Anirvan Ghosh

Semaphorin 3A is a chemoattractant for cortical apical dendrites

The apical dendrites of pyramidal neurons integrate inputs from various cortical layers and are central to information processing. Here we show that the growth of apical dendrites towards the pial surface is regulated by a diffusible chemoattractant present at high levels near the marginal zone. A major component of this signal is semaphorin 3A (Sema3A), which was previously characterized as a chemorepellant for cortical axons. Soluble guanylate cyclase is asymmetrically localized to the developing apical dendrite, and is required for the chemoattractive effect of Sema3A. Thus the asymmetric localization of soluble guanylate cyclase confers distinct Sema3A responses to axons and dendrites. These observations reveal a mechanism by which a single chemotropic signal can pattern both axons and dendrites during development.

ACTIVITY-DEPENDENT REGULATION OF DENDRITIC GROWTH AND PATTERNING

One of the most remarkable features of the developing brain is its ability to undergo structural change in response to experience. Among the cellular elements that show this kind of plasticity are dendrites, which are the components that receive and process synaptic information. Recent observations indicate that calcium signalling in neurons can regulate dendritic growth and remodelling by several mechanisms, and these mechanisms are likely to be key mediators of structural plasticity in the developing brain.

Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis

To identify molecules that regulate the transition of dividing neuroblasts to terminally differentiated neurons in the CNS, conditions have been developed that allow the neuronal differentiation of cortical precursor cells to be examined in vitro. In these cultures, the proliferation of undifferentiated precursor cells is controlled by basic fibroblast growth factor (bFGF). The proliferative effects of bFGF do not preclude the action of signals that promote differentiation, since addition of neurotrophin-3 (NT-3) antagonizes the proliferative effects of bFGF and enhances neuronal differentiation. In addition, blocking NT-3 function with neutralizing antibodies leads to a marked decrease in the number of differentiated neurons, without affecting the proliferation of cortical precursors or the survival of postmitotic cortical neurons. These observations suggest that bFGF and NT-3, by their distinct effects on cell proliferation and differentiation, are key regulators of neurogenesis in the CNS.

Regulation of Dendritic Growth and Remodeling by Rho, Rac, and Cdc42

The acquisition of cell type-specific morphologies is a central feature of neuronal differentiation and has important consequences for nervous system function. To begin to identify the underlying molecular mechanisms, we have explored the role of Rho-related GTPases in the dendritic development of cortical neurons. Expression of dominant negative mutants of Rac or Cdc42, the Rho-inhibitory molecule C3 transferase, or the GTPase-activating protein RhoGAP p190 causes a marked reduction in the number of primary dendrites in nonpyramidal (multipolar) neurons and in the number of basal dendrites in neurons with pyramidal morphologies. Conversely, the expression of constitutively active mutants of Rho, Rac, or Cdc42 leads to an increase in the number of primary and basal dendrites. In cortical cultures, as in vivo, dendritic remodeling leads to an apparent transformation from pyramidal to nonpyramidal morphologies over time. Strikingly, this shift in favor of nonpyramidal morphologies is also inhibited by the expression of dominant negative mutants of Cdc42 and Rac and by RhoGAP p190. These observations indicate that Rho, Rac, and Cdc42 play a central role in dendritic development and suggest that differential activation of Rho-related GTPases may contribute to the generation of morphological diversity in the developing cortex.

Calcium Regulation of Dendritic Growth via CaM Kinase IV and CREB-Mediated Transcription

We report that CaM kinase IV and CREB play a critical role in mediating calcium-induced dendritic growth in cortical neurons. Calcium-dependent dendritic growth is suppressed by CaM kinase inhibitors, a constitutively active form of CaM kinase IV induces dendritic growth in the absence of extracellular stimulation, and a kinase-dead form of CaM kinase IV suppresses dendritic growth induced by calcium influx. CaM kinase IV activates the transcription factor CREB, and expression of a dominant negative form of CREB blocks calcium- and CaM kinase IV-induced dendritic growth. In cortical slice cultures, dendritic growth is attenuated by inhibitors of voltage-sensitive calcium channels and by dominant negative CREB. These experiments indicate that calcium-induced dendritic growth is regulated by activation of a transcriptional program that involves CaM kinase IV and CREB-mediated signaling to the nucleus.

Nuclear Notch1 signaling and the regulation of dendritic development

To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-domain constructs transfected into cortical neurons were present in multiple phosphorylated forms, localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an important regulatory influence on the specification of dendritic morphology in neurons.

Regulation of Dendritic Development by Neuron-Specific Chromatin Remodeling Complexes

The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53bs role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.

LRRTM2 Interacts with Neurexin1 and Regulates Excitatory Synapse Formation

We identify the leucine-rich repeat transmembrane protein LRRTM2 as a key regulator of excitatory synapse development and function. LRRTM2 localizes to excitatory synapses in transfected hippocampal neurons, and shRNA-mediated knockdown of LRRTM2 leads to a decrease in excitatory synapses without affecting inhibitory synapses. LRRTM2 interacts with PSD-95 and regulates surface expression of AMPA receptors, and lentivirus-mediated knockdown of LRRTM2 in vivo decreases the strength of evoked excitatory synaptic currents. Structure-function studies indicate that LRRTM2 induces presynaptic differentiation via the extracellular LRR domain. We identify Neurexin1 as a receptor for LRRTM2 based on affinity chromatography. LRRTM2 binds to both Neurexin 1alpha and Neurexin 1beta, and shRNA-mediated knockdown of Neurexin1 abrogates LRRTM2-induced presynaptic differentiation. These observations indicate that an LRRTM2-Neurexin1 interaction plays a critical role in regulating excitatory synapse development.

Regulation of Cortical Dendrite Development by Slit-Robo Interactions

Slit proteins have previously been shown to regulate axon guidance, branching, and neural migration. Here we report that, in addition to acting as a chemorepellant for cortical axons, Slit1 regulates dendritic development. Slit1 is expressed in the developing cortex, and exposure to Slit1 leads to increased dendritic growth and branching. Conversely, inhibition of Slit-Robo interactions by Robo-Fc fusion proteins or by a dominant-negative Robo attenuates dendritic branching. Stimulation of neurons transfected with a Met-Robo chimeric receptor with Hepatocyte growth factor leads to a robust induction of dendritic growth and branching, suggesting that Robo-mediated signaling is sufficient to induce dendritic remodeling. These experiments indicate that Slit-Robo interactions may exert a significant influence over the specification of cortical neuron morphology by regulating both axon guidance and dendritic patterning.

Regulation of CBP-Mediated Transcription by Neuronal Calcium Signaling

The transcription factor CREB is involved in mediating many of the long-term effects of activity-dependent plasticity at glutamatergic synapses. Here, we show that activation of NMDA receptors and voltage-sensitive calcium channels leads to CREB-mediated transcription in cortical neurons via a mechanism regulated by CREB-binding protein (CBP). Recruitment of CBP to the promoter is not sufficient for transactivation, but calcium influx can induce CBP-mediated transcription via two distinct transactivation domains. CBP-mediated transcription is stimulus strength-dependent and can be induced by activation of CaM kinase II, CaM kinase IV, and protein kinase A, but not by activation of the Ras-MAP kinase pathway. These observations indicate that CBP can function as a calcium-sensitive transcriptional coactivator that may act as a regulatory switch for glutamate-induced CREB-mediated transcription.