IGF-1 promotes b-amyloid production by a secretase-independent mechanism Wataru Araki a, * , Hideaki Kume a , Akiko Oda a,b , Akira Tamaoka a,b , Fuyuki Kametani c a Department of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan b Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan c Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Setagaya, Tokyo 156-8585, Japan article info Article history: Received 19 December 2008 Available online 22 January 2009 Keywords: Alzheimer’s disease b-Amyloid IGF-1 Neuroblastoma Phosphorylation abstract b-Amyloid peptide (Ab) is generated via the sequential proteolysis of b-amyloid precursor protein (APP) by b- and c-secretases, and plays a crucial role in the pathogenesis of Alzheimer’s disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Ab. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of b-C-termi- nal fragment (b-CTF) and secreted Ab. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of b-CTF-expressing cells with IGF-1 increased the levels of b-CTF and secreted Ab. The IGF-1-induced aug- mentation of b-CTF was observed in the presence of c-secretase inhibitors, but not in cells expressing b- CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Ab production through a secretase-independent mechanism involving APP phosphorylation. Ó 2009 Elsevier Inc. All rights reserved. Cerebral accumulation of b-amyloid peptide (Ab) is a major neuropathological hallmark of Alzheimer’s disease (AD) [1].Ab is produced by the sequential cleavage of the transmembrane amyloid precursor protein (APP) by b-secretase and c-secretase, which have been identified as b-site APP cleaving enzyme 1 (BACE1) and prese- nilin 1 (or presenilin 2) complex, respectively [2]. In the amyloido- genic processing pathway, b-secretase cleavage of APP generates secreted APP (sAPP)-b (sAPP-b) and b-C-terminal fragment (b-CTF), the latter of which is then cleaved intramembranously by c-secre- tase to produce Ab. By comparison, non-amyloidogenic a-secretase processing of APP within the Ab sequence generates sAPP-a and a-CTF, precluding Ab production [1]. IGF-1 is a well-characterized trophic factor that regulates cell growth and survival. IGF-1 and its receptors are expressed in the brain, and IGF-1 is actively transported across the blood-brain bar- rier [3,4]. IGF-1 is known to exert neuroprotective effects and to modulate neuronal activity [5]. Several recent studies have impli- cated the IGF-1/insulin system in the AD pathomechanism [6,7]. In one study, insulin was found to stimulate Ab release from neu- rons [8]. In other works, abnormalities in insulin and IGF-1 gene expression were detected in AD brains [9], and serum IGF-1 levels were found to be reduced in AD patients [10,11]. However, it re- mains unclear whether IGF-1 plays a specific role in the modula- tion of Ab production. Here, we sought to clarify this issue, using human neuroblastoma SH-SY5Y cells expressing Swedish mutant APP (swAPP) or b-CTF. Our data suggest that IGF-1 promotes Ab production through a secretase-independent mechanism involving APP phosphorylation. Materials and methods cDNA constructs. APP b-CTF cDNA was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), as described previously [12,13]. APP b-CTF cDNA with the Thr668Ala mutation (APP695 isoform numbering) was generated using the GeneEditor TM in vitro muta- genesis system (Promega. Madison, WI, USA) according to the manufacturer’s instructions. Cell culture and transfection. Human neuroblastoma SH-SY5Y cells were cultured as described previously [14]. SH-SY5Y cells sta- bly expressing swAPP were established previously [14]. SH-SY5Y cells were transfected with APP b-CTF or the Thr668Ala mutant APP b-CTF cDNA by the calcium phosphate method, and stable transformants were selected with 400 lg/mL G418. Antibodies and chemicals. A rabbit polyclonal antibody specific for the C-terminus of APP was described previously [15]. A rabbit polyclonal antibody (P-Thr668 antibody) specific for the Thr668- phosphorylated APP (P-APP) was obtained from Cell Signaling Technology (Beverly, MA, USA). A rabbit polyclonal antibody against BACE1 and a mouse monoclonal antibody against b-actin were purchased from Chemicon (Temecula, CA, USA) and Sigma (St. Louis, MO, USA), respectively. IGF-1 was obtained from Invitro- gen, and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylgly- cine t-butyl ester (DAPT) [16] and L-685,458 [17] were from Calbiochem (San Diego, CA, USA). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.01.044 * Corresponding author. Fax: +81 423 46 1747. E-mail address: araki@ncnp.go.jp (W. Araki). Biochemical and Biophysical Research Communications 380 (2009) 111–114 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc