N-terminal Domain of Amyloid-ß Impacts Fibrillation and Neurotoxicity.

Alzheimer's disease is characterized by the presence of distinct amyloid-ß peptide (Aß) assemblies with diverse sizes, shapes, and toxicity. However, the primary determinants of Aß aggregation and neurotoxicity remain unknown. Here, the N-terminal amino acid residues of Aß42 that distinguished between humans and rats were substituted. The effects of these modifications on the ability of Aß to aggregate and its neurotoxicity were investigated using biochemical, biophysical, and cellular techniques. The Aß-derived diffusible ligand, protofibrils, and fibrils formed by the N-terminal mutational peptides, including Aß42(R5G), Aß42(Y10F), and rat Aß42, were indistinguishable by conventional techniques such as size-exclusion chromatography, negative-staining transmission electron microscopy and silver staining, whereas the amyloid fibrillation detected by thioflavin T assay was greatly inhibited in vitro. Using circular dichroism spectroscopy, we discovered that both Aß42 and Aß42(Y10F) generated protofibrils and fibrils with a high proportion of parallel ß-sheet structures. Furthermore, protofibrils formed by other mutant Aß peptides and N-terminally shortened peptides were incapable of inducing neuronal death, with the exception of Aß42 and Aß42(Y10F). Our findings indicate that the N-terminus of Aß is important for its fibrillation and neurotoxicity.

Endocytosis Is a Key Mode of Interaction between Extracellular ß-Amyloid and the Cell Membrane.

Interactions between amyloid-ß peptide (Aß) and the cell membrane include interaction with membrane lipids and binding to membrane receptors, both of which are considered to be the toxicity mechanisms of Aß. However, it is unclear whether both mechanisms lead to cytotoxicity. Thus, we aimed to analyze these two mechanisms of Aß42 interaction with cell membranes under different Aß aggregation states. To this end, model membrane experiments were conducted. Quantitative analysis of Aß42 monomers or oligomers bound to the membrane of neuro-2a cells was also performed, and laser confocal microscopy was employed to assess endocytosis of FITC-Aß42 monomers or oligomers by neuro-2a cells. We found that the binding capacity of Aß42 to membrane lipids was weak and that the amount of Aß42 bound to membrane lipids was low. Moreover, clathrin-mediated endocytosis of Aß42 oligomers by neuro-2a cells was observed. Endocytosis serves as a key mode of interaction between extracellular Aß42 and neurons. These findings provide insights into the mechanisms underlying Aß oligomer metabolism.

LncRNA-MALAT1 promotes neovascularization in diabetic retinopathy through regulating miR-125b/VE-cadherin axis.

Background: Diabetic retinopathy (DR) is currently the leading cause of blindness and visual disability in adults with diabetes mellitus (DM). Neovascularization has been identified as an important clinical property in DR, however, the exact mechanisms in DR neovascularization are still unclear and need further elucidation.Methods: Quantitative real-time PCR (qRT-PCR) was conducted to detect the expression level of long non-coding RNA (lncRNA)-metastasis associated lung adenocarcinoma transcript 1 (MALAT1), miR-125b and vascular endothelial-cadherin (VE-cadherin) in human retina microvascular endothelial cells (hRMECs) treated with high glucose (HG). Luciferase assay was used to detect interaction of MALAT1 with miR-125b and miR-125b with VE-cadherin. MTT assay, transwell assay, tube formation assay and vascular permeability assay were conducted to detect the cell viability, migration tube formation ability and permeability of hRMECs, respectively. ELISA was used to examine the release of VE-cadherin and vascular endothelial growth factor (VEGF). Western blotting was used to access the protein expression of VE-cadherin, VEGF, ß-catenin, matrix metalloproteinase (MMP) 2 (MMP2) and MMP9.Results: MALAT1 and VE-cadherin were up-regulated while miR-125b was down-regulated in hRMECs treated with HG. MALAT1 could competitively bind to miR-125b against VE-cadherin at the site of 3'-untranslated region (3'-UTR), leading to the up-regulation of VE-cadherin. Knockdown of MALAT1 inhibited the proliferation, migration, tube formation and vascular permeability of hRMECs induced by HG through up-regulating miR-125b. Furthermore, we found the deletion of MALAT1 suppressed the VE-cadherin/ß-catenin complex and neovascularization related proteins expression, which was up-regulated by HG.Conclusion: Knockdown of MALAT1 inhibited cell proliferation, migration and angiogenesis of hRMECs via suppressing the VE-cadherin/ß-catenin complex through targeting miR-125b. Inhibition of MALAT1 may serve as a potential target for anti-angiogenic therapy for DR.

