Fig 2: Summary figure of the WIB results. IL-18 may induce OS due to increase of antioxidative PRX3, -6, and BLVRA in SH-SY5Y cells. 14-3-3? (modulator of apoptosis; intracellular trafficking; cell cycle control; signal transduction; and metabolism), 14-3-3e (anti-apoptotic; a component of the prion/amyloid deposits of Gerstmann-Straüssler-Scheinker disease). 14-3-3e can bind PRX6, translocating PRX6 to lysosomes when MAPK is activated. Both 14-3-3? and -e can prevent a-synuclein inclusion formation. 14-3-3 proteins can complex with tau and GSK3ß promoting tau phosphorylation and NFT formation. 14-3-3e can increase MMP2. TIMP2 (MMP inactivator), MMP14 (a physiological activator for pro-MMP2 complexed with TIMP2). IL-18 inducible MMP2 and MMP9 are also expressed in SH-SY5Y cells. MMP2, -9 and soluble MMP14 can participate in Aß degradation. Increased PRX6 is linked with increased MMP9 and uPA, and decreased TIMP2 expression. uPA and tPA are expressed in SH-SY5Y cells, and both, via plasminogen, increase protease plasmin, also able degrading Aß and regulating MMP levels. Active plasmin can remodel directly and indirectly, via activation of MMPs, the ECM. ENOG (glycolysis; a neurotrophic-like factor promoting growth, differentiation, survival, and regeneration of neurons). The neurotrophic activity is mediated by the C-terminus which activates PI3K/Akt and MAPK/ERK. Cathepsin X, which activity increases during aging and interacts also with Aß-plaques, cleaves the C-terminus of ENOG and ENOA, impairing the survival and neuritogenesis of neuronal cells. DJ-1 (scavenges H2O2; modulates Cu/Zn-SOD; a redox-sensitive chaperone; prevents a-SNC aggregation; detectable in tau inclusions; loss of function leads to neurodegeneration, likely mediated by mitochondria). DDAH (regulates apoptosis; controls cellular methylarginine concentration by hydrolyzing NOS inhibitors ADMA and MMA). DDAH can increase NOS activity, NO production and resultant increase in nitrosative stress. NO also regulates learning and memory. ONOO- can activate MMPs. ENOA (glycolysis; plasminogen receptor). Interaction of plasminogen with ENOA enhances its activation to plasmin by tPA or uPA. Modifications of ENOA can lead to its catalytic inactivation, which can alter its role as a plasminogen receptor, effecting ECM remodeling, neuronal survival and Aß degradation. SEPT2 (interacts with Aß; linked to pathological features of Aß plaques and NFTs; filament-forming cytoskeletal GTPase that can modulate cytokinesis, cell division, exocytosis, and membrane trafficking). Generally, Septins form hetero-oligomeric complexes which assemble into filaments, mediated by the GTPase activity. CRMP2 (mediates Sem3A signaling in the nervous system; interacts e.g., with tubulin, Numb, kinesin1, and Sra1; promotes axon elongation; phosphorylated by several Ser/Thr kinases (ROCK: Thr555, Cdk5: Ser522, GSK3ß: Ser518, Thr514,509). Ser522-phosphorylation is essential for sequential phophorylations by GSK3ß at Ser518 and Thr514,509. These phosphorylation steps are required in Sem3A signaling. The phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb. CRMP2 hyperphosphorylation is an early event in AD progression and occurs downstream of APP, but neither excessive Aß42 peptide nor neurotoxicity alone are sufficient to promote it. CRMP2 hyperphosphorylated at Thr509 and Ser518,522 is also present in NFTs. IL-18 can induce Cdk5 and GSK3ß and caspase-3 (Ojala et al., 2008) as well as APP, BACE-1, and PS-1 (Sutinen et al., 2012) marked as smaller font. Interactions are shown with dashed lines.

Fig 3: Western immunoblot verification of the selected targets. (A) IL-18 induced our apoptosis marker caspase-1 in a concentration dependent manner, with APP level reduced in 72 h treated cells. DDAH2 and SEPT2 levels are increased as well as processed MMP14 (54 kD) and precursor MMP14 (above, 66 kD, propeptide 64 kD). The 30 kD form may be some a non-typical cleavage form of MMP14 (antibody detects aa 511–582 at C-terminus). CRMP2 P-Ser522 specific antibody detected a faint concentration dependent band (>). CRMP2 P-Thr514 also increase (>), with the intensity of CRMP2 different to that of CRMP-2 P-Ser522. Also an additional band about 30 kD was detectable. All the results are from the same membrane. (B) CRMP2 also showed increased phosphorylation at Thr555 at 24 and 48 h time-points, after which it reduced, as detected by specific antibody. The band about 30 kD, was evident only at 24 h. Tubulin shows some co-regulation. Non-specific band indicates evenness of sample loading. (C) CRMP2 was studied also with non-phosphor specific antibody after 150 ng/ml IL-18 treatment. CRMP2 (isoform 1: 62 kD, isoform 2: 58 kD) were unaffected, but high MW-form above was IL-18 responsive (>), which may be a modified form of CRMP2. CRMP is phosphorylatable at several sites but other modifications may also exist. Actin was used as a loading control. (D) IL-18 at 150 ng/ml also increased TIMP2 levels in the culture medium of SH-SY5Y cells vs. untreated control.