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Blood-based biomarkers for Alzheimer’s disease – an in vitro proof of concept
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Alzheimer’s disease (AD), the main cause of dementia, is a fatal neurodegenerative disease, being one of the great healthcare challenges of the 21st century. It is characterized by the imbalance between the production and clearance of Amyloid-β (Aβ) species, being neuronal death caused by its accumulation. Structured Aβ oligomers are viewed as more toxic than fibrils, existing in two isoforms, leading to distinct toxicity profiles. Advances in drug targeting to the central nervous system (CNS) are crucial, since the blood-brain barrier (BBB) is selective, limiting drug penetration. Therefore, cell-translocating peptides (CTPs), like PepH3, that effectively translocates the BBB, are a promising tool. Aiming to be tested as diagnosis tools to detect toxic Aβ42 oligomers, PepH3 was coupled to a therapeutic single-domain antibody (sdAb) anti-Aβ42, creating VLx-PepH3 and VHH-PepH3. First, oligomers were produced as 14 kDa tetramers, showcasing a heterogeneous size distribution. Aβ42 took 10 hours to reach equilibrium plateau, having a t50 of 6.45±0.32h. The produced oligomers showed toxicity towards bEnd.3 cells, having an IC50 of 8.65±1.41 µM. Secondly, therapeutic effect was evaluated. VHH-PepH3 was efficient in both disturbing Aβ42’s aggregation, reducing t50 to 6.39±0.35h, and protecting bEnd.3 cells against Aβ42-induced toxicity. Whereas VLx-PepH3 significantly protected bEnd.3 cells, but didn’t inhibit Aβ42’s aggregation, increasing t50 to 6.93±0.12h. Binding affinity to Aβ42 was determined by ELISA and SPR, demonstrating that VHH-PepH3 had a higher affinity with a KD of 14.2 nM whilst VLx-PepH3 was weakly bound to the oligomers, having a KD of 230 000 nM. Concerning BBB translocation, VLx-PepH3 translocated at 5 and 24 hours, 20.06%±19.01 and 44.15%±24.41, respectively, whilst VHH-PepH3 was unable to cross it. To be implemented as a diagnosis tool, VLx-PepH3 should accomplish reversible translocation through the BBB, which wasn’t corroborated through the experiments. Thus, both molecules need further development for their application in AD’s diagnosis.
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cell-penetrating peptides Aβ42 oligomers blood-brain barrier single-domain antibody Alzheimer’s disease