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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., (© 2024. The Author(s).)

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-β-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology. Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ . We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions. Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-β structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature. Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-β structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

The misfolding and aggregation of α-synuclein is the general hallmark of a group of devastating neurodegenerative pathologies referred to as synucleinopathies, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In such conditions, a range of different misfolded aggregates, including oligomers, protofibrils, and fibrils, are present both in neurons and glial cells. Growing experimental evidence supports the proposition that soluble oligomeric assemblies, formed during the early phases of the aggregation process, are the major culprits of neuronal toxicity; at the same time, fibrillar conformers appear to be the most efficient at propagating among interconnected neurons, thus contributing to the spreading of α-synuclein pathology. Moreover, α-synuclein fibrils have been recently reported to release soluble and highly toxic oligomeric species, responsible for an immediate dysfunction in the recipient neurons. In this review, we discuss the current knowledge about the plethora of mechanisms of cellular dysfunction caused by α-synuclein oligomers and fibrils, both contributing to neurodegeneration in synucleinopathies., Competing Interests: None

Natural aminosterols are promising drug candidates against neurodegenerative diseases, like Alzheimer and Parkinson, and one relevant protective mechanism occurs via their binding to biological membranes and displacement or binding inhibition of amyloidogenic proteins and their cytotoxic oligomers. We compared three chemically different aminosterols, finding that they exhibited different (i) binding affinities, (ii) charge neutralizations, (iii) mechanical reinforcements, and (iv) key lipid redistributions within membranes of reconstituted liposomes. They also had different potencies (EC 50 ) in protecting cultured cell membranes against amyloid-β oligomers. A global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane effects. The analysis correlates aminosterol-mediated protection with well-defined chemical moieties, including the polyamine group inducing a partial membrane-neutralizing effect (79 ± 7%) and the cholestane-like tail causing lipid redistribution and bilayer mechanical resistance (21 ± 7%), linking quantitatively their chemistry to their protective effects on biological membranes.

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., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Safety data regarding BNT162b2 in cancer patients (CPs) are scarce. Herein we report the side effects (SEs), the adverse events (AEs), and the patient-reported outcomes (PROs) following BNT162b2 administration in CPs treated at the San Luigi Gonzaga University Hospital. All CPs who agreed to participate in our vaccination campaign received BNT162b2 and were included in the descriptive analysis. An anonymous questionnaire investigating the occurrence of SEs/AEs and PROs was administered to the study population 21 days after the first dose. Pearson's chi-squared test was used to estimate the risk of experiencing SEs/AEs according to selected variables. A total of 997 patients were included in the study: 62.0% had stage IV cancer, and 68.8% were receiving an active treatment, of whom 15.9% were receiving immunotherapy. SEs/AEs were recorded in 37.1% of cases after the first dose and in 48.5% of cases after the second dose. The most common SEs were muscle pain/local rash (27.9% and 28%, after the first and second dose, respectively). Patients older than 70 years showed lower risk of SEs/AEs, while women showed a higher risk. Before receiving the vaccine, 18.2% of patients felt fearful and/or insecure about the vaccination. After the first dose, 57.5% of patients changed their feelings positively. Our data support the short-term safety of BNT162b2 in CPs, regardless of disease stage and concurrent treatments. Overall, the vaccination showed a positive impact on quality of life.

