Stabilization of neurites in cerebellar granule cells by transglutaminase activity: Identification of midkine and galectin-3 as substrates

The formation of covalent isopeptide cross-links between cell surface prote in molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. e t nl. (1995) Neuroscience 65, 1063-1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebella r cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryoni c dorsal rhombic lip, which contain granule neuron precursors. A possible b iological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-0 07. Inhibition of cross-linking activity did not influence the number of ne urites formed by granule neurons, but caused the destabilization of neurite s during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formati on (bifurcation). Inactivation of cross-linking activity prevented the form ation of fascicles between neurites only when cells were cultured on a biom atrix surface. Two glial proteins involved in cell-extracellular matrix int eractions, midkine and galectin-3, were identified as putative substrates f or granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possibl e non-pathological role for transglutaminase in the control of axon collate ral branching by developing granule neurons in the cerebellar cortex is dis cussed.