Endosomal pH favors shedding of membrane-inserted amyloid-ß peptide.

Amyloid-ß peptides (Aßs) are generated in a membrane-embedded state by sequential processing of amyloid precursor protein (APP). Although shedding of membrane-embedded Aß is essential for its secretion and neurotoxicity, the mechanism behind shedding regulation is not fully elucidated. Thus, we devised a Langmuir film balance-based assay to uncover this mechanism. We found that Aß shedding was enhanced under acidic pH conditions and in lipid compositions resembling raft microdomains, which are directly related to the microenvironment of Aß generation. Furthermore, Aß shedding efficiency was determined by the length of the C-terminal membrane-spanning region, whereas pH responsiveness appears to depend on the N-terminal ectodomain. These findings indicate that Aß shedding may be directly coupled to its generation and represents an unrecognized control mechanism regulating the fate of membrane-embedded products of APP processing.

Attenuation of periostin in retinal Müller glia by TNF-α and IFN-γ.

AIM: To investigate the regulation and mechanisms of periostin expression in retinal Müller glia, and to explore the relevance to retinal neovascularization. METHODS: The oxygen-induced retinopathy (OIR) mouse model and the human Moorfield/Institute of Ophthalmology-Müller 1 (MIO-M1) cell line were used in the study. Immunofluorescence staining was used to determine the distribution and expression of periostin and a Müller glial cell marker glutamine synthetase (GS). Cytokines TNF-α and IFN-γ were added to stimulate the MIO-M1 cells. ShRNA was used to knockdown periostin expression in MIO-M1 cells. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was conducted to assess the mRNA expression of periostin. RESULTS: Immunofluorescence staining showed that periostin was expressed by MIO-M1 Müller glia. GS-positive Müller glia and periostin increased in OIR retinas, and were partially overlaid. The stimulation of TNF-α and IFN-γ reduced the mRNA expression of periostin significantly and dose-dependently in MIO-M1 cells. Knockdown of periostin reduced mRNA expression of vascular endothelial growth factor A (VEGFA) in MIO-M1 cells, while VEGFA expression was not changed in periostin knock-out OIR retinas. CONCLUSION: Müller glia could be one of the main sources of periostin in the retina, and might contribute to the pathogenesis of retinal neovascularization. Proinflammatory cytokines TNF-α and IFN-γ attenuate the periostin expression in retinal Müller glia, which provides a potential and novel method in treating retinal neovascular diseases.

C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

Previous studies have reported the pathogenic role of C-reactive protein (CRP) during diabetic kidney disease (DKD) in human CRP transgenic and Crp-/- mice. However, because humans and mice have inverse acute phase expression patterns of CRP and serum amyloid P component, this could lead to the inaccurate evaluation of CRP function with the above-mentioned CRP transgenic mouse. But different from mice, rats have the same acute phase protein expression pattern as human, which might avoid this problem and be a better choice for CRP function studies. To dispel this doubt and accurately define the role of CRP during diabetic nephropathy, we created the first Crp-/- rat model, which we treated with streptozocin to induce DKD for in vivo studies. Moreover, an established cell line (human kidney 2) was used to further investigate the pathologic mechanisms of CRP. We found that CRP promotes epithelial-mesenchymal transition (EMT) through Wnt/ß-catenin and ERK1/2 signaling, which are dependent on CRP binding to FcγRII on apoptotic cells. By promoting EMT, CRP was demonstrated to accelerate the development of DKD. We thus present convincing evidence demonstrating CRP as a therapeutic target for DKD treatment.-Zhang, L., Shen, Z.-Y., Wang, K., Li, W., Shi, J.-M., Osoro, E. K., Ullah, N., Zhou, Y., Ji, S.-R. C-reactive protein exacerbates epithelial-mesenchymal transition through Wnt/ß-catenin and ERK signaling in streptozocin-induced diabetic nephropathy.