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 (Aβ 42 O) show membrane-binding capacity and trigger the disruption of Ca 2+ 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 Ca 2+ homeostasis in living neurons exposed to Aβ 42 O. We show that both exogenous and endogenous S1P rescued neuronal Ca 2+ dyshomeostasis induced by toxic Aβ 42 O in primary rat cortical neurons and human neuroblastoma SH-SY5Y cells. Further analysis revealed a strong neuroprotective effect of S1P 1 and S1P 4 receptors, and to a lower extent of S1P 3 and S1P 5 receptors, which activate the G i -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 Aβ 42 O. Our findings disclose underlying mechanisms of S1P neuronal protection against harmful Aβ 42 O, suggesting that S1P and its signalling axis can be considered promising targets for therapeutic approaches for AD., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are associated with deposition of cytosolic inclusion bodies of TAR DNA-binding protein 43 (TDP-43) in brain and motor neurons. We induced phase separation of purified full-length TDP-43 devoid of large tags using a solution-jump method, and monitored it with an array of biophysical techniques. The tetramethylrhodamine-5-maleimide- or Alexa488-labeled protein formed rapidly (<1 min) apparently round, homogeneous and 0.5-1.0 μm wide assemblies, when imaged using confocal fluorescence, bright-field, and stimulated emission depletion microscopy. The assemblies, however, had limited internal diffusion, as assessed with fluorescence recovery after photobleaching, and did not coalesce, but rather clustered into irregular bunches, unlike those formed by the C-terminal domain. They were enriched with α-helical structure, with minor contributions of β-sheet/random structure, had a red-shifted tryptophan fluorescence and did not bind thioflavin T. By monitoring with turbidimetry both the formation of the spherical species and their further clustering under different experimental conditions, we carried out a multiparametric analysis of the two phenomena. In particular, both processes were found to be promoted by high protein concentrations, salts, crowding agents, weakly by reducing agents, as the pH approached a value of 6.0 from either side (corresponding to the TDP-43 isoionic point), and as the temperature approached a value of 31°C from either side. Important differences were found with respect to the TDP-43 C-terminal domain. Our multiparametric results also provide explanations to some of the solubility data obtained on full-length TDP-43 that were difficult to explain following the multiparametric analysis acquired on the C-terminal domain., (© 2022 The Protein Society.)

Amyloid fibril formation plays a central role in the pathogenesis of a number of neurodegenerative diseases, including Alzheimer and Parkinson diseases. Transient prefibrillar oligomers forming during the aggregation process, exhibiting a small size and a large hydrophobic surface, can aberrantly interact with a number of molecular targets on neurons, including the lipid bilayer of plasma membranes, resulting in a fatal outcome for the cells. By contrast, the mature fibrils, despite presenting generally a high hydrophobic surface, are endowed with a low diffusion rate and poorly penetrate the interior of the lipid bilayer. However, increasing evidence shows that both intracellular α-synuclein fibrils, as well and as extracellular amyloid-β and β2-microglobulin fibrils, can release oligomers over time that quickly diffuse to reach the membrane of the neighboring cells. The persistent leakage of harmful oligomers from fibrils triggers an ongoing cascade of events resulting in a sustained injury to neurons and glia and also provides aggregates with the ability to cross biological membranes and diffuse between cells or cellular compartments., (© 2022 The Authors. BioEssays published by Wiley Periodicals LLC.)

Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic Ca 2+ levels, flowing from the extracellular space, and production of reactive oxygen species (ROS). Here we investigated the relationship between the increase in Ca 2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ 42 -derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca 2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca 2+ than ROS levels, and a lag phase in the latter process. A Ca 2+ -deprived cell medium prevented the increase of intracellular Ca 2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca 2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca 2+ causes the production of ROS in cells challenged by aberrant protein oligomers., (© 2022. The Author(s).)

A number of neurodegenerative conditions are associated with the formation of cytosolic inclusions of TDP-43 within neurons. We expressed full-length TDP-43 in a motoneuron/neuroblastoma hybrid cell line (NSC-34) and exploited the high-resolution power of stimulated emission depletion microscopy to monitor the changes of nuclear and cytoplasmic TDP-43 levels and the formation of various size classes of cytoplasmic TDP-43 aggregates with time. Concomitantly, we monitored oxidative stress and mitochondrial impairment using the MitoSOX and MTT reduction assays, respectively. Using a quantitative biology approach, we attributed neuronal dysfunction associated with cytoplasmic deposition component to the formation of the largest inclusions, independently of stress granules. This is in contrast to other neurodegenerative diseases where toxicity is attributed to small oligomers. Using specific inhibitors, markers, and electron microscopy, the proteasome and autophagy were found to target mainly the largest deleterious inclusions, but their efficiency soon decreases without full recovery of neuronal viability.

