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- Rofo, Fadi, et al.
(författare)
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Enhanced neprilysin-mediated degradation of hippocampal A beta 42 with a somatostatin peptide that enters the brain
- 2021
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Ingår i: Theranostics. - : IVYSPRING INT PUBL. - 1838-7640. ; 11:2, s. 789-804
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Tidskriftsartikel (refereegranskat)abstract
- Background: Aggregation of the amyloid-beta (A beta) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading A beta, with its activity enhanced by the neuropeptide somatostatin (SST). SST levels are decreased in the brains of AD patients. The poor delivery of SST over the blood-brain barrier (BBB) and its extremely short half-life of only 3 min limit its therapeutic significance.Methods: We recombinantly fused SST to a BBB transporter binding to the transferrin receptor. Using primary neuronal cultures and neuroblastoma cell lines, the ability of the formed fusion protein to activate neprilysin was studied. SST-scFv8D3 was administered to mice overexpressing the A beta-precursor protein (A beta PP) with the Swedish mutation (APPswe) as a single injection or as a course of three injections over a 72 h period. Levels of neprilysin and A beta were quantified using an Enzyme-linked immunosorbent assay (ELISA). Distribution of SST-scFv8D3 in the brain, blood and peripheral organs was studied by radiolabeling with iodine-125.Results: The construct, SST-scFv8D3, exhibited 120 times longer half-life than SST alone, reached the brain in high amounts when injected intravenously and significantly increased the brain concentration of neprilysin in APPswe mice. A significant decrease in the levels of membrane-bound A beta 42 was detected in the hippocampus and the adjacent cortical area after only three injections.Conclusion: With intravenous injections of our BBB permeable SST peptide, we were able to significantly increase the levels neprilysin, an effect that was followed by a significant and selective degradation of membrane-bound A beta 42 in the hippocampus. Being that membrane-bound A beta triggers neuronal toxicity and the hippocampus is the central brain area in the progression of AD, the study has illuminated a new potential treatment paradigm with a promising safety profile targeting only the disease affected areas.
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| 2. |
- Rofo, Fadi, et al.
(författare)
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Novel multivalent design of a monoclonal antibody improves binding strength to soluble aggregates of amyloid beta
- 2021
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Ingår i: Translational Neurodegeneration. - : BioMed Central (BMC). - 2047-9158. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Amyloid-beta (A beta) immunotherapy is a promising therapeutic strategy in the fight against Alzheimer's disease (AD). A number of monoclonal antibodies have entered clinical trials for AD. Some of them have failed due to the lack of efficacy or side-effects, two antibodies are currently in phase 3, and one has been approved by FDA. The soluble intermediate aggregated species of A beta, termed oligomers and protofibrils, are believed to be key pathogenic forms, responsible for synaptic and neuronal degeneration in AD. Therefore, antibodies that can strongly and selectively bind to these soluble intermediate aggregates are of great diagnostic and therapeutic interest. Methods: We designed and recombinantly produced a hexavalent antibody based on mAb158, an A beta protofibril-selective antibody. The humanized version of mAb158, lecanemab (BAN2401), is currently in phase 3 clinical trials for the treatment of AD. The new designs involved recombinantly fusing single-chain fragment variables to the N-terminal ends of mAb158 antibody. Real-time interaction analysis with LigandTracer and surface plasmon resonance were used to evaluate the kinetic binding properties of the generated antibodies to A beta protofibrils. Different ELISA setups were applied to demonstrate the binding strength of the hexavalent antibody to A beta aggregates of different sizes. Finally, the ability of the antibodies to protect cells from A beta-induced effects was evaluated by MTT assay. Results: Using real-time interaction analysis with LigandTracer, the hexavalent design promoted a 40-times enhanced binding with avidity to protofibrils, and most of the added binding strength was attributed to the reduced rate of dissociation. Furthermore, ELISA experiments demonstrated that the hexavalent design also had strong binding to small oligomers, while retaining weak and intermediate binding to monomers and insoluble fibrils. The hexavalent antibody also reduced cell death induced by a mixture of soluble A beta aggregates. Conclusion: We provide a new antibody design with increased valency to promote binding avidity to an enhanced range of sizes of A beta aggregates. This approach should be general and work for any aggregated protein or repetitive target.
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| 3. |
- Rofo, Fadi, et al.
(författare)
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Wide-Ranging Effects on the Brain Proteome in a Transgenic Mouse Model of Alzheimer's Disease Following Treatment with a Brain-Targeting Somatostatin Peptide
- 2021
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 12:13, s. 2529-2541
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimer’s disease is the most common neurodegenerative disorder characterized by the pathological aggregation of amyloid-β (Aβ) peptide. A potential therapeutic intervention in Alzheimer’s disease is to enhance Aβ degradation by increasing the activity of Aβ-degrading enzymes, including neprilysin. The somatostatin (SST) peptide has been identified as an activator of neprilysin. Recently, we demonstrated the ability of a brain-penetrating SST peptide (SST-scFv8D3) to increase neprilysin activity and membrane-bound Aβ42 degradation in the hippocampus of mice overexpressing the Aβ-precursor protein with the Swedish mutation (APPswe). Using LC–MS, we further evaluated the anti-Alzheimer’s disease effects of SST-scFv8D3. Following a triple intravenous injection of SST-scFv8D3, the LC–MS analysis of the brain proteome revealed that the majority of downregulated proteins consisted of mitochondrial proteins regulating fatty acid oxidation, which are otherwise upregulated in APPswe mice compared to wild-type mice. Moreover, treatment with SST-scFv8D3 significantly increased hippocampal levels of synaptic proteins regulating cell membrane trafficking and neuronal development. Finally, hippocampal concentrations of growth-regulated α (KC/GRO) chemokine and degradation of neuropeptide-Y were elevated after SST-scFv8D3 treatment. In summary, our results demonstrate a multifaceted effect profile in regulating mitochondrial function and neurogenesis following treatment with SST-scFv8D3, further suggesting the development of Alzheimer’s disease therapies based on SST peptides.
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