Rapid Changes in Phospho-MAP/Tau Epitopes during Neuronal Stress: Cofilin-Actin Rods Primarily Recruit Microtubule Binding Domain Epitopes Ineka T. Whiteman1,2, Laurie S. Minamide3, De Lian Goh1,2, James R. Bamburg3, Claire Goldsbury1,2* 1 The Brain and Mind Research Institute, University of Sydney, Sydney, Australia, 2 Bosch Institute, School of Medical Sciences, University of Sydney, Sydney, Australia, 3 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America Abstract Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer’s disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies - AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422 - raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD. Citation: Whiteman IT, Minamide LS, Goh DL, Bamburg JR, Goldsbury C (2011) Rapid Changes in Phospho-MAP/Tau Epitopes during Neuronal Stress: Cofilin- Actin Rods Primarily Recruit Microtubule Binding Domain Epitopes. PLoS ONE 6(6): e20878. doi:10.1371/journal.pone.0020878 Editor: Mark P. Mattson, National Institute on Aging Intramural Research Program, United States of America Received March 30, 2011; Accepted May 11, 2011; Published June 28, 2011 Copyright: 2011 Whiteman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Sir Zelman Cowen Universities Fund, The Judith Jane Mason and Harold Stannett Williams Memorial Foundation, The Rebecca Cooper Foundation, The NHMRC, Sydney Medical School and the National Institutes of Health grant NS40371 (JRB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: claire.goldsbury@sydney.edu.au Introduction Neuronal histopathological hallmarks of AD include neurofi- brillary tangles (NFT) and neuropil threads both comprised of hyperphosphorylated microtubule-associated protein (MAP) tau. NFTs and neuropil threads assemble in cell bodies and neurites respectively. Comprising a reported .85% of end-stage cortical tau pathology, neuropil threads correlate with cognitive decline [1–6]. The major known function of tau, like other MAPs, is its stabilization and regulation of microtubule (MT) dynamics necessary for neurite outgrowth, morphogenesis and since tau is predominantly an axonal protein, it plays an important role in facilitating MT-dependent axonal transport (for reviews see [7,8]). Tau can also interact with the plasma membrane and may play roles in relaying signals to the cytoskeleton from the cell surface or scaffolding signaling complexes [9]. The MT directed activity of tau is regulated by phosphorylation/dephosphorylation cycles, such that phosphorylation at specific sites detaches tau from MTs and allows MT depolymerization, while tau dephosphorylation enables it to bind and stabilize MT via its MT binding domain (MTBD) [10,11]. In AD, tau is hyperphosphorylated, the MT network is destabilized and tau self-assembles into paired helical filaments (PHFs) that form the NFT and neuropil thread structures. Over 20 phosphorylation sites have been characterized for tau, two of these are located in the MTBD at two ‘KXGS’ amino acid motifs corresponding to residues S262 and S356 [11– 13]. Phosphorylation of these KXGS motifs is one of the earliest markers of AD pathology, readily detectable in neuropil threads with the monoclonal antibody 12E8 that recognizes these conserved motifs in both tau as well as in other MAPs [14]. In the case of tau, phosphorylation of the MTBD sites has been shown to induce MT disassembly whereby the new unbound pool of tau is susceptible to self-assembly into PHFs [12,15–17]. Neuropil threads generally precede the appearance of extensive NFTs, suggesting tau first accumulates in neurites during the development of AD pathology before the proliferation of cell body NFTs [1,4,18,19]. An in vitro cell model for neuropil thread assembly may therefore help mimic early cellular events relevant to the disease mechanism. To this end, we recently demonstrated in primary neuronal cell culture and organotypic slice culture that mitochondrial dysfunction initiates formation of 12E8-positive neuritic inclusions that co-localize with actin depolymerizing factor (ADF)/cofilin-actin rods (AC rods) [20]. These inclusions bear some morphological resemblance to inclusions observed in AD brain which also include punctuate and rod-like linear arrays of cofilin and actin aggregates throughout the neuropil [21] (reviewed in [22]). The relevance of this model lies in an increasing body of evidence that implicates disrupted energy metabolism and mitochondrial dysfunction in AD (for reviews see [23–27]). Reduced mitochondrial function and increased oxidative stress are reported to occur early in disease progression, suggesting that these factors could play an integral role in initiating pathological PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20878