The role of microglia in the progression of glaucomatous neurodegeneration- a review.

Glaucoma is a serious leading cause of irreversible blindness worldwide. Reducing intraocular pressure (IOP) does not always stop glaucomatous neurodegeneration and the optic nerve may continue to be damaged in the normal IOP. Microglial activity has been recognized to play essential roles in pathogenesis of the central nervous system (CNS) as well as retinal ganglion cell (RGC) survival. The relationship between the neurodegeneration and the microglia cells in glaucoma is very complicated and still remains unclear. In the present review, we summarize the recent studies of mechanisms of microglia in glaucoma neurodegeneration, which might provide new ways to treat glaucoma.

Dissecting the behaviour of ß-amyloid peptide variants during oligomerization and fibrillation.

The oligomerization and fibrillation of ß-amyloid (Aß) peptides are important events in the pathogenesis of Alzheimer's disease. However, the motifs within the Aß sequence that contribute to oligomerization and fibrillation and the complex interplay among these short motifs are unclear. In this study, the oligomerization and fibrillation abilities of the Aß variants Aß1-28, Aß1-36, Aß11-42, Aß17-42, Aß1-40 and Aß1-42 were examined by thioflavin T fluorescence, western blotting and transmission electron microscopy. Compared with two C-terminal-truncated peptides (i.e. Aß1-28 and Aß1-36), Aß11-42, Aß17-42 and Aß1-42 had stronger abilities to form oligomers. This indicated that amino acids 37-42 strengthen the ß-hairpin structure of Aß. Both Aß1-42 and Aß1-40 could form fibres, but Aß17-42 formed irregular fibres, suggesting that amino acids 1-17 were essential for Aß fibre formation. Aß1-28 and Aß1-36 exhibited weak oligomerization and fibrillation, implying that they formed an unstable ß-hairpin structure owing to the incomplete C-terminal region. Intermediate peptides were likely to form a stable structure, consistent with previous results. This work explains the roles and interplay among motifs within Aß during oligomerization and fibrillation. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Bis(sulfosuccinimidyl) suberate (BS3) crosslinking analysis of the behavior of amyloid-ß peptide in solution and in phospholipid membranes.

The structure and state of amyloid-ß peptide (Aß) oligomers often need to be checked by reliable experimental methods. Electrophoresis is a commonly applied measurement method. However, due to the presence of detergents, oligomers are easily broken during electrophoresis, which makes it very hard to accurately assess Aß aggregate states. In the current study, bis(sulfosuccinimidyl) suberate (BS3) was used to cross-link Aß1-42 oligomers prior to electrophoresis. When compared to a previously reported Aß cross-linking agent, glutaraldehyde, it was quite apparent that BS3 is more suitable for detecting intra-membrane Aß oligomers and extra-membrane Aß oligomers states. As such, our findings provide an efficient method for analyzing Aß proteins or other proteins that are easily aggregated in solution and in phospholipid membranes.

BACE1 in the retina: a sensitive biomarker for monitoring early pathological changes in Alzheimer's disease.

