Involvement of endoplasmic reticulum stress in amyloid β _(1-42)-induced Alzheimer’s like neuropathological process in rat brain

Highlights

• Aβ_(1-42) significantly increases AChE activity, induces of ER stress and apoptosis.

• Aβ_(1-42) treatment causes increase in oxidative stress and neuronal degeneration.

• Aβ_(1-42) treatment also results in activation of microglial cells.

• Salubrinal attenuates Aβ_(1-42)-induced ER and oxidative stress and apoptosis.

• Salubrinal attenuates Aβ_(1-42)-induced neurodegeneration and microglial activation.

Abstract

Amyloid-β (Aβ) accumulation in the brain is a pathological hallmark of Alzheimer’s disease (AD). Endoplasmic reticulum (ER) stress has been implicated in aetiology of neurodegenerative disorders. We studied the involvement of ER stress in Aβ-induced neuronal degeneration in rat brain to correlate it with cellular and molecular modifications in Aβ-induced Alzheimer’s like neuropathological process. Aβ _(1-42) (5 μg) was administered by bilateral intracerebroventricular (icv) injection in the brain of adult male Wistar rats. Acetylcholinesterase (AChE) activity and histological alterations were observed in different brain regions. ER stress-associated proteins- glucose regulated protein-78 (GRP78), eukaryotic translation initiation factor-2α (eIF2α) and growth arrest and DNA damage-inducible protein-153 (GADD153), neuronal marker- microtubule associated protein-2 (MAP-2) and microglial protein- ionized calcium binding adaptor molecule-1 (Iba-1) were measured by western blot. Reduced glutathione (GSH), nitrite level and levels of caspase-12 and caspase-3 were also measured. ER stress inhibitor, salubrinal (1 mg/kg, intraperitoneally, ip) was used to assess the specific role of ER stress. Aβ _(1-42)-induced increase in AChE activity, GRP78 and GADD protein levels, dephosphorylation of eIF2-α and caspase-12 and caspase-3 levels and decrease in GSH and MAP-2 levels were attenuated by salubrinal. Increase in Iba-1 protein and nitrite levels after Aβ _(1-42) administration were partially attenuated by salubrinal. Aβ _(1-42)-induced histological alterations were correlated with findings of ER stress. Results of present study implicate ER stress as a potential molecular mechanism in Aβ-induced Alzheimer’s like neuropathology which could serve as surrogate biomarker for study of AD progression and efficacy of therapeutic interventions for AD management.

Introduction

Amyloid-β (Aβ) peptide accumulation in the brain is a pathological hallmark of Alzheimer’s disease (AD) (Selkoe, 1997; Yalcin et al., 2016) and has been implicated in the impairment of learning and memory (Chen et al., 2014). Also, evidence from clinical studies implicates that Aβ peptide formation is the major cause for early onset of AD, which is the most common cause of dementia in the elderly population (Rosenblum, 2014). AD is a progressive, irreversible neurodegenerative disease characterized clinically by cognitive loss due to death of neurons in the cerebral cortex, hippocampus and basal forebrain. The abnormal accumulation of 42 residue long amyloid beta peptide (Aβ _(1-42)) ultimately makes AD a protein misfolding disease (Knowles et al., 2014). Aβ is the proteolytic cleavage product of the amyloid precursor protein (APP) when it is cleaved by β- and γ-secretases (Zhang and Xu, 2007). Aβ _(1-42) is characterized by its high rate of aggregation and it also self-assembles in progressively higher molecular weight structures (Mohamed et al., 2016; Walsh and Teplow, 2012). Aβ peptide accumulation has toxic effects on neurons, it induces the activation of microglia in vitro (Malchiodi-Albedi et al., 2001; Rogers et al., 2002) which results in various pathological events leading to neuronal cell death in AD (Ballard et al., 2011). Immunohistochemical and histopathological studies have shown the accumulation of Aβ _(1-42) in cortex and hippocampus regions of rat brains having AD-like pathology (Singh et al., 2018).

The underlying mechanisms of Aβ-induced neurotoxicity are not yet fully understood. However, based on research so far, involvement of several pathways viz., oxidative stress, microglial activation and apoptosis has been implicated (Huang et al., 2012). Recent evidence supports the involvement of endoplasmic reticulum (ER) stress and glial activation in AD and other neurological disorders (Goswami et al., 2014, 2016; Hanlon et al., 2016; Lin et al., 2014). ER is the main organelle responsible for proper folding, maturation and transfer of newly synthesized proteins. However, accumulation of insoluble Aβ-peptides may upset the ER homeostasis resulting in ER stress (Hetz and Mollereau, 2014). This further activates a cellular response known as the unfolded protein response (UPR) which initially aims to restore normal ER function. However, prolonged stress results in activation of apoptotic factors (Hetz, 2012).

Previous studies have shown increased expression of UPR regulators, glucose regulated protein-78 (GRP78) and eukaryotic initiation factor 2α (eIF2α) in brain in AD (Hoozemans et al., 2005; Stutzbach et al., 2013). Pro-apoptotic components of UPR such as growth arrest and DNA damage-inducible gene-153 (GADD153) and caspase-12 are also observed to be elevated in AD brains (Lee et al., 2010). In mouse models Aβ-peptide-induced AD-like pathology is also reported to increase the levels of GRP78, phosphorylated eIF2α and GADD153 and cleave caspase-12 (Baleriola et al., 2014; Barbero-Camps et al., 2014; Yoon et al., 2012). In addition, Ma et al. (2013) have shown significant role of eIF2α in synapse loss and cognitive deficits in mouse model of AD.

Since the involvement of ER stress in Aβ- induced apoptosis has been demonstrated (Ferreiro et al., 2006; Nakagawa et al., 2000), inhibition of ER stress might prove to be beneficial. Salubrinal, a selective inhibitor of dephosphorylation of eIF2α, has shown the protective effects against ER stress-induced apoptosis (Goswami et al., 2014). Boyce et al. (2005) have also shown that the treatment of salubrinal, in vitro, can protect the cells from ER stress-induced apoptosis. In neurons, salubrinal can reduce the load of misfolded proteins in ER under pathological conditions (Liu et al., 2012). Huang et al. (2013) have also reported that salubrinal significantly reduced MPP⁺ (1-methyl-4-phenylpyridinium) and 6-OHDA (6-hydroxydopamine)-induced cell death in MN9D cells. Pharmacokinetic analysis of salubrinal have shown a rapid initial increase in the concentration of salubrinal in plasma, followed by a quick decrease within 24 h, and a half-life of 1.2 h in plasma (Zhang et al., 2012). Additionally, it has also been suggested that due to its ability to penetrate into the brain tissue salubrinal provides protection against neurotoxicity in vivo(Nakka et al., 2010; Sokka et al., 2007). However, in case of Aβ-induced neurotoxicity in rat brain it remains to be completely understood. As Aβ has been used as a model compound to induce AD-like symptoms and neuropathology (Walsh et al., 2002; Balducci et al., 2010), in the present study we studied role of ER stress using a suite of biomarkers of UPR in Aβ _(1-42)-induced neuropathological rat model. Besides ER stress, we also correlated our results by investigating oxidative stress and apoptosis markers. Salubrinal was used as a positive compound for mitigating the ER stress. Implicitly, we also wanted to investigate if the ER pathway has potential to serve as target for developing neurotherapeutic strategy for neurological disorders involving ER event at any of the stages.