Research ReportAmyloid beta modulation of neuronal network activity in vitro
Introduction
Alzheimer׳s disease (AD) is a neurodegenerative disorder in which the loss of synapses and neuronal apoptosis in cortex and hippocampus lead to behavioral deficits such as memory deficits and cognitive impairment (Lublin and Gandy, 2010). Amyloid plaques which consist of aggregated fibrillar amyloid beta (Aβ) peptides are considered a pathological marker for AD (Benilova et al., 2012). Aβ peptides are produced by cleavage of amyloid precursor protein (APP) generating peptides of lengths ranging from 36 to 43 amino acids (Zhao et al., 2012). The major protein constituents of AD plaques are peptides of 40 and 42 amino acids in length or Aβ40 and Aβ42, respectively (Götz et al., 2011). The longer peptide, Aβ42, has a high tendency to aggregate and it readily oligomerizes to form soluble dimers, trimers, and higher order oligomers (Citron, 2010, Mairet-Coello et al., 2013). The oligomers then aggregate further and form Aβ fibrils which constitute the amyloid plaques. However, prior work suggests that the most neurotoxic forms of Aβ are not the senile plaques or fibrillar Aβ but soluble Aβ oligomers (Bucciantini et al., 2002, Kirkitadze et al., 2002). In patients with AD, declines in cognition and memory appear correlated with increases in the fraction of soluble Aβ oligomer suggesting a dynamic equilibrium between multiple forms of the biomolecule (Rowan et al., 2003).
The exact mechanisms of neurotoxic effects of Aβ are not yet clearly understood. Formation of toxic pores in the cell membrane (Alarcón et al., 2006), increase in the intracellular oxidative stress (Nunomura et al., 2010), cell cycle re-entry (Seward et al., 2013), disrupting intracellular calcium release (Lazzari et al., 2014), and interruption of synaptic transmission (Eckert et al., 2008) are all among the proposed mechanisms. In addition, there have been numerous studies concerning the target receptor for Aβ oligomers. Aβ appears to interact with a wide range of cellular receptors including but not limited to nicotinic cholinergic receptors, glutamatergic receptors, ephrin-type B2 receptors, etc. (for review see Cheng et al., 2014; Mucke and Selkoe, 2012). With respect to synaptic transmission changes that are correlated with altered cognitive function, prior studies point to a role for N-methyl-D-aspartate (NMDA) receptors as either receptors for Aβ42 or as an intermediary of its neuroactive effects (Deng et al., 2014, Ferreira et al., 2012, Texidó et al., 2011, Yamin, 2009).
To date, various transgenic animal models and mammalian cell assays have been utilized to identify potential therapeutics for AD (Gravitz, 2011, McColl et al., 2012, Wilcock, 2010). In particular, in vitro assays are advantageous for drug screening applications because they reduce animal usage, provide initial risk assessment, and thus have the potential to accelerate the drug discovery process (Harry et al., 1998). To this end, assays ranging from mammalian cells which accumulate Aβ (Haugabook et al., 2001) to human induced pluripotent stem (iPS) cells expressing APP (Yahata et al., 2011) have been proposed. However, most of the available in vitro preparations do not provide functional endpoints that capture physiologically relevant neuronal activity. In addition, the associated experiments are not designed to assess immediate effects of the oligomer (Tamburri et al., 2013). As such, a network-level functional assay which can provide reliable, fast, and high-content measures is desirable. An approach which has gained attention and interest as a platform for neuropharmacology and neurotoxicity testing involves the use of neuronal networks on microelectrode arrays (MEAs). Murine primary cultures derived from neural tissue form spontaneously active networks on substrate-integrated MEAs. Previous work has demonstrated successful applications of this platform as a biosensor in neuropharmacology (Johnstone et al., 2010, Keefer et al., 2001, Morefield et al., 2000, Xiang et al., 2007), assessing biocompatibility of novel materials (Charkhkar et al., 2014) and basic neuroscience (Bakkum et al., 2008, Hamilton et al., 2013, Wagenaar et al., 2005). This method is well established (Gross et al., 1985) and has been validated across different laboratories (Novellino et al., 2011). Compared to single-cell techniques such as patch clamp, the MEA method is non-invasive and allows the concurrent examination of populations of neurons.
In this paper, we demonstrate that Aβ42 oligomer, but not monomer, produces significant reductions in neuronal spike activity of cultured neuronal networks. These network inhibitory effects appear to depend, either directly or indirectly, on modulation of both AMPA and NMDA mediated glutamate receptors. Moreover, two model compounds that have shown clinical promise in treating AD, methylene blue (MB) and memantine, appear to reverse or suppress the inhibitory effects of Aβ42 oligomer. These observations suggest that cultured neuronal networks may be a potentially useful platform for screening therapeutic candidates for AD.
Section snippets
Stability of the synthesized Aβ42 oligomer
As shown in Fig. 1, the synthesized Aβ42 oligomer in our work had a molecular weight of 4–85 kDa and showed high stability in saline buffer solution for at least 14 days at 4 °C and 25 °C (Duan et al., 2011, Matveeva et al., 2012). A slight shift in the oligomer size exclusion chromatography (SEC) profile was observed only after 14 days at 25 °C. Nevertheless, there was no evidence of fibrillization under the stability studies, as determined by SEC and sodium dodecyl sulfate-polyacrylamide gel
Discussion
In this work, we show that oligomeric rather than monomeric forms of Aβ42 induce electrophysiologic changes to network level activity and the observed effects are reversed by model AD therapeutic agents. Our findings are consistent with prior work demonstrating that the oligomeric form, compared to Aβ42 monomer, is the more neuroactive form as the former produced significant reductions in neuronal activity. Our results suggest that inotropic glutamate receptors, AMPA/kainate and NMDA, are both
Aβ42 synthesis
Aβ42 monomer was acquired commercially from Anaspec (Fremont, CA). Stable oligomer was produced by a process described in Barghorn et al. (2005) that has been optimized for maximal stability (Matveeva et al., 2012). In brief, Aβ42 monomer was first dissolved in DMSO (1.0 mg/44 μL), and then sonicated for 5 min. Phosphate buffered saline (PBS) and anionic surfactant sodium dodecyl sulfate (SDS) were added to the solution to achieve Aβ42 concentration of 100 μM. After incubation at 37 °C for 24 h,
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Author contributions
HC and JJP designed the research. HC, SM, and DM performed the experiments. HC and SM analyzed the data. EM, JRM, and RC synthesized, characterized, and provided the amyloid oligomer. RC and JRM provided data on the oligomer stability. JJP and NP supervised the experiments. HC wrote the paper. JJP, NP, and RC revised the manuscript.
Acknowledgments
This work was supported in part by Award No. 15-8 from the Commonwealth of Virginia׳s Alzheimer׳s and Related Diseases Research Award Fund, administered by the Virginia Center on Aging, School of Allied Health Professions, Virginia Commonwealth University.
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