Patricia F. Maness - US grants

The study of oncogenes has provided an unexpected probe for neuronal development and function. Normal cellular pp60c-src, a protein of unknown function that is homologous to the transforming protein of Rous sarcoma virus, has been shown by this laboratory to be expressed at elevated levels in fully differentiated, functional neurons and in cells of the early embryo. This proposal will investigate a function for pp60c-src in transmembrane signaling in mature neurons and in undifferentiated cells of early embryos during commitment to the neural pathway. (a) pp60c-src will be localized within the neuronal plasma membrane at functionally distinct sites, where specific ion channels are located, by biochemical fractionation of neuronal membranes isolated from the chick neural retina and by immunoperoxidase staining of retinal neurons in culture. (b) Regulation of the tyrosine-specific protein kinase activity of pp60c-src by neural protein kinases will be studied in retinal neurons stimulated in vivo with Beta-adrenergic agonists (which activate cAMP-dependen protein kinase), phorgal esters (which activate protein kinase C) and selected neurotransmitters. Phosphorylation sites within the pp60c-src molecule will be identified by peptide mapping and phosphoamino acid analysis; and changes in the tyrosine kinase activity of pp60c-src measured using a synthetic peptide substrate. (c) A novel src-related tyrosine kinase (100K) that we have identified in the electric organ of the electric eel will be characterized by peptide mapping and cDNA cloning for future determination of its coding sequence and to provide probes for its study in higher animals. The eel src protein may be a new member of the src family of tyrosine kinases or have a unique domain that facilitates electrical signaling. (d) Finally, the expression of pp60c-src in cells of the early chick embryo that are the targets of neural inducing signals will be mapped by immunocytochemical staining. The proposed study of tyrosine kinases in the nervous system is a fundamentally new approach to the study of neuronal function and differentiation, and will also provide insight into the mechanism by which the very similar but mutant protein of Rous sarcoma virus transforms cells.

The study of oncogenes has provided an unexpected probe for neuronal development and function. Normal cellular pp60c-src, a protein of unknown function that is homologous to the transforming protein of Rous sarcoma virus, has been shown by this laboratory to be expressed at elevated levels in fully differentiated, functional neurons and in cells of the early embryo. This proposal will investigate a function for pp60c-src in transmembrane signalling in mature neurons and in undifferentiated cells of early embryos during commitment to the neural pathway. (a) pp60c-src will be localized within the neuronal plasma membrane at functionally distinct sites, where specific ion channels are located, by biochemical fractionation of neuronal membranes isolated from the chick neural retina and by immunoperoxidase staining of retinal neurons in culture. (b) Regulation of the tyrosine-specific protein kinase activity of pp60c-src by neural protein kinases will be studied in retinal neurons stimulated in vivo with B-adrenergic agonists (which activate cAMP-dependent protein kinase), phorbol esters (which activate protein kinase C) and selected neurotransmitters. Phosphorylation sites within the pp60c-src molecule will be identified by peptide mapping and phosphoamino acid analysis; and changes in the tyrosine kinase activity of pp60c-src measured using a synthetic peptide substrate. (c) A novel src-related tyrosine kinase (100K) that we have identified in the electric organ of the electric eel will be characterized by peptide mapping and cDNA cloning for future determination of its coding sequence and to provide probes for its study in higher animals. The eel src protein may be a new member of the src family of tyrosine kinases or have a unique domain that facilitates electrical signalling. (d) Finally, the expression of pp60c-src in cells of the early chick embryo that are the targets of neural inducing signals will be mapped by immunocytochemical staining. The proposed study of tyrosine kinases in the nervous system is a fundamentally new approach to the study of neuronal function and differentiation, and will also provide insight into the mechanism by which the very similar but mutant protein of Rous sarcoma virus transforms cells.

