Interleukin-18 alters protein expressions of neurodegenerative diseases-linked proteins in human SH-SY5Y neuron-like cells

Abstract

Chronic inflammation and oxidative stress (OS) are present in Alzheimer's disease (AD) brains in addition to neuronal loss, Amyloid-β (Aβ) plaques and hyperphosphorylated tau-protein neurofibrillary tangles (NFTs). Previously we showed that levels of the pro-inflammatory cytokine, interleukin-18 (IL-18), are elevated in post-mortem AD brains. IL-18 can modulate the tau kinases, Cdk5 and GSK3β, as well as Aβ-production. IL-18 levels are also increased in AD risk diseases, including type-2 diabetes and obesity. Here, we explored other IL-18 regulated proteins in neuron-like SH-SY5Y cells. Differentiated SH-SY5Y cells, incubated with IL-18 for 24, 48, or 72 h, were analyzed by two-dimensional gel electrophoresis (2D-DIGE). Specific altered protein spots were chosen and identified with mass spectrometry (MS) and verified by western immunoblotting (WIB). IL-18 had time-dependent effects on the SH-SY5Y proteome, modulating numerous protein levels/modifications. We concentrated on those related to OS (DDAH2, peroxiredoxins 2, 3, and 6, DJ-1, BLVRA), Aβ-degradation (MMP14, TIMP2), Aβ-aggregation (Septin-2), and modifications of axon growth and guidance associated, collapsin response mediator protein 2 (CRMP2). IL-18 significantly increased antioxidative enzymes, indicative of OS, and altered levels of glycolytic α- and γ-enolase and multifunctional 14-3-3γ and -ε, commonly affected in neurodegenerative diseases. MMP14, TIMP2, α-enolase and 14-3-3ε, indirectly involved in Aβ metabolism, as well as Septin-2 showed changes that increase Aβ levels. Increased 14-3-3γ may contribute to GSK3β driven tau hyperphosphorylation and CRMP2 Thr514 and Ser522 phosphorylation with the Thr555-site, a target for Rho kinase, showing time-dependent changes. IL-18 also increased caspase-1 levels and vacuolization of the cells. Although our SH-SY5Y cells were not aged, as neurons in AD, our work suggests that heightened or prolonged IL-18 levels can drive protein changes of known relevance to AD pathogenesis.

The Role of Interleukin-18, Oxidative Stress and Metabolic Syndrome in Alzheimer’s Disease

“…Although the normal function of IL-18 in the brain requires further investigation, it seems to play a role in CNS development and function as an up-stream regulator of brain immune and inflammatory processes [14,39]. Due to its proinflammatory nature, IL-18 is a potential enhancer of OS [40], which is also supported by the finding that IL-18 binding protein can ameliorate OS [41]. IL-18 can enhance Aβ production [42] and the kinases Cdk5 and glycogen synthase kinase-3β (GSK-3β), which are involved in the hyperphosphorylation of tau [14], caspase-1 regulation as well as many other neurodegenerative diseases linked proteins [40], and cellular vacuolization [42].…”

“…Due to its proinflammatory nature, IL-18 is a potential enhancer of OS [40], which is also supported by the finding that IL-18 binding protein can ameliorate OS [41]. IL-18 can enhance Aβ production [42] and the kinases Cdk5 and glycogen synthase kinase-3β (GSK-3β), which are involved in the hyperphosphorylation of tau [14], caspase-1 regulation as well as many other neurodegenerative diseases linked proteins [40], and cellular vacuolization [42]. IL-18 can induce IFNγ, a significant driver of immune-inflammatory processes, as well as the primary inducer of indoleamine 2,3-dioxygenase (IDO).…”

“…Protein levels of dimethylarginine dimethylaminohydrolase 2 (DDAH2) can also be regulated by IL-18 [40]. In addition, IL-18 can moderately increase the hyperphosphorylation tau as well as modulate protein levels of the anti-apoptotic B-cell lymphoma-extra large (Bcl-xL) [42] and B-cell lymphoma 2 (Bcl-2) family of proteins.…”

“…However, in healthy young neuron-like cells, IL-18 was able to increase the expression of PRX3 [40]. In aged neurons, the impact of IL-18 can be different; with IL-18 facilitating the modifications of PRX3 protein, leading to its inactivation [50] or increasing its release.…”

Oxidative Stress and Neuronal Damages: Estrogenic Compounds, Anti-Apoptotic Factors, and Amyloidogenesis

“…As described earlier [46], the membranes were blocked with 3% non-fat milk (Valio; Finland) in Tris-buffered saline (TBS; pH 7.5)/0.1% Tween-20 (Sigma-Aldrich), and the antibodies were used at 1:400 dilution, with exception of WO-2, which was used at 0.5 µg/ml. For detection, the membranes were incubated with ECL TM donkey anti-rabbit IgG F(ab')2 peroxidase-linked detection antibody (from sheep) (GE Healthcare Life Sciences) or bovine antigoat IgG-HRP mouse/human adsorbed (sc-2384; Santa Cruz Biotech) detection antibodies.…”