Research ReportHigh content screen microscopy analysis of Aβ1–42-induced neurite outgrowth reduction in rat primary cortical neurons: Neuroprotective effects of α7 neuronal nicotinic acetylcholine receptor ligands
Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is characterized by progressive loss of memory accompanied with cholinergic neurodegeneration (Evans et al., 1989, Kar et al., 2004). One of the major pathological markers of AD is senile plaque (for a review, see Walsh and Selkoe, 2004). The intracellular plaques in the neocortex, hippocampus, and amygdala – regions involved in learning and memory – are formed by reversible aggregation of β-amyloid peptide (Aβ) in clusters. Aβ is generated from amyloid precursor protein (APP) through sequential proteolytic cleavages by secretase BACE1 at β-site and γ-secretase at down-stream amino acid residues 40 or 42, generating Aβ1–40 or Aβ1–42, respectively. There is considerable evidence indicating that Aβ, particularly Aβ1–42, is one of the major factors in AD pathogenesis (for a review, see Selkoe, 2002). Mutations around the proteolytic cleavage sites of APP have been identified in familial AD, which result in increased Aβ production (for a review, see Price and Sisodia, 1998). Other mutations linked to familial AD include presenilin 1 and 2 genes (PS1 and PS2) of the γ-secretase complex, which also lead to an increased ratio of Aβ1–42/Aβ1–40. Transgenic animals that expressed mutant forms of APP produced Aβ as well as plaques, and demonstrated deficits in cognitive and synaptic function (Spires and Hyman, 2005). Passive and active immunization against Aβ1–42 of APP transgenic animals and AD subjects reduced cognitive deficits (for a review, see Lemere et al., 2006). Collectively, these observations support the notion that Aβ1–42 plays a central role in AD pathogenesis. However, the mechanisms whereby Aβ1–42 leads to pathogenesis and cognitive impairment in AD remain to be further elucidated.
To further understand the mechanisms of Aβ-induced pathogenesis in AD, Aβ toxicity has been studied in various in vitro models by measurement of cell death after treatment with Aβ1–42 (Ren et al., 2005, Fodero et al., 2004, Kimura et al., 2005, Martin et al., 2004). However, no profound loss of neurons was detected in the transgenic mice over-expressing the mutant forms of APP that demonstrate cognitive deficit. Aβ1–42 disrupts synaptic function only in the early onset of AD (Kamenetz et al., 2003, Lambert et al., 1998, Walsh et al., 2002) and cholinergic neurons are either spared or affected only in late stages of the disease (Francis et al., 1999, Ladner and Lee, 1998). It is likely that Aβ toxicity may involve subtle and slowly progressive changes, leading to progressive synaptic failure. Indeed, it has been reported that APP transgenic (Tg2576) mice show decreased dendritic spine density, impaired hippocampal long-term potentiation (LTP) and behavioral deficits at 4–5 months of age, several months before plaque deposition (Jacobsen et al., 2006). Approaches to study these processes and mechanisms, especially at an in vitro level in a higher throughput manner, are currently limited.
In this study, the effect of Aβ1–42 on neurite outgrowth, a subtler marker of neuronal cellular function compared to cell viability, was investigated. Neurite outgrowth has been typically measured by conventional microscopy, which is labor-intensive and difficult for high-throughput analysis required for measurement of subtle changes. We employed high content screening (HCS), an advanced high-throughput image technology for cell-based image processing and analysis, which can provide direct assessment of cellular and subcellular morphology and generate diverse readouts. The data presented in this study demonstrate that a cell-based Aβ1–42 toxicity model that uses neurite outgrowth as a reliable measurement of neuronal degeneration can be utilized to measure the early neurotoxic effects of Aβ1–42. Further, this in vitro model was utilized to assess the neuroprotective effects of memantine and α7 neuronal nicotinic acetylcholine receptor (nAChR) ligands.
Section snippets
Aβ1–42 reduced neurite outgrowth in rat cortical cell cultures
Fig. 1a represents typical images of rat P0 primary cortical cultures after various treatments. Quantification of the neurons showed that Aβ1–42 significantly reduced neurite outgrowth in a concentration-dependent manner, while the scrambled control peptide did not alter the neurite outgrowth (Fig. 1b). Neither ATP nor caspase-3/7 assays revealed differences between any of the treatment groups (data not shown). While neurite outgrowth was reduced by Aβ1–42 in a concentration-dependent manner,
Discussion
Neurite outgrowth is usually measured by conventional microscopy, which is labor-intensive and impossible for high-throughput analysis that provides enough data points required for measurement of subtle changes. One of the major advantages of HCS using automated fluorescent microscopy is large-scale acquisition and analysis of cellular images (for a review, see Giuliano et al., 2003). Typically, cellular images are automatically acquired from several views in each of the multiple wells of a
Materials
Beta-amyloid (1–42) (Aβ1–42) and the scrambled control peptide (both hydroxyfluroisopropanol-treated) were purchased from rPeptide (Athens, GA). Other reagents and supplies include (sources indicated): Hanks' balanced salt solution (HBSS), phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) (Gibco Life Technologies Inc., Grand Island, NY); papain (Worthington, Lakewood, NJ); deoxyribonuclease I (DNase I) and memantine (Sigma, St. Louis, MO);
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