Benzimidazole-based fluorophores for the detection of amyloid fibrils with higher sensitivity than Thioflavin-T.

Protein aggregation into amyloid fibrils is a key feature of a multitude of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Prion disease. To detect amyloid fibrils, fluorophores with high sensitivity and better efficiency coupled with the low toxicity are in high demand even to date. In this pursuit, we have unveiled two benzimidazole-based fluorescence sensors ([C15 H15 N3 ] (C1) and [C16 H16 N3 O2 ] (C2), which possess exceptional affinity toward different amyloid fibrils in its submicromolar concentration (8 × 10-9  M), whereas under a similar concentration, the gold standard Thioflavin-T (ThT) fails to bind with amyloid fibrils. These fluorescent markers bind to α-Syn amyloid fibrils as well as amyloid fibrils forming other proteins/peptides including Aß42 amyloid fibrils. The 1 H-15 N heteronuclear quantum correlation spectroscopy nuclear magnetic resonance data collected on wild-type α-Syn monomer with and without the fluorophores (C1 and C2) reveal that there is weak or no interactions between C1 or C2 with residues in α-Syn monomer, which indirectly reflects the specific binding ability of C1 and C2 to the α-Syn amyloid fibrils. Detailed studies further suggest that C1 and C2 can detect/bind with the α-Syn amyloid fibril as low as 100 × 10-9  M. Extremely low or no cytotoxicity is observed for C1 and C2 and they do not interfere with α-Syn fibrillation kinetics, unlike ThT. Both C1/C2 not only shows selective binding with amyloid fibrils forming various proteins/peptides but also displays excellent affinity and selectivity toward α-Syn amyloid aggregates in SH-SY5Y cells and Aß42 amyloid plaques in animal brain tissues. Overall, our data show that the developed dyes could be used for the detection of amyloid fibrils including α-Syn and Aß42 amyloids with higher sensitivity as compared to currently used ThT.

Lipopolysaccharide from Gut Microbiota Modulates α-Synuclein Aggregation and Alters Its Biological Function.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

The Familial α-Synuclein A53E Mutation Enhances Cell Death in Response to Environmental Toxins Due to a Larger Population of Oligomers.

Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson's disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early onset aggressive form of PD, which delays α-Syn aggregation. When we overexpressed wild-type (WT) and A53E proteins in cells, showed neither toxicity nor aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cells expressing the A53E mutant might possess enhanced susceptibility to PD-associated toxicants compared to that of the WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and an increased production of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers being formed in cells as confirmed by a dot blot assay using amyloid specific OC and A11 antibody and using an  in vitro aggregation study. The cellular model presented here suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.

Parkinson's Disease Associated α-Synuclein Familial Mutants Promote Dopaminergic Neuronal Death in Drosophila melanogaster.

α-Synuclein (α-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson's disease (PD). In addition, familial mutations in α-Syn are shown to be one of the definite causes of PD. Here we have extensively studied familial PD associated α-Syn G51D, H50Q, and E46K mutations using Drosophila model system. Our data showed that flies expressing α-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immunofluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of α-Syn familial mutations is highly correlated with the sustained population of oligomer production and retention in mutant flies. Furthermore, this was supported by our in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation and retention could be a major cause of cell death in α-Syn familial mutants.

Comparison of Kinetics, Toxicity, Oligomer Formation, and Membrane Binding Capacity of α-Synuclein Familial Mutations at the A53 Site, Including the Newly Discovered A53V Mutation.

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to late-onset PD. In this study, we characterized for the first time the biophysical properties of A53V, including the aggregation propensities, toxicity of aggregated species, and membrane binding capability, along with those of all familial mutations at the A53 position. Our data suggest that the A53V mutation accelerates fibrillation of α-Syn without affecting the overall morphology or cytotoxicity of fibrils compared to those of the wild-type (WT) protein. The aggregation propensity for A53 mutants is found to decrease in the following order: A53T > A53V > WT > A53E. In addition, a time course aggregation study reveals that the A53V mutant promotes early oligomerization similar to the case for the A53T mutation. It promotes the largest amount of oligomer formation immediately after dissolution, which is cytotoxic. Although in the presence of membrane-mimicking environments, the A53V mutation showed an extent of helix induction capacity similar to that of the WT protein, it exhibited less binding to lipid vesicles. The nuclear magnetic resonance study revealed unique chemical shift perturbations caused by the A53V mutation compared to those caused by other mutations at the A53 site. This study might help to establish the disease-causing mechanism of A53V in PD pathology.

Cytotoxic Oligomers and Fibrils Trapped in a Gel-like State of α-Synuclein Assemblies.

