Atherosclerosis was considered to induce many vascular-related complications, such as acute myocardial infarction and stroke. Abnormal lipid metabolism and its peroxidation inducing blood–brain barrier (BBB) leakage were associated with the pre-clinical stage of stroke. Dapsone (DDS), an anti-inflammation and anti-oxidation drug, has been found to have protective effects on vascular. However, whether DDS has a protective role on brain microvessels during lipid oxidation had yet to be elucidated. We investigated brain microvascular integrity in a high-fat diet (HFD) mouse model. We designed this study to explore whether DDS had protective effects on brain microvessels under lipid oxidation and tried to explain the underlying mechanism. In our live optical study, we found that DDS significantly attenuated brain microvascular leakage through reducing serum oxidized low-density lipoprotein (oxLDL) in HFD mice (p < 0.001), and DDS significantly inhibited LDL oxidation in vitro (p < 0.001). Our study showed that DDS protected tight junction proteins: ZO-1 (p < 0.001), occludin (p < 0.01), claudin-5 (p < 0.05) of microvascular endothelial cells in vivo and in vitro. DDS reversed LAMP1 aggregation in cytoplasm, and decreased the destruction of tight junction protein: ZO-1 in vitro. We first revealed that DDS had a protective role on cerebral microvessels through preventing tight junction ZO-1 from abnormal degradation by autophagy and reducing lysosome accumulation. Our findings suggested the significance of DDS in protecting brain microvessels under lipid metabolic disorders, which revealed a novel potential therapeutic strategy in brain microvascular-related diseases.

Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na⁺ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA^4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a “denaturation temperature” of ~68°C. Titrating Ca²⁺ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na⁺ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA^4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.

Phospholipid transfer protein (PLTP) is a widely expressed lipid transfer protein participating in the transport of cholesterol and other lipids in the plasma and peripheral tissues. Recently, elevated amyloid β (Aβ) in young and aged PLTP-deficient brains had been reported. However, the role of PLTP in amyloid precursor protein (APP) processing and Alzheimer's disease (AD) pathology remains elusive. Here we first found that deficiency of PLTP accelerated memory dysfunction in APP/PS1ΔE9 AD model mice at the age of 3 months. Further characterization showed that PLTP deficiency increased soluble Aβ peptides, and intracellular accumulation of Aβ was illustrated, which might be due to disrupted APP turnover and the enhanced amyloidogenic pathway. Besides, reduced brain-derived neurotrophic factor (BDNF) was found in PLTP-deficient APP/PS1ΔE9 mice, and the BDNF level was negatively correlated with Aβ42 content, instead of Aβ40 content. In addition, autophagic dysfunction was found in the PLTP-deficient APP/PS1ΔE9 mice. Our data presented a novel model to link phospholipid metabolism to APP processing and also suggested that PLTP played an important role in Aβ metabolism and would be useful to further elucidate functions of PLTP in AD susceptibility.