Toxicity in Rat Primary Neurons through the Cellular Oxidative Stress Induced by the Turn Formation at Positions 22 and 23 of Aβ42

The 42-mer amyloid β-protein (Aβ42) aggregates to form soluble oligomers that cause memory loss and synaptotoxicity in Alzheimer’s disease (AD). Oxidative stress is closely related to the pathogenesis of AD. We previously identified the toxic conformer of Aβ42 with a turn at positions 22 and 23 (“toxic turn”) by solid-state NMR and demonstrated that a monoclonal antibody (11A1) against the toxic turn in Aβ42 mainly detected the oligomer in the brains of AD patients. Our recent study suggested that oxidative stress is a key factor of the oligomerization and cognitive impairment induced by Aβ overproduction in vivo. However, the involvement of the toxic conformer in Aβ42-induced oxidative damage remains unclear. To investigate this mechanism, we examined the levels of intracellular reactive oxygen species (ROS) and neurotoxicity in rat primary neurons using E22P-Aβ42, a mutant that induces a turn at positions 22 and 23, and E22V-Aβ42, a turn-preventing mutant. E22P-Aβ42, but not E22V-Aβ42, induced greater ROS production than Wt-Aβ42 in addition to potent neurotoxicity. Interestingly, the formation of the toxic conformer in both E22P-Aβ42 and Wt-Aβ42 probed by the 11A1 antibody preceded Aβ42-induced neurotoxicity. Trolox (a radical scavenger) and Congo red (an aggregation inhibitor) significantly prevented the neurotoxicity and intracellular ROS induced by E22P-Aβ42 and Wt-Aβ42, respectively. These results suggest that Aβ42-mediated toxicity is caused by the turn that favors toxic oligomers, which increase generation of ROS

Palladium and Platinum Nanoparticles Attenuate Aging-Like Skin Atrophy via Antioxidant Activity in Mice

<div><p>Cu-Zn superoxide dismutase (<i>Sod1</i>) loss causes a redox imbalance as it leads to excess superoxide generation, which results in the appearance of various aging-related phenotypes, including skin atrophy. Noble metal nanoparticles, such as palladium (Pd) and platinum (Pt) nanoparticles, are considered to function as antioxidants due to their strong catalytic activity. In Japan, a mixture of Pd and Pt nanoparticles called PAPLAL has been used to treat chronic diseases over the past 60 years. In the present study, we investigated the protective effects of PAPLAL against aging-related skin pathologies in mice. Transdermal PAPLAL treatment reversed skin thinning associated with increased lipid peroxidation in <i>Sod1</i>^−/− mice. Furthermore, PAPLAL normalized the gene expression levels of <i>Col1a1</i>, <i>Mmp2</i>, <i>Has2</i>, <i>Tnf-α</i>, <i>Il-6</i>, and <i>p53</i> in the skin of the <i>Sod1</i>^−/− mice. Pt nanoparticles exhibited marked SOD and catalase activity, while Pd nanoparticles only displayed weak SOD and catalase activity <i>in vitro</i>. Although the SOD and catalase activity of the Pt nanoparticles significantly declined after they had been oxidized in air, a mixture of Pd and Pt nanoparticles continued to exhibit SOD and catalase activity after oxidation. Importantly, a mixture of Pd and Pt nanoparticles with a molar ratio of 3 or 4 to 1 continued to exhibit SOD and catalase activity after oxidation, indicating that Pd nanoparticles prevent the oxidative deterioration of Pt nanoparticles. These findings indicate that PAPLAL stably suppresses intrinsic superoxide generation both <i>in vivo</i> and <i>in vitro</i> via SOD and catalase activity. PAPLAL is a potentially powerful tool for the treatment of aging-related skin diseases caused by oxidative damage.</p></div

PAPLAL improved the transcriptional profiles of skin-related genes in the skin of <i>Sod1</i>^−/− mice.

<p>(A) The relative mRNA expression levels of <i>Col1a1</i>, <i>Has2</i>, <i>Mmp2</i>, <i>Decorin</i>, <i>Ki67</i>, <i>Tnf-α</i>, <i>Il-6</i>, <i>p53</i>, and <i>Mdm2</i>. Each mRNA expression level was determined using qRT-PCR. Data are shown as the mean ± SD; *<i>p</i><0.05 vs. <i>Sod1</i>^+/+, **<i>p</i><0.01 vs. <i>Sod1</i>^+/+, ^#<i>p</i><0.05 vs. <i>Sod1</i>^−/−.</p

PAPLAL is non-toxic in wild-type mice.

<p>(A) Hematoxylin and eosin staining of the skin on the backs of <i>Sod1</i>^+/+ mice (17–20 weeks of age). E, epidermis; D, dermis. The scale bars represent 20 µm (top) or 100 µm (bottom). The thickness of the (B) epidermal and (C) dermal layers of the skin on the backs of <i>Sod1</i>^+/+ mice treated with PAPLAL (n = 5).</p

Pd nanoparticles protected the SOD and catalase activity of Pt nanoparticles against oxidative degradation <i>in vitro</i>.

<p>(A) The SOD and (B) catalase activity of 1.03 mM Pt nanoparticles and PAPLAL that had been stored at room temperature for four weeks. (C) The SOD and (D) catalase activity of Pt nanoparticles and PAPLAL that had been rotated for 24 hours in a tube in order to oxidize the nanoparticles. Data are shown as the mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01.</p

Pt nanoparticles possess SOD and catalase activity.

<p>PAPLAL includes 2.82 mM of Pd nanoparticles and 1.03 mM of Pt nanoparticles. (A) The SOD and (B) catalase activity of 2.82 mM Pd nanoparticles, 1.03 mM Pt nanoparticles, and PAPLAL. Five nM of SOD derived from bovine erythrocytes (A) and 0.2 µM of catalase derived from bovine liver tissue (B) were used as positive controls for SOD and catalase, respectively.</p

Protective effects of PAPLAL against skin atrophy in the <i>Sod1</i>-deficient mice.

<p>(A) Hematoxylin and eosin staining of the skin on the backs of the <i>Sod1</i>^+/+ and <i>Sod1</i>^−/− mice (five months of age). E, epidermis; D, dermis. The scale bars represent 20 µm (top) or 100 µm (bottom). The thickness of the (B) epidermal and (C) dermal layers of the skin on the backs of <i>Sod1</i>^+/+ and <i>Sod1</i>^−/− mice treated with PAPLAL (n = 6–8). (D) 8-isoprostane content of the skin on the backs of <i>Sod1</i>^+/+ and <i>Sod1</i>^−/− mice treated with PAPLAL (n = 5–8). 0.01× and 1× PAPLAL indicate 0.01- or 1-fold concentrations of PAPLAL, respectively. Data are shown as the mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01.</p

Pd nanoparticles protected the SOD activity of Pt nanoparticles against oxidative degradation at various molar ratios.

<p>Pd nanoparticles were added to Pt nanoparticles at various molar ratios, and the SOD activity of each mixture was measured after four weeks of storage at room temperature.</p

PAPLAL attenuates cellular damage in skin.

<p>(A) Relative number of outgrowing fibroblasts in cultured <i>Sod1</i>^+/+ and <i>Sod1</i>^−/− skin specimens. (B) LDH activity in the medium used to culture the <i>Sod1</i>^+/+ and <i>Sod1</i>^−/− skin specimens. Data are shown as the mean ± SD; *<i>p</i><0.05, **<i>p</i><0.01.</p