Neuronal differentiation of NTE-deficient embryonic stem cells Zhongyou Li a , Paul F. Szurek a , Chuantao Jiang b , Annie Pao a , Brian Bundy c , Wei-dong Le b , Allan Bradley d , Y. Eugene Yu a,e,f, * a Department of Cancer Genetics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA b Department of Neurology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA c Department of Gynecologic Oncology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA d Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK e Genetics Program, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA f New York State Center of Excellence in Bioinformatics and Life Sciences, 901 Washington Street, Buffalo, NY 14203, USA Received 25 February 2005 Available online 23 March 2005 Abstract Organophosphates induce neurological disorders. One of the enzymes inhibited by these compounds is neuropathy target ester- ase (NTE). In vitro, inhibition of NTE activity by organophosphates is correlated with inhibition of neurite initiation and reduc- tion of neurite length, supporting the hypothesis that organophosphate-induced neurological disorders are caused by inhibition of NTE activity. However, there is no direct evidence for the involvement of NTE in organophosphate-induced impairment of neu- rites in vitro. To examine the role of NTE, we have generated NTE-deficient mouse embryonic stem cells. These cells can differ- entiate into neuron-like cells. Although NTE-deficient cells exhibited a delay in neurite initiation in vitro, both the proportion of neuron-like cells which initiated neurites and the elongation of these neurites occurred at the normal rate. These results demon- strate that NTE activity is not required for neurite initiation or elongation per se, but is essential for the optimal rate of neurite initiation. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Neuropathy target esterase; Embryonic stem cells; In vitro differentiation; Neurite initiation/elongation Neurotoxic organophosphates (OPs) constitute active components of major classes of chemical warfare agents and insecticides. Human exposure to these OPs may lead to acute respiratory arrest due to the inhibition of ace- tylcholinesterase. OPs also elicit chronic neurological disorders such as OP-induced delayed neuropathy (OPIDN), characterized by axonal degeneration of the spinal cord and peripheral nerves that leads to paralysis of the lower limbs [1]. The molecular process underlying OP-induced neuropathological changes remains unknown. The two major mechanistic hypotheses are centered on neuropathy target esterase (NTE): Neuro- logical disorders are either caused by the loss/reduction of NTE activity [2–4] or gain of abnormal NTE function caused by phosphorylation and/or removal of an alkoxy group of NTE which leaves a negatively charged phos- phate in the catalytic site [1,5]. Study of OPIDN has been focused on the analysis of the cytopathic consequence to cultured neuronal cells after exposure to OPs. The correlation between the inhi- bition of NTE by some neurotoxic OPs and the com- plete inhibition of neurite outgrowth [6,7] as well as the significant reduction of neurite length [8–10] of cul- tured neuronal cells has been considered as supportive evidence for the loss of function hypothesis. This corre- lation has also been proposed as a basis for developing a screening strategy for identifying neurotoxic OPs [10]. However, there is no direct evidence for the causative role of NTE in OP-induced impairment of neurite 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.090 * Corresponding author. Fax: +1 7138451698. E-mail address: yuejin.yu@roswellpark.org (Y.E. Yu). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 330 (2005) 1103–1109 BBRC