Abstract
We developed a novel therapeutic strategy for Alzheimer’s disease (AD) exploiting the properties of a natural variant of Amyloid-β (Aβ) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aβ, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aβ1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aβ1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aβ toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aβ1-6A2VTAT(D) inhibits Aβ aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AβA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aβ1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aβ production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.
Highlights
The isotopically labelled peptide is composed of D-amino acids and has all Gly amino acids deuterated with a molecular weight of 2,418 Da, a mass increase of 10 Da compared to the native analogue
The results indicate that Aβ1-6A2VTAT(D) was largely resistant to digestion with proteinase K and bacteria proteases, a small fraction was cleaved at Gly 11 leading to formation of a peptide homologous to residues [48-57] of TAT
For the calibration curves the STD (0-30ng) and Internal Standard (IS) (20 ng) were added when the sample was suspended in 0.1 % HCOOH in water + 1 % acetonitrile in auto-sampler vials for analysis in HPLC-MS/MS. Liquid chromatography (HPLC)-mass spectrometry (MS)/MS
Summary
Samples were than centrifuged (4000 rpm, 5 min) and the supernatant collected for mass spectrometry analysis using MALDI-TOF instrument (Bruker Daltonics, MA, USA). Primary stock solutions of Aβ1-6A2VTAT(D) and the deuterated analogue (internal standard (IS)) were separately prepared with the mobile phase (HCOOH 0.1 % + ACN 1 %) at a concentration of 20 μM.
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