A multidisciplinary approach for the early diagnosis of Alzheimer’s disease and potential therapeutic applications

Alzheimer's disease (AD) is a progressive and debilitating neurodegenerative disorder responsible for the majority of dementia cases worldwide. Pathologically, AD is marked by the presence of extracellular amyloid β (Aβ) plaques and intracellular tau neurofibrillary tangles in specific vulnerable populations of neurons, thus leading to their death. Such an accumulation is reflected by cerebrospinal fluid (CSF) biomarkers, specifically by a decreased Aβ42/Aβ40 ratio and elevated total and phosphorylated tau levels. Nevertheless, these biomarkers often produce uncertain diagnoses especially in early AD and this underlines the urgent need for reliable biomarkers that can identify individuals in the preclinical phase and in the early stages of the disease, as the prospect of an effective cure in the future is inextricably linked to early diagnosis. As a proteinopathy, AD is characterized by a generic failure of the proteostasis network, which physiologically maintains proteins in a soluble non-aggregated state; in such a condition, a great number of proteins – not only Aβ and tau - lose solubility and gain a propensity to misfold and aggregate. Based on this ground, in the first part of this work we compared CSF samples extracted from AD patients and age-matched non-demented controls in a new Italian study called PRAMA to identify misfolded/aggregated/proteotoxic proteins not only of Aβ and tau, but of the entire proteome, by performing a series of biophysical analyses and cell viability readouts. Specifically, we took advantage of dynamic light scattering (DLS) to reveal the presence of large protein species in AD CSFs, that induce an abnormal influx of calcium ions across the cell membrane of neuronal cells, which demonstrates their proteotoxicity. Thus, we identified two novel promising biomarkers of AD, that can be combined with traditional ones to gain sensitivity and specificity for AD diagnosis. Despite not being the sole population of protein aggregates in the CSF, soluble Aβ oligomers represent a relevant portion of them and are widely recognized as the main agents responsible for neurotoxicity, thus their identification and quantification in the CSF can represent another valuable biomarker of AD. In this context, epitope-specific recombinant single-domain antibodies (sdAbs), composed only of variable fragments of camelid heavy chain-only Abs, have been recently proposed as promising tools for the early diagnosis and therapy for AD. Consequently, the second part of this work was devoted to the study of the ability of a conformation-sensitive sdAb called DesAb-O, a human single domain antibody based on the VH domain of the clinically approved Trastuzumab, specifically raised against a specific epitope exclusively present in Aβ42 oligomers and absent in the monomeric peptide and in mature fibrils, to selectively detect them both in vitro and in cultured neuronal cells. To this aim, we exploited an array of techniques including dot-blot, ELISA and super-resolution stimulated emission depletion (STED) microscopy. We also demonstrated the ability of DesAb-O to counteract the neurotoxicity induced by Aβ42 oligomers by monitoring its ability to inhibit their interaction with neuronal membranes and the subsequent impairment of cell viability. Importantly, DesAb-O selectively identified Aβ42 oligomers present in the CSFs of AD patients as compared to control individuals, and completely prevented the cellular dysfunction induced by the administration of AD CSFs to neuronal cells. With the aim to improve the already incredible binding avidity and specificity of DesAb-O and obtain a more accurate tool able to detect oligomers at even lower concentrations, we engineered the structure of DesAb-O by designing the dimeric antibody referred to as Dimeric-DesAb-O. This dimeric sdAb was able to markedly interfere with the Aβ42 aggregation process in Thioflavin T (ThT) assays and it showed to recognize Aβ42 oligomers with significantly higher specificity and selectivity than DesAb-O as revealed by ELISA assays. Furthermore, with the application of Transmission Electron Microscopy (TEM), dot blot and proteinase K (PK) analyses, we monitored the morphological and structural changes in the Aβ42 fibrils obtained by co-incubating the Aβ42 monomer in the absence or presence of the sdAbs. In particular, fibrils obtained when co-incubating the monomer with the Dimeric-DesAb-O showed jagged appearance with the presence of globular structures on their surface, and a reduced diameter as compared to those formed in the presence of DesAb-O or in the absence of sdAbs, resulting exceptionally thin and fragile. We also studied the ability of Dimeric-DesAb-O to counteract the toxicity induced by oligomers present in the CSFs of AD patients on neuronal cells in culture by applying the previously described techniques. Our data shows that the Dimeric-DesAb-O possesses a significantly neuroprotective capacity to neutralize toxic species in the AD CSF at much lower concentrations as compared to DesAb-O. In conclusion, with this study we identified two novel promising CSF biomarkers of AD and we demonstrated the enormous potential of sdAbs for the set-up of an immunodiagnostic test for the early diagnosis of AD, as well as for the generation of novel therapeutic approaches for AD.