Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, so far, there are no effective measures to prevent and cure this deadly condition. Ginsenoside Rg1 (Rg1) was shown to improve behavioral abnormalities in AD; however, the potential mechanisms remain unclear. In this study, we pretreated 7-month-old 3xTg-AD mice for 6 weeks with Rg1 and evaluated the effects of Rg1 on the behaviors and the protein expression of hippocampal tissues. The behavioral tests showed that Rg1 could improve the memory impairment and ameliorate the depression-like behaviors of 3xTg-AD mice. Proteomic results revealed a total of 28 differentially expressed hippocampal proteins between Rg1-treated and nontreated 3xTg-AD mice. Among these proteins, complexin-2 (CPLX2), synapsin-2 (SYN2), and synaptosomal-associated protein 25 (SNP25) were significantly downregulated in the hippocampus of 3xTg-AD mice compared with the WT mice, and the treatment of Rg1 modulated the expression of CPLX2 and SNP25 in the hippocampus of 3xTg-AD mice. The expression of CPLX2, SYN2, and SNP25 was further validated by Western blot analysis. Taken together, we concluded that Rg1 could be a potential candidate drug to improve the behavioral deficits in AD via modulating the expression of the proteins (i.e., CPLX2, SYN2, and SNP25).

Connexin43 (Cx43)-mediated gap junctions are vital in maintaining corneal endothelium homeostasis. Tumor necrosis factor-alpha (TNF-α) is among the most important inflammatory factors which cause corneal endothelial dysfunction in various eye diseases. However, the effect of TNF-α on Cx43-mediated gap junctions of the corneal endothelium remains undefined. In the current research, we determined the effect of TNF-α on gap junction intercellular communication (GJIC) in rabbit corneal endothelium. To evaluate alterations of GJIC, if any, we treated ex vivo cultured rabbit corneal endothelium with different concentrations of TNF-α (2-20 ng/ml). The localization of Cx43 was analyzed by immunostaining, while RT-qPCR and western blot were used to profile the expression of Cx43 and zonula occludens-1 (ZO-1). The association between ZO-1 and Cx43 was evaluated using immunoprecipitation and double staining. GJIC activity was determined by the scrap loading and dye transfer assay (SLDT). Our data demonstrated that a high concentration of TNF-α (10 ng/ml and 20 ng/ml) disrupts the Cx43 mediated gap junction distribution in rabbit corneal endothelium and suppresses the expression of Cx43 protein. Furthermore, rabbit corneal endothelial GJIC was inhibited due to the decreased association between the ZO-1 and Cx43 proteins. Current results demonstrate that TNF-α inhibits corneal endothelial GJIC via decreasing the association between ZO-1 and Cx43, disrupting the distribution of Cx43, and downregulating the expression of Cx43 protein. This study offers a new theoretical foundation for diagnosing and treating corneal endothelial cell decompensation induced by elevated TNF-α in various eye diseases.

A number of recent studies have implicated that autophagy was activated by reactive oxygen species (ROS). Our previous report indicated that CLOCK increased the accumulation of ROS under hypoxic conditions. In this study, we investigated the mechanisms by which CLOCK mediated endothelial damage, focusing on the involvement of oxidative damage and autophagy. Overexpression of CLOCK in human umbilical vein endothelial cells (HUVECs) showed inhibition of cell proliferation and higher autophagosome with an increased expression of Beclin1 and LC3-I/II under hypoxic conditions. In contrast, CLOCK silencing reversed these effects. Interestingly, pretreatment with 3-methyladenine (3-MA) resulted in the attenuation of CLOCK-induced cell autophagy and but did not influence the production of intracellular reactive oxygen species (ROS). Furthermore, Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt), a ROS scavenger, significantly attenuated CLOCK-induced cell autophagy. In addition, we found that overexpression of CLOCK had no significant effects on the production of ROS and expression of Beclin1 and LC3-I/II under normoxic conditions in HUVEC. In this present investigation, our results suggested a novel mechanism of action of CLOCK in HUVECs, opening up the possibility of targeting CLOCK for the treatment of vascular diseases.

The human Circadian Locomotor Output Cycle protein Kaput (CLOCK) gene was originally discovered as a regulator of essential human daily rhythms. This seemingly innocuous gene was then found to be associated with a multitude of human malignancies, via several biochemical pathways. We aimed to further investigate the role of hCLOCK in the hypoxia-oxidative stress response system at the biochemical level.

Alzheimer's disease (AD), the most common neurodegenerative disease, has no effective treatment. Dauricine (DAU), a benzyl tetrahydroisoquinoline alkaloid isolated from the root of Menispermum dauricum DC, reportedly has neuroprotective effects in cerebral ischemia. Here, we investigated the effects of DAU on N2a cells stably transfected with Swedish mutant amyloid precursor protein (N2a/APP), an AD-like cell model. ELISA and Western blot analysis revealed that DAU inhibited APP processing and reduced Aβ accumulation. In addition, DAU ameliorated tau hyperphosphorylation via PP2A, p35/25, and CDK5 pathways in N2a/APP cells. The amelioration of tau hyperphosphorylation by DAU was also validated in HEK293/Tau cells, another cell line with tau hyperphosphorylation. Proteomic analysis revealed 85 differentially expressed proteins in the lysates between the wild-type N2a cells (N2a/WT) and the N2a/APP cells in the presence or absence of DAU; these were classified into 6 main categories according to their functions: endoplasmic reticulum (ER) stress-associated proteins, oxidative stress-associated proteins, cytoskeleton proteins, molecular chaperones, mitochondrial respiration and metabolism-related proteins, and signaling proteins. Taken together, we demonstrated that DAU treatment reduces AD-like pathology, thereby suggesting that DAU has potential therapeutic utility in AD.

