Enhanced neprilysin-mediated degradation of hippocampal Aβ42 with a somatostatin peptide that enters the brain

Abstract

Background: Aggregation of the amyloid-beta (Aβ) peptide is one of the main neuropathological events in Alzheimer's disease (AD). Neprilysin is the major enzyme degrading Aβ, 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 Aβ-precursor protein (AβPP) 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 Aβ 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 Aβ42 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 Aβ42 in the hippocampus. Being that membrane-bound Aβ 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.

Modelling the efficacy of Neprilysin from various species in degrading different Amyloid-β peptides: Potential application in therapeutics of Alzheimer’s disease

“…The neprilysin-Amyloidβ (Aβ) was selected as the enzyme-substrate prototype as recent studies have indicate the potential role of developing recombinant neprilysin as a therapeutic candidate for treating Alzheimer's disease (AD). 2,6,8,9,16 Therapeutic development of neprilysin for use in treating AD will require establishing the optimal enzyme kinetics against AD specific Aβ peptides. 2,8,9 Although the association Aβ peptides in the progression of AD is well established, developing effective treatment will require specific and selective cleaving of a variety of Aβ peptides.…”

“…2,6,8,9,16 Therapeutic development of neprilysin for use in treating AD will require establishing the optimal enzyme kinetics against AD specific Aβ peptides. 2,8,9 Although the association Aβ peptides in the progression of AD is well established, developing effective treatment will require specific and selective cleaving of a variety of Aβ peptides. [14][15][16][17] Depending on the variation in the site of cleavage in the amyloid precursor protein a variety of Aβ peptides can be generated, which may impact the pathophysiology of AD differently.…”

“…The neprilysin- Amyloidβ (Aβ) was selected as the enzyme-substrate prototype as recent studies have indicate the potential role of developing recombinant neprilysin as a therapeutic candidate for treating Alzheimer’s disease (AD). 2, 6, 8, 9, 16…”

“…The enzyme kinetics of 5JMY and Amyloid β1-40 interactions are extensively reported in the literature and forms the basis of several commercially available kits to assess neprilysin activity. 1, 6, 8, 14–16 Hence the enzymatic cleaving of Amyloid β1-40 (at a substrate concentration of 0.625, 1.25, 2.5, 5, 10, 15 and 20 μM) was studied in presence of 5 nM concentration of recombinant neprilysin (5JMY). The resultant enzyme-substrate kinetic curve was used for modelling the kinetics of rest of the Amyloid β (4XFO, 5TPT, 3SGP, 2ROZ, 2YT1, 4YN0, 2KJ7, 1NMJ, 4DBB, 2BFI, 2ONX, Aβ1-40) and neprilysin (5JMY, 6GID, 4XBH and 6GHX) combinations.…”

“…The enzyme kinetics of 5JMY and Amyloid β 1-40 interactions are extensively reported in the literature and forms the basis of several commercially available kits to assess neprilysin activity. 1,6,8,[14][15][16] Hence the enzymatic cleaving of Amyloid…”