How neuronal circuitry in the brain is established during development and refined in the adult is a central unanswered question in neuroscience. Neural recognition molecules expressed on the neuronal surface are pivotal players in developing cortical circuits. Among the most relevant of these molecules to human disease are members of the NCAM and L1 family (L1, CHL1, NrCAM, Neurofascin). Each of these cell recognition molecules has established functions in axon guidance that mediate correct topographic synaptic targeting of axons, while exciting new findings reveal that they also have vital functions in regulating synaptogenesis and plasticity of cortical networks.
Importantly, mutations in neural adhesion molecule genes may contribute to susceptibility to human neuropsychiatric diseases. Abnormal expression of NCAM in the developing prefrontal cortex is associated with working memory deficits in schizoprenia, while polymorphisms and aberrant splicing of NrCAM is linked to autism spectrum disorders. Mutations in the L1 gene are involved in a form of X-linked mental retardation, and CHL1 mutation is associated with low intelligence and delayed motor development. In addition to NCAM, L1 and CHL1 polymorphisms are also associated with schizophrenia in specific human populations.
To study the normal and abnormal function of neural cell adhesion molecules in brain development and function, our laboratory uses a multidisciplinary approach to generate and analyze novel mouse genetic models of neurodevelopmental disorders. We apply a panoply of methods to determine the mechanism of action of candidate genes in normal and pathological processes, including signal transduction technology, axon targeting, imaging of living neurons in acute brain slices by confocal microscopy, as well as plasmid mutagenesis and neuronal culture assays.
As a member of the Carolina Institute for Developmental Disabilities and UNC Neuroscience Center, I have productive collaborations with outstanding UNC colleagues dedicated to unraveling the neurodevelopmental mechanisms of connectivity. We are currently collaborating closely with Dr. Paul Manis, an electrophysiologist at UNC, on optogenetics and electrophysiology, and with Dr. Sheryl Moy, Director of the mouse behavioral core on working memory and fear conditioning.
Role of NrCAM and Semaphorins in Dendritic Spine Remodeling
In our first research project we seek to illuminate a novel mechanism for dendritic spine regulation in the mammalian neocortex by immunoglobulin (Ig)-class cell adhesion molecules and secreted Semaphorins (class 3-Semas). The aims focus on NrCAM (Neuron-Glial Related Cell Adhesion Molecule), a risk factor for autism spectrum disorder (ASD), and Close Homolog of L1 (CHL1), which is implicated in intellectual disability. A new concept to be studied is that L1-CAMs function as obligate subunits of Semaphorin receptor complexes to promote spine pruning in developing cortical pyramidal neurons. The central hypothesis to be investigated is that NrCAM, Neuropilin-2 (Npn2), and the Rap-GAP PlexinA3 (PlexA3), comprise a postsynaptic holoreceptor for Sema3F in cortical pyramidal neurons, which signals through small GTPases (Rap1, Rac1, RhoA) and PDZ scaffold and cytoskeletal effector proteins (SAP102, Ankyrins, Doublecortin-like kinase1 (DCLK1) to prune excess dendritic spines in adolescence. We will focus on the medial frontal cortex (MFC), where essential circuits regulate social behavior and cognition. The overall goal is to identify Ig-CAM-linked mechanisms that govern synaptic connectivity in the mammalian neocortex, and to elucidate how their deficiency causes abnormal brain wiring relevant to neurodevelopmental disorders.
Aim 1. Developmental Regulation of NrCAM and Role of CHL1 in Dendritic Spine Remodeling
Aim 3. Molecular Mechanism of Sema3F Signaling through Small GTPases
We will test the hypothesis that Sema3F-induced holoreceptor clustering stimulates PlexA3's Rap-GAP activity, which sets in motion dual RhoA and Rac1 GTPase signaling pathways that converge at the level of the actin cytoskeleton to cause spines to collapse. Based on preliminary results, we postulate that Sema3F signaling through RhoA (Rho Kinase-Myosin II) generates contractile force that exerts tension on actin filaments assembled through Rac1 signaling (Tiam-Rac1-PAK-LIMK1-Cofilin1) to retract spines. These dual pathways will be probed using assays for GTPases, protein kinases, protein-protein interactions, and spine retraction in cortical neuron cultures. The role of intrinsic activity in Sema3F-mediated spine pruning will be evaluated by activity blockade in cortical cultures.
The outcome of these studies will have significant impact, because they will delineate a novel molecular mechanism involving IgCAMs for regulating spine and synapse development important for excitatory/inhibitory balance in frontal cortical circuits. These studies are expected to provide molecular insight into pathology associated with ASD and other neurodevelopmental disorders.
A novel transgenic mouse line was generated to identify consequences on cortical development and function of expressing soluble NCAM-EC from the neuron-specific enolase promoter in developing and mature neocortex and hippocampus. NCAM-EC transgenic mice exhibited a striking reduction in synaptic puncta of inhibitory GABAergic interneurons in the cingulate, frontal association cortex, and amygdala, but not hippocampus, as shown by decreased immunolabeling of glutamic acid decarboxylase-65 (GAD65), GAD67, and the GABA transporter GAT-1. Interneuron cell density was unaltered in the transgenic mice. Affected subpopulations of interneurons included basket interneurons evident in NCAM-EC transgenic mice intercrossed with a reporter line expressing green fluorescent protein and by parvalbumin staining. Behavioral analyses demonstrated higher basal locomoter activity of NCAM-EC mice and enhanced responses to amphetamine and MK-801 compared to wild type controls. Transgenic mice were deficient in prepulse inhibition, which was restored by clozapine but not haloperidol. Additionally, NCAM-EC mice were impaired in contextual and cued fear conditioning. These results suggested that elevated shedding of NCAM perturbs synaptic connectivity of GABAergic interneurons, and produces abnormal behaviors that may be relevant to schizophrenia and other neuropsychiatric disorders.
Another project in our lab is to address 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.