Yoichi Matsunaga

Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-beta-derived short peptides

Amyloid beta 1-42 (Abeta42) and Abeta17-42 are major constituents of diffuse plaque in brains with Alzheimers disease (AD). We demonstrate the potent cytotoxicity of Abeta42 and Abeta17-42, lesser toxicity of Abeta1-40 (Abeta40) and lack of toxicity of Abeta1-16 (Abeta16) in neuronal cells as measured by inhibition of cell proliferative response using thymidine incorporation assay and that this cytotoxicity can be reduced with Abeta16 and eight-residue Abeta derivatives such as Abeta1-8 and Abeta9-16. FACS analysis also revealed that Abeta16 could dramatically protect against the apoptosis induced by Abeta17-42 with over 80% viable cells. We determined the caspases involved in the Abeta-mediated apoptotic pathway using caspase-specific inhibitors in MTT assays. For all Abetas, the executor was caspase 3, while the initiator was caspase 9 for Abeta42 and caspase 8 for Abeta40 and Abeta17-42. Microscopic observation of lucifer-yellow-labeled neuronal cells demonstrated the occurrence of lysosomal membrane injury of the cells, corresponding to the severe cytotoxic effects of Abeta42. Our findings suggest that the apoptosis of neuronal cells due to Abeta42, Abeta40 and Abeta17-42 is mediated by the different caspase pathways and that this apoptosis can be reduced with the eight-residue Abeta-derived fragments Abeta1-8, Abeta9-16 and Abeta16.

Insights into the Roles of Cathepsins in Antigen Processing and Presentation Revealed by Specific Inhibitors

Abstract Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papainlike cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, α-fodrin, in Sjogrens syndrome and suppresses the Th-1 response and autoantibody production.

A pH-dependent conformational transition of Aβ peptide and physicochemical properties of the conformers in the glial cell

In the present study we identified the epitopes of antibodies against amyloid beta-(1-42)-peptide (Abeta1-42): 4G8 reacted with peptides corresponding to residues 17-21, 6F/3D reacted with peptides corresponding to residues 9-14, and anti 5-10 reacted with peptides corresponding to residues 5-10. The study also yielded some insight into the Abeta1-42 structures resulting from differences in pH. An ELISA study using monoclonal antibodies showed that pH-dependent conformational changes occur in the 6F/3D and 4G8 epitopes modified at pH 4.6, but not in the sequences recognized by anti 1-7 and anti 5-10. This was unique to Abeta1-40 and Abeta1-42 and did not occur with Abeta1-16 or Abeta17-42. The reactivity profile of 4G8 was not affected by blockage of histidine residues of pH-modified Abeta1-40 and Abeta1-42 with diethyl pyrocarbonate; however, the mutant [Gln(11)]Abeta1-40 abrogated the unique pH-dependence towards 4G8 observed with Abeta1-40. These findings suggest that these epitopes are cryptic at pH 4.6, and that Glu(11) is responsible for the changes. We suggest that the abnormal folding of 6F/3D epitope affected by pH masked the 4G8 epitope. A study of the binding of metal ions to Abeta1-42 suggested that Cu(2+) and Zn(2+) induced a conformational transition around the 6F/3D region at pH 7.4, but did not affect the region when it was modified at pH 4.6. However, Fe(2+) had no effect, irrespective of pH. Abeta modified at pH 4.6 appeared to be relatively resistant to proteinase K compared with Abetas modified at pH 7.4, and the former might be preferentially internalized and accumulated in a human glial cell. Our findings suggest the importance of microenvironmental changes, such as pH, in the early stage of formation of Abeta aggregates in the glial cell.

Conformational Changes Preceding Amyloid-fibril Formation of Amyloid-beta and Stefin B; Parallels in pH Dependence

Amyloid beta (A beta) protein is the key component of amyloid plaques in Alzheimers disease brain whereas stefin B is an intracellular cysteine proteinase inhibitor, broadly distributed in different tissue and recently reported to form amyloid fibrils in vitro. By reducing the pH to 4.6, the native conformation of both polypeptides are changed into less ordered metastable intermediates that are stabilized by formation of the more stable fibrils. In A beta, the Glu at position 11 was found to be responsible for the conformational change at pH 4.6. Metal ions, including copper and zinc, could also induce conformational changes of A beta at neutral pH. The acid modified A beta conformer exhibited protease K resistance, preferential internalization and accumulation in the human glial cells. In stefin B, reducing the pH to pH 3.3 results in another intermediate of the molten-globule type which also leads to amyloid fibril formation. Multiple sequence alignment revealed distinct similarities of A beta (1-42) peptide, stefin B (13 to 61 residues) and prion fragment (90 to 144 residues).

Eight-residue Aβ peptides inhibit the aggregation and enzymatic activity of Aβ42

Insoluble Aβ1–42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Aβ1–42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimers disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Aβ1–42 to inhibit the acid-induced aggregation of Aβ1–42 and identified the potent peptides to be Aβ15–22, Aβ16–23 and Aβ17–24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Aβ1–42 against casein at different pHs. Chemical modification of amino acid residues in Aβ1–42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29–42 are required to stabilize the conformer. A study of metal ions suggested that Cu2+ affected the enzymatic activity, but Zn2+ and Fe2+ did not. Interestingly, Aβ14–21 and Aβ15–22 were the only peptides that inhibited the proteolytic activity of Aβ42. Therefore, Aβ15–22 may control both aggregation of Aβ1–42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimers disease.

