Yasuhide Okamoto

Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse.

BackgroundSeveral lines of evidence indicate that memory loss represents a synaptic failure caused by soluble amyloid β (Aβ) oligomers. However, the pathological relevance of Aβ oligomers (AβOs) as the trigger of synaptic or neuronal degeneration, and the possible mechanism underlying the neurotoxic action of endogenous AβOs remain to be determined.ResultsTo specifically target toxic AβOs in vivo, monoclonal antibodies (1A9 and 2C3) specific to them were generated using a novel design method. 1A9 and 2C3 specifically recognize soluble AβOs larger than 35-mers and pentamers on Blue native polyacrylamide gel electrophoresis, respectively. Biophysical and structural analysis by atomic force microscopy (AFM) revealed that neurotoxic 1A9 and 2C3 oligomeric conformers displayed non-fibrilar, relatively spherical structure. Of note, such AβOs were taken up by neuroblastoma (SH-SY5Y) cell, resulted in neuronal death. In humans, immunohistochemical analysis employing 1A9 or 2C3 revealed that 1A9 and 2C3 stain intraneuronal granules accumulated in the perikaryon of pyramidal neurons and some diffuse plaques. Fluoro Jade-B binding assay also revealed 1A9- or 2C3-stained neurons, indicating their impending degeneration. In a long-term low-dose prophylactic trial using active 1A9 or 2C3 antibody, we found that passive immunization protected a mouse model of Alzheimers disease (AD) from memory deficits, synaptic degeneration, promotion of intraneuronal AβOs, and neuronal degeneration. Because the primary antitoxic action of 1A9 and 2C3 occurs outside neurons, our results suggest that extracellular AβOs initiate the AD toxic process and intraneuronal AβOs may worsen neuronal degeneration and memory loss.ConclusionNow, we have evidence that HMW-AβOs are among the earliest manifestation of the AD toxic process in mice and humans. We are certain that our studies move us closer to our goal of finding a therapeutic target and/or confirming the relevance of our therapeutic strategy.

Disease Modifying Therapies for Alzheimer's Disease Targeting Aβ Oligomers: Implications for Therapeutic Mechanisms

Several lines of evidence indicate that amyloid β (Aβ), particularly Aβ oligomers (AβOs), plays a causative role in Alzheimers disease. However, the mechanisms underlying the action of an anti-AβO antibody to clarify the toxic action of AβOs remain elusive. Here, we showed that the anti-AβO antibody (monoclonal 72D9) can modify the Aβ aggregation pathway. We also found that 72D9 directly sequesters both extracellular and intraneuronal AβOs in a nontoxic state. Thus, therapeutic intervention targeting AβOs is a promising strategy for neuronal protection in Alzheimers disease.

The efficacy of anti-beta-amyloid oligomers antibody in mouse models of Alzheimer's disease

IgG2a, produced from splenocytes and a predominant IgG1 antibody secretion (Th2) from cultured bone marrow cells. In DNA primed/peptide boosted and peptide prime/DNA boosted mice high levels of plasma antibodies were present but whereas the spleens or DNA primed/peptide boosted mice showed high levels of antibody producing cells; the number of antibody producing cells found in the spleens from peptide primed/ DNA boosted micewas comparable to the levels found in DNAAb42 trimer immunized mice. Thus the later immunization is likely to direct antibody producing cells into the spleens or bone marrow respectively. Conclusions: These data indicate that the bone marrow may be an important reservoir for B cells following DNAAb42 immunization and is in line with studies showing that the bone marrow represents an excellent niche for survival of long lived plasma cells and a lifetime source for antibody producing B cells which are independent of continuous antigen specific stimulation. In the context of active Ab1-42 immunization, the presence of long lived antibody secreting plasma cells might add another important fact to proceed with this DNA Ab42 immunotherapy for AD patients.

Research and development of anti-Abeta oligomers antibodies for the treatment of Alzheimer's disease

