Abstract Soluble Aβ oligomers are regarded as major neurotoxic agents in Alzheimer’s disease. Several monoclonal antibodies have been developed to target Aβ oligomers, but most of them show limited specificity binding also to monomers and fibrils. To generate an antibody with high specificity for the oligomers, we aimed to increase the efficiency and sensitivity of a human VH–derived Aβ-oligomer-specific single domain antibody, called DesAb-O. We engineered a dimeric DesAb-O variant, DiDesAb-O, which showed significantly higher binding affinity for Aβ oligomers as compared to the monomeric sdAb. DiDesAb-O selectively detected Aβ42 oligomers not only in vitro and in cultured cells using synthetic preparations, but also in the cerebrospinal fluid from Alzheimer’s patients. Moreover, it inhibited the binding of these toxic species to cellular membranes and neutralized their neurotoxicity both in cells and in patient-derived cerebrospinal fluid at lower concentrations compared to DesAb-O. These results indicate that rational dimerization of single-domain antibodies can substantially enhance target engagement and functional efficacy, providing a promising strategy for the development of improved diagnostic and therapeutic molecules for Alzheimer’s disease.

Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents in Alzheimer’s disease (AD). In the last few years, Aprile and co-workers designed and purified a single-domain antibody (sdAb), called DesAb-O, with high specificity for Aβ 1-42 oligomeric conformers. Recently, Cascella and co-workers showed that DesAb-O can selectively detect synthetic Aβ 1-42 oligomers both in vitro and in cultured cells, neutralizing their associated neuronal dysfunction. DesAb-O can also identify Aβ 1-42 oligomers in the cerebrospinal fluid (CSF) of AD patients, with respect to healthy individuals, preventing cell dysfunction induced by the administration of CSFs to neuronal cells. Given the extraodinary potentialities of this nanobody, we design a dimeric-structure of DesAb-O, with the aim to increase its avidity and affinity for toxic Aβ 1-42 oligomers. We designed the dimeric-DesAb-O structure by linking two DesAb-O monomeric domains with a flexible linker region (GGGGS) 3. Once expressed and purified the protein, we characterised its molecular weight by mass spectrometry and its secondary structure by Circular Dichroism. Then, we performed an aggregation assay to monitor its ability to interfere with the Aβ 1-42 aggregation process and a Real-Time based ELISA assay to study its binding for Aβ 1-42 oligomers. The Dimeric-DesAb-O is able to interfere with the Aβ 1-42 aggregation process to a greater extent than DesAb-O. Furthermore, the dimeric structure of DesAb-O showed a higher specificity and affinity for Aβ 1-42 oligomers compared to DesAb-O. The Dimeric-DesAb-O appears to be a promising tool for the future development of sdAbs-based immunodiagnostic tests for the early diagnosis of AD.

The aggregation of α-Synuclein (αS) into amyloid fibrils and their deposition in intraneuronal Lewy bodies are hallmark features of Parkinson's disease (PD) and other synucleinopathies. Among the molecular players implicated in αS toxicity, the cellular prion protein (PrPC) has emerged as a potential modulator of αS-neuron interactions. Using confocal microscopy, colocalization analysis and both siRNA-induced PrPC silencing and antibody-based blockade, we investigated the contribution of PrPC to αS-induced neurotoxicity in human iPSC-derived dopaminergic neurons, primary rat cortical neurons and human SH-SY5Y neuroblastoma cells. We show that PrPC facilitated the early recruitment of αS prefibrillar type B* oligomers (OB*) and short fibrils (SF) to neuronal membranes, enhancing αS-induced Ca2+ influx and membrane permeabilization. However, PrPC levels remained unchanged following prolonged exposure with OB* and SF, suggesting no feedback modulation of PrPC expression. While PrPC blockade partially inhibited the release of toxic soluble oligomers from αS fibrils, downstream cell death was only marginally reduced, indicating a limited contribution of PrPC to the final neurotoxic outcome. By contrast, extracellular Ca2+ emerged as a major driver of αS toxicity, directly promoting the membrane recruitment, internalization and cytotoxic effects of αS aggregates. Collectively, our findings indicate that while PrPC facilitates early events in αS aggregate interaction with neurons, the sustained neurotoxicity induced by αS prefibrillar oligomers and fibrils is predominantly mediated by extracellular Ca2+. This promotes aggregate-membrane interactions, membrane permeabilization, and intracellular Ca2+ dyshomeostasis, thereby establishing a vicious cycle of neuronal dysfunction and death.

