How neuronal circuitry arises in the brain during ontogeny is a central unanswered question in neuroscience.
A pioneering attempt to develop a molecular understanding of neural connectivity was the discovery of the first neural cell
adhesion molecule NCAM in the Edelman laboratory. Since then distinct classes of neural recognition molecules expressed
on the surface of developing neurons have been identified, including the L1 family (L1, Close Homolog of L1 (CHL1), NrCAM,
Neurofascin), the NCAM family (NCAM1, NCAM2), Cadherins, and integrins. These important adhesion molecules regulate
guidance of growth cones at the tips of growing axons and dendrites, as well as migration of neural stem cells to their destinations
in the brain. Many of these molecules also function in synaptogenesis, learning, and memory.
Importantly, mutations in these genes can be responsible for human neuropsychiatric disease. Mutations in the human L1 gene on the X chromosome (Xq28) leads to X-linked mental retardation termed the L1 syndrome (retardation, aphasia, spasticity, hydrocephalus, optic atrophy). Mutation in the CHL1 gene on chromosome 3 (3p26) is associated with the 3-p syndrome characterized by low intelligence and delayed motor development. Abnormal expression of NCAM in which the extracellular region is aberrantly cleaved and secreted in the brain is linked to schizoprenia. Moreover, L1 and CHL1 polymorphisms are 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 involving the creation of novel mouse genetic models. We apply a variety of methods to determine the mechanism of action of these molecules including signal transduction technology, DNA microarrays, axon growth and cell migration assays, imaging of living neurons by confocal microscopy, cytoskeletal interactions, and mouse behavioral analyses.
L1/CHL1 Interactions
Retinocollicular Topographic Mapping.
Genetically altered mice provide exciting new models for understanding neurological disorders. L1-mutant mice are excellent
models for the L1 syndrome. Mice with a total deletion of L1 display defective axon guidance both in the neocortex and retinocollicular
projection, enlarged ventricles, and optic atrophy, characteristic of the L1 syndrome. We have generated L1 knock-in mice which
express a mutated form of L1 that does not bind the actin cytoskeleton through ankyrin, which is found in L1 syndrome patients.
These mice are being exploited to elucidate the normal function of L1-cytoskeletal interactions in normal neuronal connectivity,
especially in the projection of retinal axons to the superior colliculus in the brain. Other L1 mutations are being generated to
understand how the location of different mutations in the L1 molecule causes different spectra of defects in the disease.
We are pursuing molecular biological, immunocytochemical, and biochemical approaches to elucidate the mechanism by which L1 interacts with ephrin ligands and their Eph receptor tyrosine kinases to guide retinal axons to appropriate synaptic targets in the brain. We have evidence that L1-ankyrin interaction increases integrin-dependent adhesion stimulated by ephrinB/EphB signaling. An important goal is to determine the precise molecular mechanism that governs this response, which we believe involves tyrosine phosphorylation of the ankyrin binding site FIGQY. This will include culture studies with dissociated retinal cells and retinal explants to determine their neurite outgrowth and adhesive responses to ephrinB ligands.
NrCAM knockout mice display a similar retinocollicular mapping phenotype, as shown in preliminary results. NrCAM has been recently linked to autism spectrum disorder, in which visual processing is thought to be defective. Another project is to conduct axon tracing with DiI lipophilic dye in NrCAM mutant and wild type mice to define the nature of the misprojections, and to analyze L1/NrCAM double mutants to see if each adhesion molecule guides unique populations of axons or whether they coordinately control axon guidance. Analogous retinal cell culture and biochemical studies will be performed to elucidate the signaling mechanism regulating NrCAM-directed axon guidance.
Thalamocortical Topographic Targeting to Neocortical Areas.
The neocortex is partitioned into discrete areas with unique functions and specific patterns of neuronal connections (frontal, motor,
somatosensory, visual areas). How this cortical arealization arises in development is the subject of intensive investigation. CHL1
is a potential determinant of arealization as it is unique among the L1 family of adhesion molecules in being expressed in the
neocortex in an area-specific gradient with a high caudal (visual and somatosensory) to low rostral (motor, frontal) distribution. New
studies have revealed an important new function for CHL1 in regulating thalamocortical axon guidance to the somatosensory neocortical
area. Interestingly, CHL1 serves as a co-receptor for Neuropilin-1, a receptor for the repulsive axon guidance cue Semaphorin3A,
regulating axon guidance through the intermediate target of thalamocortical axons, the ventral telencephalon. We have evidence that
L1 and NrCAM regulate thalamocortical mapping to different neocortical areas, potentially involving other repellent guidance cues
including ephrinA/EphA receptor systems. An important new project is to map and determine the molecular basis for L1 and NrCAM
mediated thalamocortical guidance, potentially identifying cell adhesion molecules as critical coreceptors for repellent axon guidance.
The extracellular region of the transmembrane protein NCAM (NCAM-EC) is shed as a soluble fragment at elevated levels in 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.
In future work, NCAM-EC mice will be extensively mapped for frontal cortical circuitry defects using novel fluorescent indicator mice labeling other populations of GABAergic interneurons and excitatory pyramidal neurons. For this purpose we are generating new strains of mice expressing Green Fluorescent Protein in these subpopulations using recombineering with Bacterial Artificial Chromosome technology. In addition DNA microarray studies will be used to identify alterations in gene expression in the frontal cortical areas of NCAM transgenic compared to control mice. Biochemical studies are being actively pursued to identify the enzyme responsible for normal and aberrant cleavage of the NCAM-EC.
Translations research studies on a large DNA sample of human schizophrenic patients suggests that specific polymorphisms in the NCAM gene are associated with neurocognitive defects. Analyses of NCAM, CHL1, and L1 genes using single nucleotide polymorphisms (SNPs) in normal and schizophrenic populations are underway to determine if these genes are candidates for schizophrenia vulnerability.