The study of oncogenes has provided an unexpected probe for neuronal development and function. Normal cellular pp60c-src, a protein of unknown function that is homologous to the transforming protein of Rous sarcoma virus, has been shown by this laboratory to be expressed at elevated levels in fully differentiated, functional neurons and in cells of the early embryo. This proposal will investigate a function for pp60c-src in transmembrane signaling in mature neurons and in undifferentiated cells of early embryos during commitment to the neural pathway. (a) pp60c-src will be localized within the neuronal plasma membrane at functionally distinct sites, where specific ion channels are located, by biochemical fractionation of neuronal membranes isolated from the chick neural retina and by immunoperoxidase staining of retinal neurons in culture. (b) Regulation of the tyrosine-specific protein kinase activity of pp60c-src by neural protein kinases will be studied in retinal neurons stimulated in vivo with Beta-adrenergic agonists (which activate cAMP-dependen protein kinase), phorgal esters (which activate protein kinase C) and selected neurotransmitters. Phosphorylation sites within the pp60c-src molecule will be identified by peptide mapping and phosphoamino acid analysis; and changes in the tyrosine kinase activity of pp60c-src measured using a synthetic peptide substrate. (c) A novel src-related tyrosine kinase (100K) that we have identified in the electric organ of the electric eel will be characterized by peptide mapping and cDNA cloning for future determination of its coding sequence and to provide probes for its study in higher animals. The eel src protein may be a new member of the src family of tyrosine kinases or have a unique domain that facilitates electrical signaling. (d) Finally, the expression of pp60c-src in cells of the early chick embryo that are the targets of neural inducing signals will be mapped by immunocytochemical staining. The proposed study of tyrosine kinases in the nervous system is a fundamentally new approach to the study of neuronal function and differentiation, and will also provide insight into the mechanism by which the very similar but mutant protein of Rous sarcoma virus transforms cells.

DESCRIPTION: (Applicant 's abstract) Tyrosine-specific protein kinases are abundant in synaptic layers of the vertebrate neural retina but their function remains a central unanswered question in vision research. We recently made the important discovery that tyrosine (Tyr) phosphorylation of specific proteins (106, 78, 40 Kd and others) associated with terminals of retinal neurons and surrounding Muller glial processes in the chick neural retina is dramatically stimulated in response to illumination. The response is rapid, nontransient, and readily reversible. Our immediate goal is to define the proximal components of the pathway of signal transduction mediated by Tyr kinases in response to light (the neurotransmitter signals, the Tyr kinases activated by light, and the substrates of kinase action) with the long range goal of elucidating their function in the photic response. Our working hypothesis is that Tyr dephosphorylation of substrates is related to neurotransmitter signalling or reuptake in retinal neurons or glia. We will apply a new tool, phosphotyrosine (PTyr) antibodies to analyze the PTyr response in the chick retina by immunocytochemical, biochemical, and molecular biological techniques. We will (1) determine the optimal illumination conditions pathways, (2) use immunoelectron microscopy to localize the subcellular sites of the substrates and to test the postulate that the PTyr response is related to coated vesicle dynamics involved in membrane recycling, (3) identify specific neurotransmitters as signals for Tyr dephosphorylation or phosphorylation in the outer and inner plexiform layers, (4) generate monolconal antibodies to study the structure and function of retinal substrates, and (5) identify novel Tyr kinases responsible for light-dependent phosphorylation of substrates in the retina by PTyr antibody screening of a retinal cDNA library. This research investigates an unexplored areas of protein modification in the retina, which will contribute to our understanding of normal retinal function and vision.

