from the cell soma, including both rough and smooth endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Dendrites also have satellite Golgi-like cisternal stacks known as Golgi outposts that have no membranous connections with the somatic Golgi. Golgi outposts presumably serve both general and specific local trafficking needs, and could mediate membrane traffic required for polarized dendritic growth during neuronal differentiation. Recent findings suggest that dendritic growth, but apparently not axonal growth, relies very much on classical exocytic traffic, and is affected by defects in components of both the early and late secretory pathways. Within dendrites, localized processes of recycling endosome-based exocytosis regulate the growth of dendritic spines and postsynaptic compartments. Emerging membrane traffic processes and components that contribute specifically to dendritic growth are discussed.