Protein misfolding is a general hallmark of protein deposition diseases, such as Alzheimer's disease or Parkinson's disease, in which different types of aggregated species (oligomers, protofibrils and fibrils) are generated by the cells. Despite widespread interest, the relationship between oligomers and fibrils in the aggregation process and spreading remains elusive. A large variety of experimental evidences supported the idea that soluble oligomeric species of different proteins might be more toxic than the larger fibrillar forms. Furthermore, the lack of correlation between the presence of the typical pathological inclusions and disease sustained this debate. However, recent data show that the β-sheet core of the α-Synuclein (αSyn) fibrils is unable to establish persistent interactions with the lipid bilayers, but they can release oligomeric species responsible for an immediate dysfunction of the recipient neurons. Reversibly, such oligomeric species could also contribute to pathogenesis via neuron-to-neuron spreading by their direct cell-to-cell transfer or by generating new fibrils, following their neuronal uptake. In this Review, we discuss the various mechanisms of cellular dysfunction caused by αSyn, including oligomer toxicity, fibril toxicity and fibril spreading., (© 2022. The Author(s).)

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer's and Parkinson's diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer's and Parkinson's diseases., Competing Interests: DB and MZ are inventors in a patent for the use of aminosterols in the treatment of Parkinson’s disease. DB and MZ are co-founders of Enterin Inc. and serve as the President and CSO, respectively, of the company. MV, TPJK, and JH are co-founders, and BM and MP are employees of Wren Therapeutics Ltd., which is independently pursuing inhibitors of protein misfolding and aggregation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Limbocker, Staats, Chia, Ruggeri, Mannini, Xu, Perni, Cascella, Bigi, Sasser, Block, Wright, Kreiser, Custy, Meisl, Errico, Habchi, Flagmeier, Kartanas, Hollows, Nguyen, LeForte, Barbut, Kumita, Cecchi, Zasloff, Knowles, Dobson, Chiti and Vendruscolo.)

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson's disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder that is characterized by amyloid β-protein deposition in senile plaques, neurofibrillary tangles consisting of abnormally phosphorylated tau protein, and neuronal loss leading to cognitive decline and dementia. Despite extensive research, the exact mechanisms underlying AD remain unknown and effective treatment is not available. Many hypotheses have been proposed to explain AD pathophysiology; however, there is general consensus that the abnormal aggregation of the amyloid β peptide (Aβ) is the initial event triggering a pathogenic cascade of degenerating events in cholinergic neurons. The dysregulation of calcium homeostasis has been studied considerably to clarify the mechanisms of neurodegeneration induced by Aβ. Intracellular calcium acts as a second messenger and plays a key role in the regulation of neuronal functions, such as neural growth and differentiation, action potential, and synaptic plasticity. The calcium hypothesis of AD posits that activation of the amyloidogenic pathway affects neuronal Ca 2+ homeostasis and the mechanisms responsible for learning and memory. Aβ can disrupt Ca 2+ signaling through several mechanisms, by increasing the influx of Ca 2+ from the extracellular space and by activating its release from intracellular stores. Here, we review the different molecular mechanisms and receptors involved in calcium dysregulation in AD and possible therapeutic strategies for improving the treatment.

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-β structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.

Alzheimer's disease, which is the most common form of dementia, is characterized by the aggregation of the amyloid β peptide (Aβ) and by an impairment of calcium homeostasis caused by excessive activation of glutamatergic receptors (excitotoxicity). Here, we studied the effects on calcium homeostasis caused by the formation of Aβ oligomeric assemblies. We found that Aβ oligomers cause a rapid influx of calcium ions (Ca 2+ ) across the cell membrane by rapidly activating extrasynaptic N -methyl-d-aspartate (NMDA) receptors and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We also observed, however, that misfolded oligomers do not interact directly with these receptors. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralize the oligomer-induced activation of the NMDA receptors, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a mechanism of toxicity of Aβ oligomers in Alzheimer's disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDA and AMPA receptors.