DESCRIPTION: Three sets of findings stand out as being particularly significant. (1) In one series of experiments it was shown that axons from cerebellar cells (90 percent granule cells) in which Src has been knocked out extend axons about 1/2 as fast as wild type cells on L1 coated nitrocellulose. Cerebellar neurons from Fyn or Yes knockout mice have normal growth rates on L1. Conversely, NCAM dependent growth of Fyn- cerebellar cells grown on NCAM expressing fibroblasts is impaired, while growth of Src- and Yes- cells is unaffected. These findings suggest that normal L1 mediated outgrowth specifically requires Src function, while NCAM mediated outgrowth specifically requires Fyn- function. A second set of findings which amplify these results is the demonstration by immunoprecipitation and western blotting of complex formation between L1 and Src from a presumptive growth cone preparation, and between NCAM and Fyn from postnatal cerebellum and other sources. The demonstration of physical contact between these CAMS and specific kinases lends weight to the hypothesis that the kinases may be activated by Cam function. (3) Perhaps most interesting of all is the finding that olfactory epithelial axons in mice doubly mutant for Src- and Fyn- are less fasciculated and more scattered in their trajectories than axons in either single mutant alone or in wild type. These results support the hypothesis that Src and Fyn are required for normal pathfinding, but can partially compensate for one another. These accomplishments lead the field in rigor and in significance. The proposal has four specific aims. Work described in the first specific aim is a continuation of the current studies characterizing the association between Src, Fyn, L1 , and NCAM. Thus far there is good evidence that the kinases associate with their respective CAMS, but little information about whether this association is modulated by Cam-Cam binding, or whether such binding would activate the kinases. One objective is to determine if homophilic binding (or 'triggering') of the CAMS recruits the kinases into a complex, and if they thereby become activated. The basic strategy is similar to the work already accomplished, except now physical association will be compared in cells that are exposed to an appropriate Cam ligand to those that are not, and added to the assay will be a determination of kinase phosphorylation. A second objective is to identify the regions of Src and Fyn that mediate binding to CAMS. The strategy proposed is to use co-transfection of various kinase constructs along with the appropriate Cam in neuroblastoma cells, and then assay for physical associations by immunoprecipitation. A final objective is to further explore the possibility that L1 associates with Fyn by binding in cis to NCAM which in turn is bound to Fyn. The second specific aim proposes to examine in greater detail the possibility that Fak associates with CAMS and is activated when they bind their ligands. Again, the basic strategy is to use immunoprecipitation to characterize who is bound to whom, and antibodies against phosphotyrosine to determine if Fac is activated. An additional set of experiments is proposed to determine what other cytoskeletal components are organized by Cam-Cam binding using immunoprecipitation of Cam treated Cos cells cotransfected with NCAM and Fyn. In further experiments, beads coated with CAMS would be assayed for their ability to cluster Fak, Fyn, Tensin, and others. In specific aim 3 it is proposed to examine the axonal trajectories of retinal ganglion cell axons in mice with L1, Fyn, or Src knockouts, or combinations of these knockouts. First, the overall structures of the retina, chiasm, and superior colliculus would be checked for gross defects. Retinal axons would be labeled with DiI and traced through these structures to ascertain if any defects in the trajectories are present. In specific aim 4 it is proposed to further examine the expression pattern of a novel receptor kinase (REK, most closely related to Tyro) that is expressed in retinal ganglion cell axons and in the cerebellum. A second objective is to determine if REK can function as a homophilic adhesion molecule, as preliminary experiments suggest. It is also hoped that REK kinase activity can be activated by homophilic binding.

DESCRIPTION: Several X-linked mental retardation syndromes including hydrocephalus, MASA syndrome, agenesis of the corpus callosum and corticopinal tract and spastic paraparesis are caused by mutations in the gene encoding L1, a neural cell adhesion molecule. L1 is a neuronal transmembrane glycoprotein that directs axon growth through homophilic L1-L1 binding and functions in long term potentiation and learning. Extra-cellular Ig and FN III domains mediate homophilic binding, and the cytoplasmic domain mediates signal transduction. Using gene knockout mice, the investigator has established that L1 signals axon growth through a member of the src family of tyrosine kinases (pp60 c-src and p59 fyn), to which it binds. The hypothesis is that adhesive interactions of L1 in neuronal growth cones activate intracellular signaling pathways modulated by non-receptor src family tyrosine kinases that culminate in normal axon growth or guidance. Naturally occuring mutations in the L1 gene may interfere with src kinase signaling, resulting in axonal growth defects in individuals with the L1 disease complex. Mapping of human mutations in the L1 gene has revealed lesions distributed throughout L1, although it is not known how such mutations disrupt L1 function. Information of the signaling pathways activated normally by L1 will help in the understanding of the basic mechanism underlying axonal growth and guidance in CNS development and into the molecular mechanism of X-linked mental retardation. Specific aims are : 1. To characterize the normal interaction of L1 with src family kinases in mouse brain and neuronal cultures. 2. To determine whether selected L1 mutations in the cytoplasmic and extracellular domains disrupt L1-src/fyn interactions. 3. To determine if L1 mutations cause defects in neurite outgrowth in cultures. 4. To determine if there are in vivo defects in major axon tracts (corticospinal tract, corpus callosum) in mice with an L1 gene knockout, and in L1/src, L1/fyn and L1/CAM double knockouts.

