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.
Translational research studies conducted by our lab in collaboration with human geneticists on a large DNA sample of human schizophrenic patients indicated that specific polymorphisms in the NCAM gene are associated with neurocognitive defects. In addition, the extracellular region of the transmembrane protein NCAM (NCAM-EC) is shed as a soluble fragment at elevated levels in human schizophrenic brain.
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.
We have received a new NIH R01 grant, " Molecular Mechanisms of Inhibitory Circuit Development" for 5 years, and are recruiting postdoctoral fellows to study this project. Our co-Investigator is Dr. Paul Manis, an electrophysiologist at UNC with whom we have collaborated on optogenetic studies in novel mutant mice.
The following describes the grant's specific aims:
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.