Functional and biochemical studies on neuronal Thy-1

Citation

Mahanthappa, Nagesh Kalyana (1991) Functional and biochemical studies on neuronal Thy-1. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/qa3k-hw80. https://resolver.caltech.edu/CaltechETD:etd-06212007-133431

Abstract

Thy-1, a cell surface glycoprotein, is one of the smallest members of the immunoglobulin superfamily. It is expressed abundantly in nervous systems of many species and is expressed at highest levels by long projection axons after the major period of neurite outgrowth and synaptogenesis. In this study, I demonstrate that three distinct perturbations that either remove or prevent expression of cell surface Thy-1 result in enhanced neurite outgrowth and initiation of sprouting: (i) Binding by soluble anti-Thy-1 monoclonal antibodies results in increased numbers of neurite-bearing neurons, chromaffin cells, and PC12 cells in culture. Sprouting requires multivalent binding (anti-Thy-1 Fab fragments do not give this effect), and results in shedding of Thy-1 from the cell surface. (ii) Chromaffin and PC12 cells exhibit enhanced sprouting when exposed to phosphotidylinositol-specfic phospholipase C, a treatment that removes cell surface Thy-1. This effect is blocked by anti-Thy-1 Fabs. (iii) When mutagenized and selected for Thy-l-deficiency, 90% of resultant PC12 cell mutants sprout spontaneously. These pertubations suggest that the normal function of Thy-1 is to stabilize neurites and inhibit sprouting, and that removal or prevention of Thy-1 expression disinhibits such mechanisms.

Biochemical analysis reveals that Thy-1 exists in homomultimeric forms in situ and in cultured neurons. Neuronal Thy-1 is distributed equally in monomeric, dimeric, and hexameric forms; the latter being composed of monomers and dimers. The dimer does not give rise to monomers when boiled in the presence of disulfide reducing agents and SDS. In PC12 cells, the multimeric forms of Thy-1 predominate, but treatment with agents that cause sprouting make the Thy-1 distribution more neuronal. The homology between Thy-1 and immunoglobulin variable domain allowed peptides to be synthesized that correspond to candidate sites of intermolecular association. These were tested for their effects on neurite outgrowth and Thy-1 multimerization. One peptide induces a decrease in Thy-1 multimerization, as well as enhancing outgrowth. Thus, the stabilization function of Thy-1 may be mediated by homomultimerization on the cell surface, and its removal from the surface, or dissolution into monomers and dimers, permits outgrowth and sprouting.

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