DESCRIPTION (provided by applicant): CHL1 (Close Homolog of L1) is an integrin-interacting cell recognition molecule related to the L1 cell adhesion molecule with a potential role in area-specific development of the neocortex. Mutation of the CHL1 gene (CALL) in humans is associated with the 3p-syndrome of mental retardation. CHL1 is expressed in a high caudal to low rostral gradient in cortical precursors during radial migration and in differentiating pyramidal neurons. Preliminary results show CHL1 knockout mice exhibit area- and lamina-specific abnormalities in radial migration and apical dendrite projection of pyramidal cells, and topographic mapping errors of thalamocortical axons. The hypothesis to be tested is that CHL1 modulates radial migration of cortical neurons in the mouse neocortex with consequences on dendrite projection and thalamocortical mapping. Aims are: (1) To define the area- and lamina-specific distribution of cortical neurons and their dendritic development in homozygous and heterozygous CHL1 knockout mice and to assess the interaction of CHL1 with L1 in double mutant mice. A role for CHL1 in Semaphorin 3A-induced dendritic projection and branching of pyramidal neurons will be studied in cortical slices. (2) To determine the cellular mechanism of CHL1 in radial migration of cortical neurons in the posterior neocortex by BrdU labeling in vivo and in brain slice assays by time lapse videomicroscopy. (3) To investigate the molecular mechanism of CHL1 in intracellular signaling through intermediates (Src, Rac, Pak, ERK1,2) important for adhesion dynamics. (4) To identify topographic mapping defects in the thalamocortical projection of CHL1-/- mice by axon tracing in vivo and to analyze CHL1 function in thalamic axon guidance by axon tracing in embryos and a novel telencephalic whole mount assay. This investigation can reveal new molecular determinants and novel mechanisms governing cortical area development and provide insight into the pathology associated with mental retardation.

"This award is funded under the American Recovery and Reinvestment Act of 2009
(Public Law 111-5)."

Guidance and targeting of neuronal axons from eye to brain is orchestrated through a set of cues that are being discovered. This project addresses the mechanism by which L1, a key neural cell adhesion molecule, regulates the map of retinal axons to the superior colliculus, a region of the brain that contains a topographic map of the visual world. The function of this system is to direct behavioral responses toward points in surrounding space through eye, head, and arm movements. The hypothesis to be tested is that L1 guides retinal axons to correct synaptic targets by regulating adhesion to a substrate-bound molecule, termed ALCAM, in conjuction with graded axon guidance cues ephrinB and EphB receptors. The role of L1 interactions in synaptic targeting will be studied using a novel L1 mutant mouse that lacks the ability to stabilize adhesion, and mice with genetic deletions in ALCAM and EphB genes. Axon tracing, cell adhesion and axon growth assays using retinal cells from each strain of mice will determine if L1 binds to ALCAM thus promoting ephrinB1-dependent synaptic targeting of retinal axons to proper coordinates in the brain. This project will enhance economic growth and recovery and provide exceptional educational value by scientific training of undergraduates, graduate, and postdoctoral students, and hiring of a research technician. The importance of this work is that it provides the first molecular explanation for how neurons develop reversible anchorage to form an eye-to-brain map.

Neural cell adhesion molecule NCAM (11q23) performs vital roles in learning and memory by regulating guidance of CMS axons. NCAM merits study as a schizophrenia vulnerability gene, as NCAM polymorphisms are associated with neurocognitive impairment in a schizophrenia population (CATIE). Moreover, a soluble NCAM fragment consisting of the entire extracellular region (NCAM-EC) is overexpressed in schizophrenic brain, and is released from normal neurons by proteolytic cleavage (ectodomain shedding) by a metalloprotease with properties of an ADAM (a disintegrin and metalloprotease). Excess NCAM shedding in the prefrontal cortex (RFC) was modeled in NCAM-EC transgenic mice, which revealed a striking decrease in synapses of GABAergic interneurons, including basket cells that regulate pyramidal cell output and synchrony. NCAM-EC mice display behavioral abnormalities associated with neurotransmission defects, including hyperlocomotion, stereotypies, decreased sensory gating and fear conditioning. It is hypothesized that NCAM regulates the excitatory/inhibitory balance between basket interneurons and pyramidal cells in the RFC, and that dysregulation of NCAM by excessive shedding perturbs synaptic connectivity, thus altering cortical circuitry and synchrony of pyramidal cell groups important for neurocognition. GABAergic function may be compromised in schizophrenia, but it is not known if GABAergic dysfunction reflects altered development of cortical circuitry. It will be determined if there are developmentally regulated changes in dendritic/axonal arborization and synaptogenesis of GABAergic interneurons and pyramidal neurons in normal RFC,and whether NCAM dysregulation interferes with development in NCAM-EC and null mutant mice. NCAM shedding will be assessed during development in post-mortem human brain and from individuals with schizophrenia. Cortical neuron cultures will be exploited to identify the ADAM protease(s) responsible for normal NCAM shedding, to localize the NCAM cleavage site, and to ascertain the role of NCAM shedding on neuronal process outgrowth and branching. Finally, behavioral testing in mice will assess whether NCAM-EC overexpression impairs executive functions such as working memory, decreases gamma oscillatory activity, and alters sensitivity to GABA agonists in anxiety- like behavior and sensorimotor gating. This work will assist other center investigators in understanding development of GABAergic interneurons from early differentiation (Project 4), migration (Projects 4 and 5), and establishment of connections (this project) and will characterize a molecular substrate for abnormal neurocognitive functions in patients who are at risk or in early stages of schizophrenia (Projects 1 and 3).These studies will illuminate a mechanism whereby NCAM contributes to GABAergic cortical circuitry relevant to neurocognitive function, and will explore NCAM as a pathophysiological target for schizophrenia vulnerability.