Background: The role of immune-related adverse events (irAEs), as a surrogate predictor of the efficacy of checkpoint inhibitors, has not yet been described in the setting of first-line, single-agent pembrolizumab for patients with metastatic non-small-cell lung-cancer (NSCLC) with a programmed death-ligand 1 (PD-L1) expression of ≥ 50%., Patients and Methods: We previously conducted a multicenter retrospective analysis in patients with treatment-naive metastatic NSCLC and a PD-L1 expression of ≥ 50% receiving first-line pembrolizumab. Here, we report the results of the irAE analysis and the potential correlation between irAEs and clinical outcomes., Results: A total of 1010 patients were included in this analysis; after a 6-week landmark selection, 877 (86.8%) patients were included in the efficacy analysis. Any grade irAEs (P < .0001), grade 3/4 irAEs (P = .0025), leading to discontinuation irAEs (P = .0144), multiple-site and single-site irAEs (P < .0001), cutaneous irAEs (P = .0001), endocrine irAEs (P = .0227), pulmonary irAEs (P = .0479), and rheumatologic irAEs (P = .0018) were significantly related to a higher objective response rate. Any grade irAEs (P < .0001), single-site irAEs (P < .0001), multiple-site irAEs (P = .0005), cutaneous irAEs (P = .0042), endocrine irAEs (P < .0001), gastrointestinal irAEs (P = .0391), and rheumatologic irAEs (P = .0086) were significantly related to progression-free survival. Any grade irAEs (P < .0001), single-site irAEs (P < .0001), multiple-site irAEs (P = .0003), cutaneous irAEs (P = .0002), endocrine irAEs (P = .0001), and rheumatologic irAEs (P = .0214) were significantly related to overall survival., Conclusions: This study confirms the feasibility and the safety of first-line, single-agent pembrolizumab, in a large, real-world cohort of patients with NSCLC with PD-L1 expression ≥ 50%. The occurrence of irAEs may be a surrogate of clinical activity and improved outcomes in this setting., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most prevalent form of dementia and soluble amyloid β (Aβ) oligomers are thought to play a critical role in AD pathogenesis. Cellular prion protein (PrP C ) is a high-affinity receptor for Aβ oligomers and mediates some of their toxic effects. The N -terminal region of PrP C can interact with Aβ, particularly the region encompassing residues 95-110. In this study, we identified a soluble and unstructured prion-derived peptide (PrP 107-120 ) that is external to this region of the sequence and was found to successfully reduce the mitochondrial impairment, intracellular ROS generation and cytosolic Ca 2+ uptake induced by oligomeric Aβ 42 ADDLs in neuroblastoma SH-SY5Y cells. PrP 107-120 was also found to rescue SH-SY5Y cells from Aβ 42 ADDL internalization. The peptide did not change the structure and aggregation pathway of Aβ 42 ADDLs, did not show co-localization with Aβ 42 ADDLs in the cells and showed a partial colocalization with the endogenous cellular PrP C . As a sequence region that is not involved in Aβ binding but in PrP self-recognition, the peptide was suggested to protect against the toxicity of Aβ 42 oligomers by interfering with cellular PrP C and/or activating a signaling that protected the cells. These results strongly suggest that PrP 107-120 has therapeutic potential for AD.

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer's disease and α-synuclein (αS) in Parkinson's disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.

