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A defining characteristic of the vertebrate nervous system is the specificity with which its diverse cell types interact, especially at specialized sites of adhesive contact called synapses. It is the exquisite specificity of neuronal circuitry that underlies the brain’s unique ability to store and process information. Conversely, disruptions in the formation of this circuitry appear to precipitate such genetic developmental disorders as autism and mental retardation. Thus, elucidating the genetic basis of the specificity of neuronal interactions, the primary long-term goal of my laboratory, will greatly advance our basic scientific understanding of the most intricate system in all of biology, while providing a platform on which future therapeutic interventions might be based. My laboratory has taken a candidate gene approach, focusing on cell adhesion molecules, particularly the γ -Protocadherin (γ -Pcdh) family of cadherin-like proteins and the immunoglobulin superfamily (IgSF) member ALCAM.
Protocadherin Gene Clusters
Three large clusters of cadherin-related genes (Protocadherin-α, -β, and -γ) lie in a tandem array on a single chromosome in mammals. The γ cluster, on which we focus, consists of 22 "variable" exons, each of which encodes the extracellular, transmembrane, and partial cytoplasmic domains of a single protocadherin isoform. Each variable exon is spliced to a set of three "constant" exons which encode a shared C-terminal domain. Thus, a variety of adhesive specificities may link into a common signaling pathway.
Our work has shown that γ-protocadherins are expressed in the nervous system during development, and are found at a subset of synapses. Mice in which the entire γ-protocadherin locus is deleted lack voluntary movements and reflexes and die at birth due to massive apoptosis of spinal interneurons and concomitant loss of synapses. We have genetically dissociated these two phenotypes, using mice in which apoptosis is blocked, to show that control of synapse development is a primary function of γ-protocadherins. Our work indicates that when mice lack the γ-protocadherins, synaptic density and activity is reduced, and this leads to an exacerbation of an underlying pattern of developmental cell death amongst molecularly distinct spinal interneuron populations.
We are addressing a wide range of ongoing, critical questions:
1) Do the γ-protocadherins act exclusively or primarily as homophilic adhesion molecules, or do they engage in heterophilic interactions and/or act as signaling molecules??We recently showed that the γ-protocadherins promiscuously form cis-tetramers that then interact in a strictly homophilic manner in trans.
2) What roles to the γ-protocadherins play in in the cerebral cortex, which matures only after birth, when the constitutive mutants die??Our ongoing work has found that the γ-protocadherins regulate dendritic arborization by inhibiting a FAK/PKC kinase cascade.
3) What is the function of the γ-protocadherins in astrocytes, glial cells that express multiple members of the family? We have shown that the γ-protocadherins are neuronal-glial adhesion molecules and that astrocytic contacts stabilize nascent synapses during development.
4) Does the diversity of the γ-protocadherin family provide a molecular code that underlies the specificity of synaptic interactions?
To answer these questions, we are utilizing new conditional mutant mice and several transgenic lines expressing the Cre recombinase to disrupt γ-protocadherin function in specific cell types at discrete developmental stages. We are also employing a variety of biochemical techniques to characterize the molecular interactions of the γ-protocadherins in neurons and in other cell types.
We hope to use insights gained from studying the functions of the γ-protocadherins to improve our general understanding of the ways in which synapse formation and function might go awry in a number of developmental disorders such as autism and mental retardation.
The immunoglobulin superfamily molecule ALCAM is expressed by subsets of neurons and has been implicated in the control of numerous developmental and pathological processes. To test hypotheses about ALCAM function, we have disrupted its gene via homologous recombination. In mice lacking ALCAM, both motor neuron and retinal ganglion cell axons fasciculate poorly and are occasionally misdirected. In addition, ALCAM mutant retinae exhibit dysplasias with photoreceptor ectopias that resemble the retinal folds observed in some human retinopathies (Figure 3). This appears to be due to loss of ALCAM in the choroid, a pigmented vascular tissue that lies behind the neural retina. Because ALCAM has been associated with melanoma metastasis, we hypothesize that defects in choroidal melanocyte adhesion and/or motility underlie the mutant phenotypes. We now are determining the specific cellular defect and examining its relationship to ocular development and disease processes. We are also searching for ALCAM's heterophilic binding partners and intracellular signaling pathways.
With collaborators at the University of North Carolina, we have recently characterized a role for ALCAM in the targeting of retinal axons to their termination zones in two brain targets, the superior colliculus and the lateral geniculate nucleus. ALCAM knockout mice exhibit defects in both of these retinal projections, due to disrupted trans-interaction with another member of the immunoglobulin superfamily, L1. (see Fig. 5)