Regulation of lymphocyte proliferation by eosinophils via chymotrypsin-like protease activity and adhesion molecule interaction.

We investigated the regulatory mechanisms responsible for release of eosinophil cationic protein (ECP) from eosinophils activated by platelet‐activating factor (PAF) and monitored intra‐cellular pH (pHi) changes using a pH‐sensitive fluorescent probe. We also explored the mechanisms by which eosinophils suppress T‐lymphocyte proliferation induced by phytohaemagglutinin (PHA). In these experiments, a separated culture to investigate the ECP‐mediated pathway and a coculture to identify the adhesion molecules involved in eosinophil‐lymphocyte interactions were employed. Chymostatin (1×10−6 M) inhibited ECP release by about 50% via stimulation by PAF or recombinant interleukin 5(rIL‐5) plus IgG. PAF (1×10−7 M) raised eosinophil pHi from 6.9 to 7.3 within 20 s and pretreatment of these cells with chymostatin (1×10−6 M), but not with leupeptin or E64‐d, completely prevented this increase. Calcium ionophore A23187 (1×10−7 M) induced ECP release and raised pHi to within a range similar to that of PAF, however, chymostatin had no effect on either. Chymostatin reversed ECP‐mediated suppression of PHA‐induced T‐lymphocyte proliferation in separated cultures, but not in cocultures. In coculture, eosinophils exhibited the same level of suppression of both CD4+ and CD8+ T‐cell proliferation in response to PHA. Monoclonal antibodies against CD11a, CD18 and CD54, but not CD11b, restored eosinophil suppression of T‐lymphocyte proliferation which was chymostatin‐resistant in coculture. Eosinophils were unable to suppress the proliferative response to lymphocytes to anti‐CD3 stimulation. In conclusion, chymostatin specifically inhibited both the eosinophil pHi increase and ECP release induced by PAF. Eosinophils regulate PHA‐induced T‐lymphocyte proliferation via the ECP‐mediation associated with chymotrypsin‐like protease activity. These cells also control interactions with lymphocyte between adhesion molecules, CD11a, CD18 and CD54.

Vizantin Inhibits Endotoxin-Mediated Immune Responses via the TLR 4/MD-2 Complex

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1β induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride–conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1β and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.

Reversal of temperature-induced conformational changes in the amyloid-beta peptide, Aβ40, by the β-sheet breaker peptides 16–23 and 17–24

Background and purpose:  Aggregates of the protein amyloid‐beta (Aβ) play a crucial role in the pathogenesis of Alzheimers disease (AD). Most therapeutic approaches to AD do not target Aβ, so determination of the factor(s) that facilitate aggregation and discovering agents that prevent aggregation have great potential therapeutic value.

Effects of 8-Residue Beta Sheet Breaker Peptides on Aged Aβ40 – Induced Memory Impairment and Aβ40 Expression in Rat Brain and Serum Following Intraamygdaloid Injection

Amyloidβ-protein (Aβ) assembly into toxic fibrillar structures is seminal in development of senile plaques, the pathological hallmark of Alzheimers disease. Blocking this process could have a therapeutic value. β-sheet breaker peptides (βSBP) decrease Aβ fibrillogenesis and neurotoxicity by preventing or dissolving misfolded Aβ aggregates. The present study investigated the effects of βSBPs on Aβ40-related neuropathology, memory impairment in 8-armed radial maze and expression of Aβ40 in brain and serum. Aβ40 was injected into amygdaloid nucleus followed 8 days later by octapeptideβSBPs 15-22, 16-23 and 17-24. Aβ40 was detected not only in amygdala, but also in serum. Aβ40 induced cellular changes in amygdala and additionally in hippocampus. Aβ40 decreased correct choices, whereas increased errors (both number of arms revisited and total number of revisits) and latency of completing the maze test. The βSBPs decreased Aβ40-induced pathological changes, memory impairment and Aβ40 expression in serum. The βSBP15-22 distinctively decreased the total errors on day 14. The present results show that octapeptide βSBPs corrected Aβ40-induced memory impairment, and support investigation of βSBPs as a promising treatment of diseases characterized by neurodegeneration and memory impairment such as Alzheimers disease.

Novel neurotrophic phenylbutenoids from Indonesian ginger Bangle, Zingiber purpureum.

Two new curcuminoids 1 and 2, and a new phenylbutenoid dimer 3, were isolated from Bangle (Zingiber purpureum). Their structures were determined on the basis of comprehensive spectroscopic data and their biogenetic pathway. Compounds 1 and 2 are the first example of curcumin coupled with phenylbutenoid. Compounds 1 and 2 promoted neurite outgrowth of NGF-mediated PC12 cells at concentrations ranging from 1 to 10 μM. In addition, compound 1 was found to accelerate the prevention of Aβ42 aggregation.