Intravenously Injected Amyloid-ß Peptide With Isomerized Asp7 and Phosphorylated Ser8 Residues Inhibits Cerebral ß-Amyloidosis in AßPP/PS1 Transgenic Mice Model of Alzheimer's Disease.

Cerebral ß-amyloidosis, an accumulation in the patient's brain of aggregated amyloid-ß (Aß) peptides abnormally saturated by divalent biometal ions, is one of the hallmarks of Alzheimer's disease (AD). Earlier, we found that exogenously administrated synthetic Aß with isomerized Asp7 (isoD7-Aß) induces Aß fibrillar aggregation in the transgenic mice model of AD. IsoD7-Aß molecules have been implied to act as seeds enforcing endogenous Aß to undergo pathological aggregation through zinc-mediated interactions. On the basis of our findings on zinc-induced oligomerization of the metal-binding domain of various Aß species, we hypothesize that upon phosphorylation of Ser8, isoD7-Aß loses its ability to form zinc-bound oligomeric seeds. In this work, we found that (i) in vitro isoD7-Aß with phosphorylated Ser8 (isoD7-pS8-Aß) is less prone to spontaneous and zinc-induced aggregation in comparison with isoD7-Aß and intact Aß as shown by thioflavin T fluorimetry and dynamic light scattering data, and (ii) intravenous injections of isoD7-pS8-Aß significantly slow down the progression of institutional ß-amyloidosis in AßPP/PS1 transgenic mice as shown by the reduction of the congophilic amyloid plaques' number in the hippocampus. The results support the role of the zinc-mediated oligomerization of Aß species in the modulation of cerebral ß-amyloidosis and demonstrate that isoD7-pS8-Aß can serve as a potential molecular tool to block the aggregation of endogenous Aß in AD.

Phosphorylation of the Amyloid-Beta Peptide Inhibits Zinc-Dependent Aggregation, Prevents Na,K-ATPase Inhibition, and Reduces Cerebral Plaque Deposition.

The triggers of late-onset sporadic Alzheimer's disease (AD) are still poorly understood. Impairment of protein phosphorylation with age is well-known; however, the role of the phosphorylation in ß-amyloid peptide (Aß) is not studied sufficiently. Zinc-induced oligomerization of Aß represents a potential seeding mechanism for the formation of neurotoxic Aß oligomers and aggregates. Phosphorylation of Aß by Ser8 (pS8-Aß), localized inside the zinc-binding domain of the peptide, may significantly alter its zinc-induced oligomerization. Indeed, using dynamic light scattering, we have shown that phosphorylation by Ser8 dramatically reduces zinc-induced aggregation of Aß, and moreover pS8-Aß suppresses zinc-driven aggregation of non-modified Aß in an equimolar mixture. We have further analyzed the effect of pS8-Aß on the progression of cerebral amyloidosis with serial retro-orbital injections of the peptide in APPSwe/PSEN1dE9 murine model of AD, followed by histological analysis of amyloid burden in hippocampus. Unlike the non-modified Aß that has no influence on the amyloidosis progression in murine models of AD, pS8-Aß injections reduced the number of amyloid plaques in the hippocampus of mice by one-third. Recently shown inhibition of Na+,K+-ATPase activity by Aß, which is thought to be a major contributor to neuronal dysfunction in AD, is completely reversed by phosphorylation of the peptide. Thus, several AD-associated pathogenic properties of Aß are neutralized by its phosphorylation.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

Peripherally Applied Synthetic Tetrapeptides HAEE and RADD Slow Down the Development of Cerebral ß-Amyloidosis in AßPP/PS1 Transgenic Mice.

Two tetrapeptides, HAEE and RADD, which are ionic-complementary to the primary zinc recognition site of amyloid-ß (Aß), have been reported to inhibit zinc-induced dimerization of the Aß metal-binding domain and slow Aß aggregation in vitro. In the present study, we investigate the impact of HAEE and RADD on the development of cerebral ß-amyloidosis in a mouse model of Alzheimer's disease. We have found chronic intravenous administration of each peptide results in significant decrease of amyloid plaque burden in the treated mice.

Catalytic antibodies in clinical and experimental pathology: human and mouse models.

Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.