Modulation of Aβ42-induced toxic effects on the cultured neuronal network activity by extracellular matrix stiffness

Abstract

Alzheimer’s disease (AD) is the most common neurodegenerative disease that usually begins with short-term memory loss, gradually progresses to cognitive dysfunction and causes loss of body function and eventual death. Mutations in the APP gene encoding the Aβ precursor protein (APP) are known to cause early-onset AD and suggest that Aβ is a major factor in AD development. Enzyme complexes, such as α-, β- and γ-secretases, catalyze various cleavage pathways to produce a variety of Aβ isoforms of different lengths. These Aβ peptides have proven toxic to the brain and accumulate in AD to form cerebral plaques. The main isoform of Aβ present in these plaques is the 42 amino acid variant known as Aβ42. A previous study revealed that changes in the stiffness of the extracellular matrix (ECM) can induce remodeling of the cytoskeleton of neurons in the brain tissues of AD patients, leading to changes in the morphology and function of neurons. ECM stiffness is unique to each specific tissue, and resident cells have developed to function optimally in microenvironments with specific ECMs. Brain tissues are reported to have a Young’s modulus of elasticity between 0.1 and 16 kPa. In patients with AD, a decrease in the elasticity of brain tissues was detected. Interestingly, the ECM is known to play an important role in cytoskeleton remodeling and neuronal function, and a stiff ECM has been reported to promote actin polymerization and stress fiber formation, whereas a soft ECM triggers actin depolymerization. However, it remains uncertain whether alterations in ECM stiffness in the AD brain contribute to Aβ-induced toxicity, particularly considering that Aβ is recognized to cause neuronal toxicity by disrupting the actin cytoskeleton, which leads to subsequent synaptic and dendritic abnormities. As such, the present study aimed to investigate the effects of substrate stiffness on Aβ-induced toxicity to the neuronal network in cultured neurons. Hippocampal neurons cultured on soft and stiff substrates were assessed for cell viability by MTT assay. When the neuronal cultures were exposed to 1 μM Aβ42 for 48 h, there was a significant decrease in the viability of the cells cultured on the stiff substrates, but there was no significant effect on the viability of the neuronal cultured on the soft substrates. In addition, the influence of Aβ42 on the number of synapses within the cultured neuronal network was analyzed using confocal immunofluorescence imaging. This analysis revealed that Aβ42 exposure induced a decrease in synaptic formation in cultured neurons, which was dependent on substrate stiffness. To evaluate the effect of substrate stiffness on Aβ42-induced toxicity to synaptic transmission in the cultured neuronal network, spontaneous Ca2+ oscillations were examined in neurons cultured on substrates with different stiffness treated with Aβ42. The percentage of neurons with spontaneous Ca2+ oscillations was significantly greater in neurons cultured on stiff substrates than in those cultured on soft substrates. After Aβ42 exposure, the percentage of neurons with spontaneous Ca2+ oscillations was significantly decreased in neurons cultured on the stiff substrates. Exposure to Aβ42 only slightly influenced the percentage of spontaneous Ca2+ oscillations in neurons cultured on the soft substrate. The amplitude and frequency of spontaneous Ca2+ oscillations were significantly greater in neurons cultured on the stiff substrates than in those cultured on the soft substrates. After exposure to Aβ42, the amplitude and frequency of spontaneous Ca2+ oscillations were significantly reduced in neurons cultured on stiff substrates. However, exposure to Aβ42 had only a weak influence on the amplitude and frequency of spontaneous Ca2+ oscillations in neurons cultured on soft substrates. To further investigate the effects of substrate stiffness on synapse function following exposure to Aβ42, spontaneous postsynaptic currents were recorded in DIV14-16 neurons cultured on stiff and soft substrates. The percentage of neurons with spontaneous postsynaptic currents was considerably greater in neurons cultured on the stiff substrates than in those cultured on soft substrates.

1. Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease that usually begins with short-term memory loss, gradually progresses to cognitive dysfunction and causes loss of body function and eventual death [1]. Mutations in the APP gene encoding the Aβ precursor protein (APP) are known to cause early-onset AD and suggest that Aβ is a major factor in AD development [2]. Enzyme complexes, such as α-, β- and γ-secretases, catalyze various cleavage pathways to produce a variety of Aβ isoforms of different lengths [3]. These Aβ peptides have proven toxic to the brain and accumulate in AD to form cerebral plaques. The main isoform of Aβ present in these plaques is the 42 amino acid variant known as Aβ42 [4].

A previous study revealed that changes in the stiffness of the extracellular matrix (ECM) can induce remodeling of the cytoskeleton of neurons in the brain tissues of AD patients, leading to changes in the morphology and function of neurons [5]. ECM stiffness is unique to each specific tissue, and resident cells have developed to function optimally in microenvironments with specific ECMs [6]. Brain tissues are reported to have a Young’s modulus of elasticity between 0.1 and 16 kPa. In patients with AD, a decrease in the elasticity of brain tissues was detected [7]. Interestingly, the ECM is known to play an important role in cytoskeleton remodeling and neuronal function [8], and a stiff ECM has been reported to promote actin polymerization and stress fiber formation, whereas a soft ECM triggers actin depolymerization [9]. However, it remains uncertain whether alterations in ECM stiffness in the AD brain contribute to Aβ-induced toxicity, particularly considering that Aβ is recognized to cause neuronal toxicity by disrupting the actin cytoskeleton, which leads to subsequent synaptic and dendritic abnormities [10]. As such, the present study aimed to investigate the effects of substrate stiffness on Aβ-induced toxicity to the neuronal network in cultured neurons.