The aberrant aggregation of α-synuclein (αS) into amyloid fibrils is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson's disease. Although the structural properties of mature amyloids of αS are currently understood, the nature of transient protofilaments and fibrils that appear during αS aggregation remains elusive. Using solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, we here characterized intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids adopting the structure of pathologically observed aggregates. These transient amyloid intermediates, which induce significant levels of cytotoxicity when incubated with neuronal cells, were found to be stabilized by a small core in an antiparallel β-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. In contrast, mature amyloids that subsequently appear during the aggregation showed different structural and biological properties, including low levels of cytotoxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. The characterization of these two fibrillar forms of αS, and the use of antibodies and designed mutants, enabled us to clarify the role of critical structural elements endowing intermediate amyloid species with the ability to interact with membranes and induce cytotoxicity.

Background Amyloid-β 42 (Aβ 42 ) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer’s disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aβ 42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aβ 42 oligomers. Methods We investigated the ability of DesAb-O to selectively detect preformed Aβ 42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. Results DesAb-O was found to selectively detect synthetic Aβ 42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aβ 42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. Conclusions Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.

Introduction: Alzheimer’s disease (AD) is a progressive debilitating neurological disorder representing the most common neurodegenerative disease worldwide. Although the exact pathogenic mechanisms of AD remain unresolved, the presence of extracellular amyloid-β peptide 1-42 (Aβ1-42) plaques in the parenchymal and cortical brain is considered one of the hallmarks of the disease.Methods: In this work, we investigated the Aβ1-42 fibrillogenesis timeline up to 48 h of incubation, providing morphological and chemo-structural characterization of the main assemblies formed during the aggregation process of Aβ1-42, by atomic force microscopy (AFM) and surface enhanced Raman spectroscopy (SERS), respectively.Results: AFM topography evidenced the presence of characteristic protofibrils at early-stages of aggregation, which form peculiar macromolecular networks over time. SERS allowed to track the progressive variation in the secondary structure of the aggregation species involved in the fibrillogenesis and to determine when the β-sheet starts to prevail over the random coil conformation in the aggregation process.Discussion: Our research highlights the significance of investigating the early phases of fibrillogenesis to better understand the molecular pathophysiology of AD and identify potential therapeutic targets that may prevent or slow down the aggregation process.

High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the β-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion.

The aberrant aggregation of specific peptides and proteins is the common feature of a range of more than 50 human pathologies, collectively referred to as protein misfolding diseases [...]

Extracellular amyloid β (Aβ) plaques and intracellular neurofibrillary tangles of the hyperphosphorylated tau protein are the main hallmarks of Alzheimer’s disease (AD). Small, soluble oligomers, rather than mature fibrils, are the major neurotoxic agents. The heterogeneous structures and the transient nature of these oligomers make their isolation and characterization very challenging. Single domain Abs (sdAbs), composed only of a variable domain of the heavy chain with high specificity and affinity, appear as promising tools for an early diagnosis and therapy for AD. Performing an in vitro and in vivo screening of different sdAbs, we selected those targeting Aβ 42 oligomers or fibrils with high specificity using dot-blot, ELISA assay and the super resolution stimulated emission depletion (STED) microscopy. The potential of sdAbs was also investigated in preventing Aβ 42 oligomer-induced cytotoxicity in cultured neuronal cells. Then, the sdAbs were used to selectively detect Aβ 42 species in the cerebrospinal fluid (CSF) of AD patients and control subjects and to neutralize their associated harmful effects following cell exposure to CSF samples. sdAbs can selectively detect Aβ 42 oligomers or fibrils both in vitro and in cultured cells, preventing the neurotoxicity induced by small oligomers. Moreover, sdAbs can significantly identify Aβ 42 oligomers in the CSF of AD patients counteracting their induced toxicity in our cell models. All these data provide a solid foundation for the development of sdAbs-based immunodiagnostic tools that can selectively detect toxic aggregates in human body fluids for an early differential diagnosis of protein deposition diseases, such as AD. Furthermore, our results contribute to the improvement of the current therapeutic approaches against AD. The study was supported by Airalzh (AGYR 2020 to R.C.).

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aβ (Aβ42 O) show membrane-binding capacity and trigger the disruption of Ca2+ homeostasis, a causative event in neuron degeneration. Since bioactive lipids have been recently proposed as potent protective agents against Aβ toxicity, we investigated the involvement of sphingosine 1-phosphate (S1P) signalling pathway in Ca2+ homeostasis in living neurons exposed to Aβ42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca2+ dyshomeostasis induced by toxic Aβ42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P1 and S1P4 receptors, and to a lower extent of S1P3 and S1P5 receptors, which activate the Gi -dependent signalling pathways, thus resulting in the endocytic internalization of the extrasynaptic GluN2B-containing N-methyl-D-aspartate receptors (NMDARs). Notably, the S1P beneficial effect can be sustained over time by sphingosine kinase-1 overexpression, thus counteracting the down-regulation of the S1P signalling induced by Aβ42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aβ42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD.