07-886 Sigma-AldrichAnti-phospho-PKR (Thr451) Antibody

Serum amyloid A (SAA) proteins are known to be surrogate markers of sepsis, but their pathogenic roles remain poorly elucidated. Here we provide evidence to support a possible role of SAA as a pathogenic mediator of lethal sepsis. In a subset of septic patients for which serum high mobility group box 1 (HMGB1) levels paralleled the clinical scores, some anti-HMGB1 antibodies detected a 12-kDa protein belonging to the SAA family. In contrast to the most abundant SAA1, human SAA induced double-stranded RNA-activated protein kinase R (PKR) expression and HMGB1 release in the wild-type, but not toll-like receptor 4/receptor for advanced glycation end products (TLR4/RAGE)-deficient, macrophages. Pharmacological inhibition of PKR phosphorylation blocked SAA-induced HMGB1 release, suggesting an important role of PKR in SAA-induced HMGB1 release. In animal models of lethal endotoxemia and sepsis, recombinant SAA exacerbated endotoxemic lethality, whereas SAA-neutralizing immunoglobulins G (IgGs) significantly improved animal survival. Collectively, these findings have suggested SAA as an important mediator of inflammatory diseases. Highlights of this study include: human SAA is possibly only expressed in a subset of septic patients; SAA induces HMGB1 release via TLR4 and RAGE receptors; SAA supplementation worsens the outcome of lethal endotoxemia; whereas SAA-neutralizing antibodies confer protection against lethal endotoxemia and sepsis.

Emerging evidence suggests that dysregulated translation through phosphorylation of eukaryotic initiation factor-2α (eIF2α) may contribute to Alzheimer's disease (AD) and related memory impairments. However, the underlying mechanisms remain unclear. Here, we crossed knockout mice for an eIF2α kinase (GCN2: general control nonderepressible-2 kinase) with 5XFAD transgenic mice, and investigated whether GCN2 deletion affects AD-like traits in this model. As observed in AD brains, 5XFAD mice recapitulated significant elevations in the β-secretase enzyme BACE1 and the CREB repressor ATF4 concomitant with a dramatic increase of eIF2α phosphorylation. Contrary to expectation, we found that GCN2(-/-) and GCN2(+/-) deficiencies aggravate rather than suppress hippocampal BACE1 and ATF4 elevations in 5XFAD mice, failing to rescue memory deficits as tested by the contextual fear conditioning. The facilitation of these deleterious events resulted in exacerbated β-amyloid accumulation, plaque pathology and CREB dysfunction in 5XFAD mice with GCN2 mutations. Notably, GCN2 deletion caused overactivation of the PKR-endoplasmic reticulum-related kinase (PERK)-dependent eIF2α phosphorylation pathway in 5XFAD mice in the absence of changes in the PKR pathway. Moreover, PERK activation in response to GCN2 deficiency was specific to 5XFAD mice, since phosphorylated PERK levels were equivalent between GCN2(-/-) and wild-type control mice. Our findings suggest that GCN2 may be an important eIF2α kinase under the physiological condition, whereas blocking the GCN2 pathway under exposure to significant β-amyloidosis rather aggravates eIF2α phosphorylation leading to BACE1 and ATF4 elevations in AD.

The pathogen- and damage-associated molecular patterns (for example, bacterial endotoxin and adenosine 5'-triphosphate [ATP]) activate the double-stranded RNA-activated protein kinase R (PKR) to trigger the inflammasome-dependent high mobility group box 1 (HMGB1) release. Extracellular ATP contributes to the inflammasome activation through binding to the plasma membrane purinergic P2X7 receptor (P2X7R), triggering the opening of P2X7R channels and the pannexin-1 (panx-1) hemichannels permeable for larger molecules up to 900 daltons. It was previously unknown whether panx-1 channel blockers can abrogate lipopolysaccharide (LPS)-induced PKR activation and HMGB1 release in innate immune cells. Here we demonstrated that a major gancao (licorice) component (glycyrrhizin, or glycyrrhizic acid) derivative, carbenoxolone (CBX), dose dependently abrogated LPS-induced HMGB1 release in macrophage cultures with an estimated IC50 ≈ 5 μmol/L. In an animal model of polymicrobial sepsis (induced by cecal ligation and puncture [CLP]), repetitive CBX administration beginning 24 h after CLP led to a significant reduction of circulating and peritoneal HMGB1 levels, and promoted a significant increase in animal survival rates. As did P2X7R antagonists (for example, oxidized ATP, oATP), CBX also effectively attenuated LPS-induced P2X7R/panx-1 channel activation (as judged by Lucifer Yellow dye uptake) and PKR phosphorylation in primary peritoneal macrophages. Collectively, these results suggested that CBX blocks LPS-induced HMGB1 release possibly through impairing PKR activation, supporting the involvement of PKR in the regulation of HMGB1 release.

The β-secretase enzyme BACE1 (β-site amyloid precursor protein-cleaving enzyme 1), which initiates amyloid-β (Aβ) production, is an excellent therapeutic target for Alzheimer's disease (AD). However, recent evidence raises concern that BACE1-inhibiting approaches may encounter dramatic declines in their abilities to ameliorate AD-like pathology and memory deficits during disease progression. Here, we used BACE1 haploinsufficiency as a therapeutic relevant model to evaluate the efficacy of partial inhibition of this enzyme. Specifically, we crossed BACE1(+/-) mice with 5XFAD transgenic mice and investigated the mechanisms by which Aβ accumulation and related memory impairments become less sensitive to rescue by BACE1(+/-) reduction. Haploinsufficiency lowered BACE1 expression by ∼50% in 5XFAD mice regardless of age in concordance with reduction in gene copy number. However, profound Aβ plaque pathology and memory deficits concomitant with BACE1 equivalent to wild-type control levels remained in BACE1(+/-)·5XFAD mice with advanced age (15-18 months old). Therefore, BACE1 haploinsufficiency is not sufficient to block the elevation of BACE1 expression (approximately twofold), which is also reported to occur during human AD progression, in 5XFAD mice. Our investigation revealed that PERK (PKR-endoplasmic reticulum-related kinase)-dependent activation of eIF2α (eukaryotic translation initiation factor-2α) accounts for the persistent BACE1 upregulation in BACE1(+/-)·5XFAD mouse brains at 15-18 months of age. Moreover, BACE1 haploinsufficiency was also no longer able to prevent reduction in the expression of neprilysin, a crucial Aβ-degrading enzyme, in 5XFAD mice with advanced age. These findings demonstrate that partial BACE1 suppression cannot attenuate deleterious BACE1-elevating or neprilysin-reducing mechanisms, limiting its capabilities to reduce cerebral Aβ accumulation and rescue memory defects during the course of AD development.