Because of a lack of sensitive biomarkers, the diagnosis of Alzheimer's disease (AD) cannot be made prior to symptom manifestation. Therefore, it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD. While brain lesions are a major feature of AD, retinal pathological changes also occur in patients. In this study, we investigated the temporal changes in ß-site APP-cleaving enzyme 1 (BACE1) expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD. APP/PS-1 transgenic mice, 3, 6 and 8 months of age, were used as an experimental group, and age-matched C57/BL6 wild-type mice served as the control group. In the Morris water maze test, there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages. Compared with wild-type mice, no changes in learning or memory abilities were detected in transgenic mice at 3 months of age. However, compared with wild-type mice, the escape latency was significantly increased in transgenic mice at 6 months, starting on day 3, and at 8 months, starting on day 2, during Morris water maze training. In addition, the number of crossings of the target area was significantly decreased in transgenic mice. The learning and memory abilities of transgenic mice were further worsened at 8 months of age. Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3, 6 or 8 months or in transgenic mice at 3 months, but they were clearly found in the entorhinal cortex, hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months. BACE1 expression was not detected in the retina of wild-type mice at 3 months, but weak BACE1 expression was detected in the ganglion cell layer, inner plexiform layer and outer plexiform layer at 6 and 8 months. In transgenic mice, BACE1 expression in the ganglion cell layer was increased at 3 months, and BACE1 expression in the ganglion cell layer, inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months, compared with age-matched wild-type mice. Taken together, these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits. Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice, and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.

Tibetan medicine "RNSP" in treatment of Alzheimer disease.

Alzheimer disease (Alzheimer Disease, AD) is one of the most common type in senile dementia. Its main pathological features were that a large number of senile plaques gathered in brain extracellular and tangles fibrosis appeared in nerve cells. Currently, the pathogenesis of AD is still uncertain, and scale investigation and combined brain CT, MRI data were analyzed mainly for clinical diagnosis. Mitigation and improvement of the nervous system activity to interfere with the subsequent behavior of the patients are the main methods for treatment. In clinical no drug can really prevent and cure AD. From the view point of Tibetan medicine studies, Tibetan medicine RNSP has effect on improving memory and repairing the neurons in the brain. In this study, we combined the characteristics of AD pathology, pathogenesis, diagnosis and treatment methods to explore the feasibility of Tibetan medicine RNSP for the treatment of AD to provide new ideas for the diagnosis and treatment of AD.

New options for uveitis treatment.

Uveitis is one of the most important causes of blindness worldwide. Its etiology and pathogenesis are complicated and have not been well understood. The treatment for uveitis is predominantly based on steroids and immunosuppressants. However, systemic side effects limit their clinical application. With the advancement of molecular biology, some intravitreal implants and biologic agents have been used for the treatment of uveitis. Additionally, novel techniques such as gene therapy and RNA interference are being studied for using as uveitis therapy. This paper reviews recent advances in uveitis treatment.

Alpha lipoic acid protects lens from H(2)O(2)-induced cataract by inhibiting apoptosis of lens epithelial cells and inducing activation of anti-oxidative enzymes.

OBJECTIVE: To determine whether alpha lipoic acid (LA) can effectively protect lenses from hydrogen peroxide (H2O2)-induced cataract. METHODS: Lens from adult Sprague-Dawley rats were cultured in 24-well plates and treated without or with 0.2 mM of H2O2, 0.2 mM of H2O2 plus 0.5 mM, 1.0 mM, or 2.0 mM of LA for 24 h. Cataract was assessed using cross line grey scale measurement. Superoxide dismutase (SOD), glutathione (GSH-Px), lactate dehydrogenase (LDH), and malondialdehyde (MDA) activity or level in lens homogenates was measured. Apoptosis of lens epithelial cells in each group were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay. RESULTS: A total of 0.2 mM of H2O2 induced obvious cataract formation and apoptosis in lens' epithelial cells, but 0.5-2.0 mM of LA could block the effect of 0.2 mM H2O2 in inducing cataract and apoptosis. Furthermore, 0.2 mM of H2O2 significantly decreased SOD, GSH-Px, and LDH activity and significant increased MDA level in the lens, but 0.5-2.0 mM of LA blocked the effect of 0.2 mM H2O2. One mM of LA was found to be the most effective. CONCLUSIONS: LA can protect lens from H2O2-induced cataract. LA exerts protective effects through inhibition of lens' epithelial cell apoptosis and activation of anti-oxidative enzymes.

Descemet membrane detachment after trabeculectomy.