Raman spectroscopy assisted by localized plasmon resonances generating effective hot spots at the gaps between intertwined silver nanowires is herein adopted to unravel characteristic molecular motifs on the surface of Aβ 42 misfolded oligomers that are critical in driving intermolecular interactions in neurodegeneration., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: The pathogenesis of Alzheimer's disease (AD) is not directly caused by the presence of senile plaques but rather by the detrimental effects exerted on neuronal cells by toxic soluble oligomers. Such species are formed early during the aggregation process of the Aβ1-42 peptide or can be released from mature fibrils. Nowadays, efficient tools for an early diagnosis, as well as pharmaceutical treatments targeting the harmful agents in samples of AD patients, are still missing., Objective: By integrating in vitro immunochemical assay with in vivo neuronal models of toxicity, we aim to understand and target the principles that drive toxicity in AD., Methods: We evaluated the specificity and sensitivity of A11 and OC conformational antibodies to target a range of pathologically relevant amyloid conformers and rescue their cytotoxic effects in neuronal culture models using a number of cellular readouts., Results: We demonstrated the peculiar ability of conformational antibodies to label pathologically relevant Aβ1-42 oligomers and fibrils and to prevent their detrimental effects on neuronal cells., Conclusion: Our results substantially improve our knowledge on the role of toxic assemblies in neurodegenerative diseases, thus suggesting new and more effective diagnostic and therapeutic tools for AD., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Deposition of soluble proteins as insoluble amyloid fibrils is associated with a number of pathological states. There is a growing interest in the identification of small molecules that can prevent proteins from undergoing amyloid fibril formation. In the present study, a series of small aromatic compounds with different substitutions of 1,3,5-triphenylbenzene have been synthesized and their possible effects on amyloid fibril formation by hen egg white lysozyme (HEWL), a model protein for amyloid formation, and of their resulting toxicity were examined. The inhibitory effect of the compounds against HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, Fourier-transform infrared spectroscopy, and cytotoxicity assays, such as the 3-(4,5-Dimethylthiazol)-2,5-Diphenyltetrazolium Bromide (MTT) reduction assay and caspase-3 activity measurements. We found that all compounds in our screen were efficient inhibitors of HEWL fibril formation and their associated toxicity. We showed that electron-withdrawing substituents such as -F and -NO 2 potentiated the inhibitory potential of 1,3,5-triphenylbenzene, whereas electron-donating groups such as -OH, -OCH 3 , and -CH 3 lowered it. These results may ultimately find applications in the development of potential inhibitors against amyloid fibril formation and its biologically adverse effects.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are progressive and fatal neurodegenerative disorders showing mislocalization and cytosolic accumulation of TDP-43 inclusions in the central nervous system. The decrease in the efficiency of the clearance systems in aging, as well as the presence of genetic mutations of proteins associated with cellular proteostasis in the familial forms of TDP-43 proteinopathies, suggest that a failure of these protein degradation systems is a key factor in the aetiology of TDP-43 associated disorders. Here we show that the internalization of human pre-formed TDP-43 aggregates in the murine neuroblastoma N2a cells promptly resulted in their ubiquitination and hyperphosphorylation by endogenous machineries, mimicking the post-translational modifications observed in patients. Moreover, our data identify mitochondria as the main responsible sites for the alteration of calcium homeostasis induced by TDP-43 aggregates, which, in turn, stimulates an increase in reactive oxygen species and, finally, caspase activation. The inhibition of TDP-43 proteostasis in the presence of selective inhibitors against the proteasome and macroautophagy systems revealed that these two systems are both severely involved in TDP-43 accumulation and have a strong influence on each other in neurodegenerative disorders associated with TDP-43.

The self-assembly of proteins into structured fibrillar aggregates is associated with a range of neurodegenerative diseases, including Alzheimer's and Parkinson's diseases, in which an important cytotoxic role is thought to be played by small soluble oligomers accumulating during the aggregation process or released by mature fibrils. As the structural characteristics of such species and their links with toxicity are still not fully defined, we have compared six examples of preformed misfolded protein oligomers with different β-sheet content, as determined using Fourier transform infrared spectroscopy, and with different toxicity, as determined by three cellular readouts of cell viability. The results show the absence of any measurable correlation between the nature of their secondary structure and their cellular toxicity, both when comparing the six types of oligomers as a group and when comparing species in subgroups characterized by either the same size or the same exposure of hydrophobic moieties.