Depression is one of the most common neuropsychiatric disorders. Although the pathogenesis of depression is still unknown, environmental risk factors and genetics are implicated. Copper (Cu), a cofactor of multiple enzymes, is involved in regulating depression-related processes. Depressed patients carrying the apolipoprotein ε4 allele display more severe depressive symptoms, indicating that ApoE4 is closely associated with an increased risk of depression. The study explored the effect of low-dose Cu exposure and ApoE4 on depression-like behavior of mice and further investigates the possible mechanisms. The ApoE4 mice and wild-type (WT) mice were treated with 0.13 ppm CuCl2 for 4 months. After the treatment, ApoE4 mice displayed obvious depression-like behavior compared with the WT mice, and Cu exposure further exacerbated the depression-like behavior of ApoE4 mice. There was no significant difference in anxiety behavior and memory behavior. Proteomic analysis revealed that the differentially expressed proteins between Cu-exposed and nonexposed ApoE4 mice were mainly involved in the Ras signaling pathway, protein export, axon guidance, serotonergic synapse, GABAergic synapse, and dopaminergic synapse. Among these differentially expressed proteins, immune response and synaptic function are highly correlated. Representative protein expression changes are quantified by western blot, showing consistent results as determined by proteomic analysis. Hippocampal astrocytes and microglia were increased in Cu-exposed ApoE4 mice, suggesting that neuroglial cells played an important role in the pathogenesis of depression. Taken together, our study demonstrated that Cu exposure exacerbates depression-like behavior of ApoE4 mice and the mechanisms may involve the dysregulation of synaptic function and immune response and overactivation of neuroinflammation.

Vascular endothelial dysfunction is considered critical development in the progression of cardiovascular events and is associated with vascular damage and oxidative stress. Previous studies have shown that profilin-1 could be induced by advanced glycation end products (AGEs) and contributes to vascular hyperpermeability; however, the mechanisms are not fully understood. In this study, we sought to assess whether reactive oxygen species (ROS) were involved in profilin-1-mediated RhoA/ROCK1 signaling. Treatment with AGEs significantly induced the expression of profilin-1 and ROS production in human umbilical vein endothelial cells (HUVECs), whereas knockdown of profilin-1 diminished AGE-induced RhoA and ROCK1 activation and ROS production. Moreover, ectopic overexpression of profilin-1 also increased RhoA and ROCK1 activation and ROS production under low AGE concentration. Furthermore, blockage of RhoA/ROCK1 with the inhibitors CT04 and Y27632 significantly attenuated the profilin-1-mediated cell damage and ROS production. Additionally, ROS inhibition resulted in a significant decrease in profilin-1-mediated RhoA/ROCK1 expression, suggesting that the regulation of RhoA/ROCK1 signaling was partly independent of ROS. Taken together, these results suggested that the RhoA/ROCK1 pathway activated by excessive ROS is responsible for profilin-1-mediated endothelial damage.

The deleterious effects of aging on the brain remain to be fully elucidated. In the present study, proteomic changes of young (4-month) and aged (16-month) B6129SF2/J male mouse hippocampus and cerebral cortex were investigated by using nano liquid chromatography tandem mass spectrometry (NanoLC-ESI-MS/MS) combined with tandem mass tag (TMT) labeling technology. Compared with the young animals, 390 hippocampal proteins (121 increased and 269 decreased) and 258 cortical proteins (149 increased and 109 decreased) changed significantly in the aged mouse. Bioinformatic analysis indicated that these proteins are mainly involved in mitochondrial functions (FIS1, DRP1), oxidative stress (PRDX6, GSTP1, and GSTM1), synapses (SYT12, GLUR2), ribosome (RPL4, RPS3), cytoskeletal integrity, transcriptional regulation, and GTPase function. The mitochondrial fission-related proteins FIS1 and DRP1 were significantly increased in the hippocampus and cerebral cortex of the aged mice. Further results in the hippocampus showed that ATP content was significantly reduced in aged mice. A neurotrophin brain-derived neurotrophic factor (BNDF), a protein closely related with synaptic plasticity and memory, was also significantly decreased in the hippocampus of the aged mice, with the tendency of synaptic protein markers including complexin-2, synaptophysin, GLUR2, PSD95, NMDAR2A, and NMDAR1. More interestingly, 8-hydroxydeoxyguanosine (8-OHdG), a marker of DNA oxidative damage, increased as shown by immunofluorescence staining. In summary, we demonstrated that aging is associated with systemic changes involving mitochondrial dysfunction, energy reduction, oxidative stress, loss of neurotrophic factor, synaptic proteins, and ribosomal proteins, as well as molecular deficits involved in various physiological/pathological processes.