Descemet's membrane detachment (DMD) can be a potentially serious complication of intraocular surgery or ocular trauma. The cause is not very clear. We are trying to remind an awareness of the spectrum of DMD resulting from trabeculectomy by presenting a case of extensive DMD after trabeculectomy which was successfully repaired.

Selective laser trabeculoplasty.

The introduction of selective laser trabeculoplasty (SLT) provided a new choice for the reduction of intraocular pressure (IOP) in eyes with open angle glaucoma (OAG) and ocular hypertension (OHT). SLT was demonstrated equally as effective as topical medical therapy and argon laser trabeculoplasty (ALT) to lower IOP. It is a potentially repeatable procedure because of the lack of coagulation damage to the trabecular meshwork (TM) and also effect in patients with previously failed ALT. SLT can be used to treat patients with OAG, pseudoexfoliation glaucoma, pigmentary glaucoma, normal-tension glaucoma, OHT, juvenile glaucoma, pseudophakic and aphakic glaucoma. Furthermore, SLT can be considered as a primary treatment option in patients who cannot tolerate or are noncompliant with medications, while not interfering with the success of future surgery. Its safety profiles include mild and transient inflammation, ocular pain and a small risk of moderate IOP elevations after the procedure. SLT is a safe and effective means of IOP reduction in eyes with OAG and OHT.

Intra-membrane oligomerization and extra-membrane oligomerization of amyloid-ß peptide are competing processes as a result of distinct patterns of motif interplay.

Soluble oligomers of amyloid-ß peptide (Aß) are emerging as the primary neurotoxic species in Alzheimer disease, however, whether the membrane is among their direct targets that mediate the downstream adverse effects remains elusive. Herein, we show that multiple soluble oligomeric Aß preparations, including Aß-derived diffusible ligand, protofibril, and zinc-induced Aß oligomer, exhibit much weaker capability to insert into the membrane than Aß monomer. Aß monomers prefer incorporating into membrane rather than oligomerizing in solution, and such preference can be reversed by the aggregation-boosting factor, zinc ion. Further analyses indicate that the membrane-embedded oligomers of Aß are derived from rapid assembly of inserted monomers but not due to the insertion of soluble Aß oligomers. By comparing the behavior of a panel of Aß truncation variants, we demonstrate that the intra- and extra-membrane oligomerization are mutually exclusive processes that proceed through distinct motif interplay, both of which require the action of amino acids 37-40/42 to overcome the auto-inhibitory interaction between amino acids 29-36 and the N-terminal portion albeit via different mechanisms. These results indicate that intra- and extra-membrane oligomerization of Aß are competing processes and emphasize a critical regulation of membrane on the behavior of Aß monomer and soluble oligomers, which may determine distinct neurotoxic mechanisms.

A redox switch in C-reactive protein modulates activation of endothelial cells.

C-reactive protein (CRP) has been implicated in the regulation of inflammation underlying coronary artery disease; however, little is known about the molecular mechanisms responsible for the expression of its pro- or anti-inflammatory activities. Here, we have identified the intrasubunit disulfide bond as a conserved switch that controls the structure and functions of CRP. Conformational rearrangement in human pentameric CRP to monomeric CRP (mCRP) is the prerequisite for this switch to be activated by reducing agents, including thioredoxin. Immunohistochemical analysis revealed 36-79% colocalization of thioredoxin and mCRP in human advanced coronary atherosclerotic lesions. Nonreduced mCRP was largely inert in activating human coronary artery endothelial cells (HCAECs), whereas reduced or cysteine-mutated mCRP evoked marked release of IL-8 and monocyte chemoattractant protein-1 from HCAECs, with ~50% increase at a concentration of 1 µg/ml. Reduced mCRP was ~4 to 40-fold more potent than mCRP in up-regulating adhesion molecule expression, promoting U937 monocyte adhesion to HCAECs, and inducing cytokine release from rabbit arteries ex vivo and in mice. These actions were primarily due to unlocking the lipid raft interaction motif. Therefore, expression of proinflammatory properties of CRP on endothelial cells requires sequential conformational changes, i.e., loss of pentameric symmetry followed by reduction of the intrasubunit disulfide bond.