The aggregation of α-synuclein, a protein involved in neurotransmitter release at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, most notably Parkinson's disease. Intraneuronal inclusion bodies, primarily composed of α-synuclein fibrils, are the major histopathological hallmarks of these disorders, although small oligomeric assemblies are believed to play a crucial role in neuronal impairment. We have probed the mechanism of neurotoxicity of α-synuclein oligomers isolated in vitro using antibodies targeting the N-terminal region of the protein and found that the presence of the antibody resulted in a substantial reduction of the damage induced by the aggregates when incubated with primary cortical neurons and neuroblastoma cells. We observed a similar behavior in vivo using a strain of C. elegans overexpressing α-synuclein, where the aggregation process itself is also partially inhibited as a result of incubation with the antibodies. The similar effects of the antibodies in reducing the toxicity of the aggregated species formed in vitro and in vivo provide evidence for a common origin of cellular impairment induced by α-synuclein aggregates.

Nucleophosmin 1 (NPM1) is a nucleus-cytoplasm shuttling protein ubiquitously expressed and highly conserved. It is involved in many cellular processes and its gene is mutated in ~ 50-60% of Acute Myeloid Leukemia (AML) patients. These mutations cause its cytoplasmic mislocation and accumulation (referred to as NPM1c+) and open the door to rational targeted therapy for AML diseases with mutated NPM1. Currently, there is limited knowledge on the mechanism of action of NPM1c+ and on structural determinants of the leukemogenic potential of AML mutations. Numerous previous studies outlined an unexpected amyloid-like aggregation tendency of several regions located in the C-terminal domain that, in wild-type form, fold as a three-helical-bundle. Here, using a combination of different techniques including Thioflavin T fluorescence, congo red absorbance, CD spectroscopy, Scanning Electron Microscopy (SEM) and wide-angle X-ray scattering on a series of peptides bearing mutations, we evidence that the amyloidogenicity of NPM1 mutants is directly linked to AML. Noticeably, AML point mutations strongly affect the amyloid cytotoxic effects in neuroblastoma cells and the morphologies of deriving fibrils. This study paves the way to deepen our understanding of AML-associated NPM1 mutants, and could help to break new ground for the identification of novel drugs targeting NPM1c+ for treatment of AML., (© 2019 Federation of European Biochemical Societies.)

Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Bemporad et al.)

Transient oligomeric species formed during the aggregation process of the 42-residue form of the amyloid-β peptide (Aβ 42 ) are key pathogenic agents in Alzheimer's disease (AD). To investigate the relationship between Aβ 42 aggregation and its cytotoxicity and the influence of a potential drug on both phenomena, we have studied the effects of trodusquemine. This aminosterol enhances the rate of aggregation by promoting monomer-dependent secondary nucleation, but significantly reduces the toxicity of the resulting oligomers to neuroblastoma cells by inhibiting their binding to the cellular membranes. When administered to a C. elegans model of AD, we again observe an increase in aggregate formation alongside the suppression of Aβ 42 -induced toxicity. In addition to oligomer displacement, the reduced toxicity could also point towards an increased rate of conversion of oligomers to less toxic fibrils. The ability of a small molecule to reduce the toxicity of oligomeric species represents a potential therapeutic strategy against AD.

The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1., (Copyright © 2018. Published by Elsevier Inc.)

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy., (© FASEB.)

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions., Competing Interests: M.Z. is the inventor on a patent application that has been filed related to the compound described in this paper. The other authors declare no conflict of interest.

