Received for publication February 16, 2006.
Revised March 15, 2006.
Accepted for publication March 16, 2006.

METHYLISOTHIAZOLINONE, A NEUROTOXIC BIOCIDE, DISRUPTS THE ASSOCIATION OF SRC FAMILY TYROSINE KINASES WITH FOCAL ADHESION KINASE IN DEVELOPING CORTICAL NEURONS

Abstract

Methylisothiazolinone (MIT) is a biocide widely used in industrial and cosmetic products with potential as a neurotoxicant. We previously reported that short acute exposures to relatively high concentrations of MIT (100 µM) lead to widespread and selective neuronal death in vitro. To evaluate the biological properties of chronic exposures to MIT, freshly dissociated rat cortical neurons were continuously exposed to low concentrations (0.1 - 3 µM) of the biocide in serum-containing media. Although we observed minimal effects on cell viability, MIT induced a dramatic inhibition of neurite outgrowth. Immunoblotting and immunoprecipitation experiments revealed that focal adhesion kinase (FAK) phosphorylation was primarily affected by the MIT treatment. The phosphorylation level at tyrosine 576 and 861 of FAK was significantly decreased and likely contributed to the overall reduction of tyrosine phosphorylation of this protein. MIT inhibited Src-family kinases (SFKs) in cell free assays, and led to the physical dissociation of FAK from the signaling complexes that it normally forms with c-Src and Fyn in developing neurons. High-density neuronal cultures were then employed to increase cell-to-cell contact. This approach resulted in an overall enhancement of SFKs and FAK phosphorylation and could overcome the deficits induced by MIT. This study suggests that a disruption of FAK-SFK complexes due to SFK inhibition leads to FAK dysfunction, with detrimental effects to immature neurons. Prolonged exposure to low levels of MIT and related compounds may have damaging consequences to the developing nervous system.

Key words: Src family kinases, biocide, cortical neuron, focal adhesion kinase, methylisothiazolinone, neurotoxicity