fold, respectively. These observations strongly suggest that TUDCA should be further evaluated as a potential therapeutic strategy in AD. In this study, we hypothesize that TUDCA reduces Aß toxicity by interfering with its production and/or accumulation. We further propose that one potential mechanism is associated with modulation of Aß endocytosis, which may play a critical part in learning/memory processes. Methods: Cell culture. Human SH-SY5Y cells were pretreated with 100 mM TUDCA (Sigma) for 12 h, and then incubated with either 10 mM Aß (1-40) or Aß (1-42) for 1-24h. Immunofluorescence. Cells were fixed and immunostained with a 1:1 mixture of anti-Aß antibody 6E10 and 4G8 (1:1000, Covance) followed by staining with anti-mouse Alexa Fluor 568-conjugated secondary antibody (1:200; Invitrogen), and with the actin dye Alexa Fluor 488-conjugated phalloidin (1:5000; Invitrogen). Semi-quantitative RT-PCR. Total RNA from SH-SY5Y cells was isolated with Trizol (Invitrogen) and subjected to RT-PCR with the Superscript II kit (Invitrogen). PCR was performed with primers for human LR11, CTGF, and ß-actin as internal control. Transgenic mice and TUDCA treatment. Male APP/PS1 double-transgenic mice and wild-type littermates were randomly assigned into four groups: TUDCA-treated transgenic mice, untreated (control) transgenic mice, TUDCA-treated wild-type mice, and untreated (control) wild-type mice. TUDCAtreated groups received a diet supplemented with 0.4% TUDCA, sodium salt. Treatment was started when the mice were 2 months old and was continued for 6 months. At the end of the treatment period animals were tested in several well-established learning and memory tasks, after which brains were processed for immunohistochemistry studies. Learning and memory tests. Morris water maze, contextual fear learning, social preference, and passive avoidance test. Immunohistochemistry. Paraffin-embedded coronal brain sections were deparaffined, rehydrated and endogenous peroxidase quenched with hydrogen peroxide. They were then incubated for 60 min in blocking buffer and subsequently in appropriately diluted primary antibodies overnight at 4 C. After rinsing, the primary antibody was developed by incubating with anti-mouse cyanine 2 (Cy2)-conjugated secondary antibody (Jackson) for 1h at room temperature or by incubating with anti-mouse biotinylated secondary antibody (Santa Cruz Biotechnology), for 30 min at room temperature, plus extravidin-HRP-conjugated (Santa Cruz Biotechnology) for 30 min at room temperature. This was followed by DAB (Sigma) using the instructions of the manufacturer for peroxidase labeling. Primary antibodies used were: mouse monoclonal anti-Ab (1:1000; 6E10; Signet) and mouse monoclonal anti-GFAP (1:400; GA4; Chemicon). Thioflavin (0.05%; Sigma) staining was performed for 8 min. Results: Exposure of SH-SY5 Y cells to Ab resulted in increased levels of apoptosis, which correlated with Ab entry into the cell as assessed by confocal microscopy. Importantly, TUDCA pretreatment markedly reduced apoptosis and intracellular Ap. In addition, TUDCA increased LR11 expression while decreasing CTGF mRNA. In vivo, APP/PS1 male mice were fed a diet containing TUDCA for a period of 6 months, and tested at 8 months of age on several behavior tasks measuring learning and memory. TUDCA rescued different memory types in transgenic mice compared to control transgenic mice (e.g. p < 0.001 in the passive avoidance test). No differences were observed in control and TUDC A-treated wild-type mice. We further tested the effect of TUDCA on Ab deposition in APP/PS1 mice. Thioflavin staining and Ab immunohistochemistry studies demonstrated a clear abundance of Ab plaques in the brains of control transgenic mice, whereas Ab deposits were absent in both control and TUDCA-treated wild-type mice. Importantly, results obtained showed a marked reduction of Ab plaque number in hippocampus and frontal cortex in brains of transgenic mice treated with TUDCA compared to control transgenic mice (p 1⁄4 0.003 and p 1⁄4 0.015, respectively). Further, activated astrocytes were visualized in APP/PS1 brain sections by GFAP immunofluorescence. GFAP staining was significantly reduced in TUDCA-fed transgenic mice compared to control transgenic mice (p 1⁄4 0.031), suggesting that inflammation is also modulated by TUDCA. No differences were observed between TUDCA-treated transgenic mice, TUDCA-treated wild-type mice and control wild-type mice, and all were significantly different from control transgenic mice. Conclusions: Although TUDCA has been shown neuroprotective properties in several models of disease, including neuronal exposure to Ab, the therapeutic role of TUDCA in AD pathology and dementia has not yet been ascertained. Our findings indicate that TUDCA treatment inhibits accumulation of Ab deposits in APP/PS1 double-transgenic mice. Furthermore, TUDCA significantly reduced the inflammatory process characteristic of the transgenic AD mouse model, as assessed by the decreased levels of astrocytic activation in TUDCA-fed APP/PS1 mice. These observations correlated with a clear rescue of different memory types in TUDCA-treated transgenic mice compared to control transgenic mice. While future studies are needed to address the exact TUDCA neuroprotective mechanism, our in vitro results showed a marked inhibition of Ab endocytosis by TUDCA. Also, the modulation of proteins known to be involved in APP processing suggests a role for TUDCA in decreasing Ab production. Characterization of the exact mechanism involved in TUDCA inhibition of Ab accumulation is likely to provide new perspectives for modulation of Ab-induced toxicity.