DESCRIPTION (provided by applicant): The long-range goal of this project is to identify mechanisms regulating synaptic connectivity in the mammalian neocortex and to elucidate how their deficiency causes abnormal brain wiring relevant to neurodevelopmental disorders such as autism spectrum disorders (ASDs). This application seeks to elucidate a novel mechanism for spine and synapse regulation involving NrCAM (Neuron-Glial Related Cell Adhesion Molecule), a risk factor in ASD, which is important for development of excitatory circuits in the neocortex. A new concept to be studied is that NrCAM regulates spine development by interacting with repellent guidance molecules Semaphorin3F (Sema3F), Neuropilin-2 (Npn-2), and PlexinA3 (PlexA3), and that NrCAM deficiency leads to hyperexcitability in neocortical circuits. The central hypothesis to be investigated is that NrCAM/Npn- 2/PlexA3 comprises a receptor complex for Sema3F that constrains or remodels dendritic spines of pyramidal neurons in the neocortex for appropriate excitatory balance. Aims to be addressed are as follows: Aim 1: A role for NrCAM in constraining spine and excitatory synapse formation will be investigated by analyzing dendritic spines and synapses of pyramidal neurons in prefrontal and sensory cortical areas of wild type (WT) and NrCAM null mice, and localizing NrCAM to synaptic sites. A double heterozygote analysis will be undertaken to investigate the postulated genetic interaction of NrCAM with Npn-2, PlexA3, and Sema3F to regulate spine morphogenesis in vivo. Aim 2: To determine if NrCAM loss induces hyperexcitability of pyramidal neurons, excitatory responses will be measured in star pyramidal neurons, the principal target of thalamocortical input to visual cortex (layer 4), in visual cortical slices of WT and NrCAM null mutant mice by whole cell recordings, and the effect of Sema3F treatment on excitatory responses in WT and null mutant slices will be compared. Aim 3: A cell autonomous, postsynaptic mechanism for NrCAM will be investigated for Sema3F-induced spine morphogenesis mediated by interaction with Npn-2 and PDZ adaptors. Genetic rescue of spine morphogenesis will be analyzed in star pyramidal neurons of NrCAM null embryos electroporated in utero with WT and NrCAM binding mutants for Npn-2 and PDZ adaptors, and in Sema3F-treated neuronal cultures. The outcome of these studies is expected to have an important positive impact, because it will delineate novel molecular mechanisms of spine morphogenesis that control sensory cortical connectivity and function, and may provide insight into molecular mechanisms targeted in ASDs. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because it seeks to define molecular determinants that regulate development of neural connectivity in the mammalian brain. The research is relevant to the mission of NIH in illuminating basic mechanisms of neurodevelopment important for understanding inherited brain disorders, and especially relevant to sensory abnormalities in autism and related syndromes, termed Autism Spectrum Disorders, for which NrCAM, Neuropilin-2, and Semaphorins are candidate risk factors.

