Reduction of mint-1, mint-2, and APP overexpression in okadaic acid-treated neurons SeungYong Yoon a,d , JungEun Choi a , JuHee Haam a , Han Choe b and DongHou Kim a,c Departments of a Anatomy and Cell Biology, b Physiology, c Institute for Biomacromolecules, University of Ulsan College of Medicine and d Department of Forensic Medicine, National Institute of Scienti¢c Investigation, Seoul, Korea Correspondence to Dr DongHou Kim, MD, PhD, Department of Anatomy and Cell Biology,University of Ulsan College of Medicine, 388 -1PoongNap-Dong, SongPa-Gu, Seoul 138 -736, Korea Tel: + 82 2 3010 4272; fax: + 82 2 3010 8063; e-mail: dhkim@amc.seoul.kr Received 1 September 2007; accepted 4 September 2007 Treatment of neurons with okadaic acid, a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neur- onal death, and to create a research model of Alzheimer’s disease. Amyloid precursor protein (APP) is the precursor protein of the b-amyloid peptide that accumulates in extracellular plaques in Alzheimer’s disease. Several studies have shown that mint-1 (munc18 -interacting protein 1) and mint-2 bind to theYENPTYmotif in the cytoplasmic domain of APP and inhibit processing of APP to b-amyloid peptide. Here, we report that, upon neurodegeneration with okadaic acid, mint-1 and mint-2 levels were reduced by pro- teolytic cleavage, and that these changes were followed by increases in APP levels. We also show that the mint-1 and mint-2 cleavage and APP overexpression were prevented by calpain inhibi- tor-I and inhibitor-II. These results indicate that mint cleavage might play a role in the pathophysiology of Alzheimer’s disease. NeuroReport 18:1879^1883  c 2007 Wolters Kluwer Health | Lippincott Williams & Wilkins. Keywords: Alzheimer’s disease, amyloid precursor protein, calpain, munc18 -interacting protein, okadaic acid Introduction Alzheimer’s disease is characterized by the progressive loss of neurons and synapses, together with two pathologic characteristics: neurofibrillary tangles and senile plaques [1]. Neurofibrillary tangles are intraneuronal accumulations of paired helical filaments composed of abnormally hyper- phosphorylated tau protein. Their presence in Alzheimer’s disease suggests that the aberrant phosphorylation of tau might be a critical step in the progress of neurodegeneration in Alzheimer’s disease. The extracellular senile plaques are mainly composed of b-amyloid peptide (Ab), which is derived from amyloid precursor protein (APP) as a result of intracellular proteolytic processing [1]. Inhibiting the devel- opment of these two pathologies might thus represent treatment strategies; however, the precise relationship between these two pathologic characteristics remains unclear. The role of APP as the precursor protein of Ab is well established, and the balance between Ab generation and degradation is critically important to understand the pathophysiology of Alzheimer’s disease. By contrast, the physiologic functions of APP and its mechanisms of cytoplasmic regulation remain unclear. The cytoplasmic region of APP functions as a docking site for an adaptor protein that allows APP trafficking and endocytosis [2]. APP adaptor proteins with phosphotyrosine-binding domains can bind to the YENPTY motif in APP, and modulate its trafficking and processing to Ab or transcription regulation [3]. Interaction studies on the APP intracellular domain have identified many putative cytosolic binding partners for APP, including Fe65, munc18-interacting proteins (mint/ X11 family proteins), c-Jun N-terminal kinase-interacting proteins (JIP-1 and JIP-2), and kinesin light chain [2]. Recently, we reported that axonal transport was disrupted during okadaic acid-induced neurodegeneration, leading to APP accumulation [4]. Treatment of neurons with okadaic acid, a protein phosphatase inhibitor, has been used as a research model for Alzheimer’s disease because it induces hyperphosphorylation of tau, neuronal death, and increased Ab [5,6]. Investigating the cellular processing of APP is essential not only for understanding the function of APP, but it also has implications for the role of Ab generation. Therefore, we investigated the role of the APP adaptor proteins, and report here, for the first time, the proteolytic cleavage of mint-1 and mint-2, which is followed by APP accumulation and phosphorylation, in primary rat neurons treated with okadaic acid. Mint cleavage and APP accumu- lation and phosphorylation were prevented by calpain inhibitors. Methods Neuron culture Primary cortical neuron cultures were prepared from the brains of embryonic day-16 rat pups. Briefly, the cerebral cortices were dissected in calcium-free and magnesium-free Hank’s balanced salt solution and incubated with a 0.125% trypsin solution for 10 min at 371C. The trypsin was inactivated with Dulbecco’s modified Eagle’s medium containing 20% fetal bovine serum, and the cortical tissue was further dissociated by serial trituration using a Pasteur CLINICAL NEUROSCIENCE AND NEUROPATHOLOGY NEUROREPORT 0959-4965  c Wolters Kluwer Health | Lippincott Williams & Wilkins Vol 18 No 18 3 December 2007 1879 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.