Journal of Neuroscience Methods 175 (2008) 96–103 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth A 1–42 reduces synapse number and inhibits neurite outgrowth in primary cortical and hippocampal neurons: A quantitative analysis Nicholas A. Evans ∗ , Laura Facci, Davina E. Owen, Peter E. Soden, Stephen A. Burbidge, Rab K. Prinjha, Jill C. Richardson, Stephen D. Skaper Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline Research and Development Limited, New Frontiers Science Park, Third Avenue, Harlow CM19 5AW, Essex, United Kingdom article info Article history: Received 9 May 2008 Received in revised form 11 July 2008 Accepted 5 August 2008 Keywords: A1–42 Synaptogenesis Quantitative synapse assay Primary neurons abstract Synaptic loss represents one of the earliest signs of neuronal damage and is observed within both Alzheimer’s disease patients and transgenic mouse models of the disease. We have developed a novel in vitro assay using high content screening technology to measure changes in a number of cell physio- logical parameters simultaneously within a neuronal population. Using Hoechst-33342 to label nuclei, III-tubulin as a neuron-specific marker, and synapsin-I as an indicator of pre-synaptic sites, we have designed software to interrogate triple-labelled images, counting only those synaptic puncta associated with tubulin-positive structures. Here we demonstrate that addition of amyloid beta peptide (A 1–42 ), to either primary hippocampal or cortical neurons for 4 days in vitro has deleterious effects upon synapse formation, neurite outgrowth and arborisation in a concentration-dependent manner. Control reverse peptide showed no effect over the same concentration range. The effects of A 1–42 were inhibited by D-KLVFFA, which contains residues 16–20 of A that function as a self-recognition element during A assembly and bind to the homologous region of A and block its oligomerisation. These effects of A 1–42 on synapse number and neurite outgrowth are similar to those described within AD patient pathology and transgenic mouse models. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Alzheimer’s disease (AD) is characterised by loss of neurons, dystrophic neurites, senile/amyloid/neuritic plaques, neurofib- rillary tangles and synaptic loss. Beta-amyloid peptide (A) deposition is the major pathological feature of AD. Increasing evidence suggests that over-expression of the amyloid precursor protein (APP), and subsequent generation of the 39–43 amino acid residue, A, are central to neuronal degeneration observed in AD patients possessing familial AD mutations (Selkoe, 2001; Golde, 2003), while transgenic mice over-expressing APP develop AD-like pathology (Holcomb et al., 1989; Chapman et al., 1999; Moechars et al., 1999; Praticò et al., 2001). Most of the missense mutations in the genes encoding APP increase the production of A 1–42 , and soluble assemblies of synthetic A 1–42 are potent neurotoxins in vitro (Dahlgren et al., 2002; Maezawa et al., 2006). One of the earliest signs of AD appears to be a reduction in synaptic density (Gonatas et al., 1967; Suzuki and Terry, 1967). Sub- sequent studies (Praprotnik et al., 1996; Cash et al., 2003), described ∗ Corresponding author. Tel.: +44 1279 622452; fax: +44 1279 622555. E-mail address: Nicholas A Evans@gsk.com (N.A. Evans). a marked reduction of microtubules and accumulation of vesicles within both cell bodies and dystrophic neurites, indicative of dys- functional axonal transport mechanisms. In APP transgenic mice, synaptic perturbation (loss of synaptic puncta) precedes amyloid plaque deposition (Mucke et al., 2000). Other intracellular per- turbations, such as multivesicular bodies and aggregated A 1–42 oligomers within both endosomes and along microtubules of neu- ronal processes have been reported in APP transgenic mice and in cortical neurons cultured from these mice (Takahashi et al., 2004). Soluble oligomeric rather than fibrillar A 1–42 is now believed to be responsible for cognitive dysfunction in AD (McLean et al., 1999; Näslund et al., 2000 within Takahashi et al., 2004; Dodart et al., 2002). A 1–42 oligomers accumulate preferentially within neuronal processes and synaptic profiles rather than extracellularly (Walsh et al., 2000). Administration of anti-A antibodies may alleviate memory impairment in APP transgenic mice (Dodart et al., 2002), and internalised antibodies to the A domain of APP reduce intra- cellular A in cortical neurons cultured from APP transgenic mice and protect against synaptic alterations (Tampellini et al., 2007). There appears to be a striking correlation between synaptic loss and cognitive impairment within AD patients (Davies et al., 1987; Hamos et al., 1989; DeKosky and Scheff, 1990; Terry et al., 1991; Selkoe, 2002), which appears to be more predictive of cognitive 0165-0270/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2008.08.001