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how an appropriate balance of excitatory and inhibitory (E/I) connectivity is achieved during development of cortical networks and adjusted through synaptic plasticity for normal functioning of the cerebral cortex. Until this gap is filled, understanding neuropsychiatric disorders with GABAergic inhibitory connection deficits, such as schizophrenia and autism, will remain a mystery. The long term goal is to identify the molecular mechanisms that establish E/I balance in the prefrontal cortex, which may identify new targets for disorders where this balance is altered. The objective is to define a novel mechanism for limiting inhibitory connections between basket interneurons and the perisomatic region of pyramidal neurons in developing prefrontal cortex. The central hypothesis is that neural cell adhesion molecule NCAM, tyrosine kinase EphA3, and ADAM10 metalloprotease comprise a presynaptic receptor complex for postsynaptic ephrinA5 that promotes elimination of perisomatic synapses critical for proper prefrontal network organization and functioning, such as in working memory. Aim 1. To identify a novel molecular mechanism for limiting perisomatic basket cell innervation in the developing mouse prefrontal cortex through NCAM-dependent ephrinA5/EphA3 signaling We will identify NCAM/EphA3 binding sites, assess the ability of NCAM to stabilize EphA3 on the cell surface by inhibiting endocytosis and promoting ephrinA5-induced EphA3 kinase signaling, and define the developmental and activity-dependent regulation of ephrinA5 in mouse prefrontal cortex. Aim 2. To define presynaptic and postsynaptic functions of NCAM, ephrinA5/EphA3, and ADAM10 metalloprotease in perisomatic inhibitory synapse regulation Analysis of new conditional NCAM and ADAM10 mutant mice and cell-specific expression in brain slices will distinguish pre- versus post-synaptic functions for NCAM, ephrinA5/EphA3, and ADAM10, and test causal roles for their interactions in perisomatic synapse regulation. Dynamics of inhibitory synapse elimination will be analyzed by time-lapse two-photon microscopy in cortical slice cultures. Aim 3. To delineate the contribution of NCAM to prefrontal cortical network organization and function using optogenetic mapping and behavioral assessment of working memory. Optogenetic mapping will be performed in brain slices from NCAM null and conditional mutant mice expressing channelrhodopsin-2 from the VGAT promoter in interneurons. Working memory performance will be measured in live mice by the delayed non-match-to-sample T-maze task. The outcome of these studies is expected to have a sustained, positive impact, because it will illuminate novel molecular mechanisms of interneuronal connectivity that control cognitive function, while innovative optogenetic technology will elucidate cortical networks targeted in neurodevelopmental disorders.

Abstract Elimination of excess dendritic spines on cortical pyramidal neurons during adolescence is critical for excitatory/inhibitory (E/I) balance in adult cortical circuits, and its impairment can lead to altered spine density in autism spectrum disorders (ASD) and schizophrenia. This proposal seeks to illuminate a novel mechanism for dendritic spine remodeling in the developing mammalian frontal cortex, mediated by immunoglobulin (Ig)- class cell adhesion molecules of the L1 family and class 3 Semaphorins. Aims focus on NrCAM (Neuron-Glial Related Cell Adhesion Molecule) and Close Homolog of L1 (CHL1), which are associated with ASD, schizophrenia, and intellectual disability. The central hypothesis to be investigated is that NrCAM and CHL1 form holoreceptor complexes with Neuropilin1/2 and PlexinAs for secreted class 3 Semaphorins, which signal through Rho GTPases to prune dendritic spines of cortical pyramidal neurons during adolescence. Aim 1. Developmental Regulation of NrCAM and Role of CHL1 in Dendritic Spine Remodeling A new conditional mutant mouse (Nex1Cre-ERT2: NrCAMf/f) that inducibly deletes NrCAM from cortical pyramidal neurons will be studied to define the developmental timing of NrCAM function in dendritic spine morphogenesis, spine dynamics, and cortical excitability. A novel function for CHL1 in spine remodeling will be identified by analysis of spine/synapse morphogenesis and cortical excitability in CHL1 null mutant mice. Aim 2. Structural and Functional Interactions of the Sema3F Holoreceptor A structure-function approach using mutagenesis will be undertaken to probe an innovative molecular model of the Sema3F holoreceptor complex, in which the NrCAM extracellular domain interacts with Npn2 to induce Sema3F-induced receptor clustering and spine remodeling. NrCAM cytoplasmic domain interactions with Synapse-associated protein 102 (SAP102) and cytoskeletal adaptors (Ankyrin-B, -G, and Doublecortin-like kinase 1) will be analyzed for promoting receptor clustering and signaling at the nascent postsynaptic density in cortical neuronal cultures and in vivo. Aim 3. Molecular Mechanism of Sema3F Signaling through Small GTPases A novel dual signaling pathway will be investigated in which Sema3F-induced signaling through RhoA (Rho Kinase-Myosin II) generates contractile force that exerts tension on actin filaments assembled through Rac1 signaling (Tiam1-Rac1-PAK-LIMK1-Cofilin1) to regulate spine elimination/protrusion. The role of intrinsic activity in Sema3F-mediated spine pruning will be evaluated by activity blockade in cortical neuron cultures. This project is expected to have sustained overall impact as it will delineate a novel molecular mechanism for regulating excitatory synapse development in the frontal neocortex, advance mechanistic understanding into pathology associated with neurodevelopmental disorders, and may reveal new therapeutic targets for intervention in adolescence, a window of opportunity for influencing cortical networks.