An altered distribution of membrane gangliosides (GM), including GM1, has recently been reported in the brains of Alzheimer's disease (AD) patients. Moreover, amyloid-positive synaptosomes obtained from AD brains were found to contain high-density GM1 clusters, suggesting a pathological significance of GM1 increase at presynaptic neuritic terminals in AD. Here, we show that membrane GM1 specifically recruits small soluble oligomers of the 42-residue form of amyloid-β peptide (Aβ42), with intracellular flux of Ca2+ ions in primary rat hippocampal neurons and in human neuroblastoma cells. Specific membrane proteins appear to be involved in the early and transient influx of Ca2+ ions induced by Aβ42 oligomers with high solvent-exposed hydrophobicity (A+), but not in the sustained late influx of the same oligomers and in that induced by Aβ42 oligomers with low solvent-exposed hydrophobicity (A-) in GM1-enriched cells. In addition, A+ oligomers accumulate in proximity of membrane NMDA and AMPA receptors, inducing the early and transient Ca2+ influx, although FRET shows that the interaction is not direct. These results suggest that age-dependent clustering of GM1 within neuronal membranes could induce neurodegeneration in elderly people as a consequence of an increased ability of the lipid bilayers to recruit membrane-permeabilizing oligomers. We also show that both lipid and protein components of the plasma membrane can contribute to neuronal dysfunction, thus expanding the molecular targets for therapeutic intervention in AD.

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer's disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca(2+) across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an α-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) are two clinically distinct neurodegenerative conditions sharing a similar histopathology characterized by the nuclear clearance of TDP-43 and its associated deposition into cytoplasmic inclusions in different areas of the central nervous system. Given the concomitant occurrence of TDP-43 nuclear depletion and cytoplasmic accumulation, it has been proposed that TDP-43 proteinopathies originate from either a loss-of-function (LOF) mechanism, a gain-of-function (GOF) process, or both. We have addressed this issue by transfecting murine NSC34 and N2a cells with siRNA for endogenous murine TDP-43 and with human recombinant TDP-43 inclusion bodies (IBs). These two strategies allowed the depletion of nuclear TDP-43 and the accumulation of cytoplasmic TDP-43 aggregates to occur separately and independently. Endogenous and exogenous TDP-43 were monitored and quantified using both immunofluorescence and Western blotting analysis, and nuclear functional TDP-43 was measured by monitoring the sortilin 1 mRNA splicing activity. Various degrees of TDP-43 cytoplasmic accumulation and nuclear TDP-43 depletion were achieved and the resulting cellular viability was evaluated, leading to a quantitative global analysis on the relative effects of LOF and GOF on the overall cytotoxicity. These were found to be ∼55% and 45%, respectively, in both cell lines and using both readouts of cell toxicity, showing that these two mechanisms are likely to contribute apparently equally to the pathologies of ALS and FTLD-U., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein misfolded oligomers are considered the most toxic species amongst those formed in the process of amyloid formation and the molecular basis of their toxicity, although not completely understood, is thought to originate from the interaction with the cellular membrane. Here, we sought to highlight the molecular determinants of oligomer-membrane interaction by atomic force microscopy. We monitored the interaction between multiphase supported lipid bilayers and two types of HypF-N oligomers displaying different structural features and cytotoxicities. By our approach we imaged with unprecedented resolution the ordered and disordered lipid phases of the bilayer and different oligomer structures interacting with either phase. We identified the oligomers and lipids responsible for toxicity and, more generally, we established the importance of the membrane lipid component in mediating oligomer toxicity. Our findings support the importance of GM1 ganglioside in mediating the oligomer-bilayer interaction and support a mechanism of oligomer cytotoxicity involving bilayer destabilization by globular oligomers within GM1-rich ordered raft regions rather than by annular oligomers in the surrounding disordered membrane domains., Competing Interests: There is no conflict of interest.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

It is well established that cytotoxic Aβ oligomers are the key factor that triggers the initial tissue and cell modifications eventually culminating in the development of Alzheimer's disease. Aβ1-42 oligomers display a high degree of polymorphism, and several structurally different oligomers have been described. Amongst them, two types, recently classified as A+ and A-, have been shown to possess similar size but distinct toxic properties, as a consequence of their biophysical and structural differences. Here, we have investigated by means of single molecule tracking the oligomer mobility on the plasma membrane of living neuroblastoma cells and the interaction with the ganglioside GM1, a component of membrane rafts. We have found that A+ and A- oligomers display a similar lateral diffusion on the plasma membrane of living cells. However, only the toxic A+ oligomers appear to interact and alter the mobility of GM1. We have also studied the lateral diffusion of each kind of oligomers in cells depleted or enriched in GM1. We found that the content of GM1 influences the diffusion of both types of oligomer, although the effect of the increased levels of GM1 is higher for the A+ type. Interestingly, the content of GM1 also affects significantly the mobility of GM1 molecules themselves., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Nucleophosmin (NPM)-1 is a multifunctional protein involved in a variety of biologic processes and has been implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments in NPM1 activities, we dissected the C-terminal domain (CTD) into its helical fragments. In this study, we observed the unexpected structural behavior of the peptide fragment corresponding to helix (H)2 (residues 264-277). This peptide has a strong tendency to form amyloidlike assemblies endowed with fibrillar morphology and β-sheet structure, under physiologic conditions, as shown by circular dichroism, thioflavin T, and Congo red binding assays; dynamic light scattering; and atomic force microscopy. The aggregates are also toxic to neuroblastoma cells, as determined using 3-(4;5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and Ca(2+) influx assays. We also found that the extension of the H2 sequence beyond its N terminus, comprising the connecting loop with H1, delayed aggregation and its associated cytotoxicity, suggesting that contiguous regions of H2 have a protective role in preventing aggregation. Our findings and those in the literature suggest that the helical structures present in the CTD are important in preventing harmful aggregation. These findings could elucidate the pathogenesis of acute myeloid leukemia (AML) caused by NPM1 mutants. Because the CTD is not properly folded in these mutants, we hypothesize that the aggregation propensity of this NPM1 region is involved in the pathogenesis of AML. Preliminary assays on NPM1-Cter-MutA, the most frequent AML-CTD mutation, revealed its significant propensity for aggregation. Thus, the aggregation phenomena should be seriously considered in studies aimed at unveiling the molecular mechanisms of this pathology., (© FASEB.)

The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.

Oxidative stress has a prominent role in life-span regulation of living organisms. One of the endogenous free radical scavenger systems is associated with glutathione (GSH), the most abundant nonprotein thiol in mammalian cells, acting as a major reducing agent and in antioxidant defense by maintaining a tight control over redox status. We have recently designed a series of novel S-acyl-GSH derivatives capable of preventing amyloid oxidative stress and cholinergic dysfunction in Alzheimer disease models, upon an increase in GSH intake. In this study we show that the longevity of the wild-type N2 Caenorhabditis elegans strain was significantly enhanced by dietary supplementation with linolenoyl-SG (lin-SG) thioester with respect to the ethyl ester of GSH, linolenic acid, or vitamin E. RNA interference analysis and activity inhibition assay indicate that life-span extension was mediated by the upregulation of Sir-2.1, a NAD-dependent histone deacetylase ortholog of mammalian SIRT1. In particular, lin-SG-mediated overexpression of Sir-2.1 appears to be related to the Daf-16 (FoxO) pathway. Moreover, the lin-SG derivative protects N2 worms from the paralysis and oxidative stress induced by Aβ/H2O2 exposure. Overall, our findings put forward lin-SG thioester as an antioxidant supplement triggering sirtuin upregulation, thus opening new future perspectives for healthy aging or delayed onset of oxidative-related diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aggregation of transthyretin (TTR) is known to be linked to the development of systemic and localized amyloidoses. It also appears that TTR exerts a protective role against aggregation of the Aβ peptide, a process linked to Alzheimer's disease. In vitro, both processes correlate with the ability of TTR to populate a monomeric state, yet a complete description of the possible conformational states populated by monomeric TTR in vitro at physiological pH is missing. Using an array of biophysical methods and kinetic tests, we show that once monomers of transthyretin are released from the tetramer, equilibrium is established between a set of conformational states possessing different degrees of disorder. A molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, kinetics, and their pathways of formation. Further subpopulations of the protein fold differently because of the occurrence of proline isomerism. The identification of conformational states unrevealed in previous studies opens the way for further characterization of the amyloidogenicity of TTR and its protective role in Alzheimer's disease.