α-Synuclein (α-Syn) aggregation is associated with Parkinson's disease (PD) pathogenesis. In PD, the role of oligomers versus fibrils in neuronal cell death is debatable, but recent studies suggest oligomers are a proximate neurotoxin. Herein, we show that soluble α-Syn monomers undergo a transformation from a solution to a gel state on incubation at high concentration. Detailed characterization of the gel showed the coexistence of monomers, oligomers, and short fibrils. In vitro, the gel was highly cytotoxic to human neuroblastoma cells. The individual constituents of the gel are short-lived species but toxic to the cells. They comprise a structurally heterogeneous population of α-helical and ß-sheet-rich oligomers and short fibrils with the cross-ß motif. Given the recent evidence of the gel-like state of the protein associated with neurodegenerative diseases, the gel state of α-Syn in this study represents a mechanistic and structural model for the in vivo toxicity of α-Syn in PD.

p53 amyloid formation leading to its loss of function: implications in cancer pathogenesis.

The transcriptional regulator p53 has an essential role in tumor suppression. Almost 50% of human cancers are associated with the loss of p53 functions, where p53 often accumulates in the nucleus as well as in cytoplasm. Although it has been previously suggested that amyloid formation could be a cause of p53 loss-of-function in subset of tumors, the characterization of these amyloids and its structure-function relationship is not yet established. In the current study, we provide several evidences for the presence of p53 amyloid formation (in human and animal cancer tissues); along with its isolation from human cancer tissues and the biophysical characterization of these tissue-derived fibrils. Using amyloid seed of p53 fragment (P8, p53(250-257)), we show that p53 amyloid formation in cells not only leads to its functional inactivation but also transforms it into an oncoprotein. The in vitro studies further show that cancer-associated mutation destabilizes the fold of p53 core domain and also accelerates the aggregation and amyloid formation by this protein. Furthermore, we also show evidence of prion-like cell-to-cell transmission of different p53 amyloid species including full-length p53, which is induced by internalized P8 fibrils. The present study suggests that p53 amyloid formation could be one of the possible cause of p53 loss of function and therefore, inhibiting p53 amyloidogenesis could restore p53 tumor suppressor functions.

Cytotoxic helix-rich oligomer formation by melittin and pancreatic polypeptide.

Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson's associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail.

The newly discovered Parkinson's disease associated Finnish mutation (A53E) attenuates α-synuclein aggregation and membrane binding.

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson's disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our in vitro study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis.

Characterization of amyloid formation by glucagon-like peptides: role of basic residues in heparin-mediated aggregation.

Glycosaminoglycans (GAGs) have been reported to play a significant role in amyloid formation of a wide range of proteins/peptides either associated with diseases or native biological functions. The exact mechanism by which GAGs influence amyloid formation is not clearly understood. Here, we studied two closely related peptides, glucagon-like peptide 1 (GLP1) and glucagon-like peptide 2 (GLP2), for their amyloid formation in the presence and absence of the representative GAG heparin using various biophysical and computational approaches. We show that the aggregation and amyloid formation by these peptides follow distinct mechanisms: GLP1 follows nucleation-dependent aggregation, whereas GLP2 forms amyloids without any significant lag time. Investigating the role of heparin, we also found that heparin interacts with GLP1, accelerates its aggregation, and gets incorporated within its amyloid fibrils. In contrast, heparin neither affects the aggregation kinetics of GLP2 nor gets embedded within its fibrils. Furthermore, we found that heparin preferentially influences the stability of the GLP1 fibrils over GLP2 fibrils. To understand the specific nature of the interaction of heparin with GLP1 and GLP2, we performed all-atom MD simulations. Our in silico results show that the basic-nonbasic-basic (B-X-B) motif of GLP1 (K28-G29-R30) facilitates the interaction between heparin and peptide monomers. However, the absence of such a motif in GLP2 could be the reason for a significantly lower strength of interaction between GLP2 and heparin. Our study not only helps to understand the role of heparin in inducing protein aggregation but also provides insight into the nature of heparin-protein interaction.

The Parkinson's disease-associated H50Q mutation accelerates α-Synuclein aggregation in vitro.

α-Synuclein (α-Syn) aggregation is directly linked with Parkinson's disease (PD) pathogenesis. Here, we analyzed the aggregation of newly discovered α-Syn missense mutant H50Q in vitro and found that this mutation significantly accelerates the aggregation and amyloid formation of α-Syn. This mutation, however, did not alter the overall secondary structure as suggested by two-dimensional nuclear magnetic resonance and circular dichroism spectroscopy. The initial oligomerization study by cross-linking and chromatographic techniques suggested that this mutant oligomerizes to an extent similar to that of the wild-type α-Syn protein. Understanding the aggregation mechanism of this H50Q mutant may help to establish the aggregation and phenotypic relationship of this novel mutant in PD.

Curcumin modulates α-synuclein aggregation and toxicity.

In human beings, Parkinson's disease (PD) is associated with the oligomerization and amyloid formation of α-synuclein (α-Syn). The polyphenolic Asian food ingredient curcumin has proven to be effective against a wide range of human diseases including cancers and neurological disorders. While curcumin has been shown to significantly reduce cell toxicity of α-Syn aggregates, its mechanism of action remains unexplored. Here, using a series of biophysical techniques, we demonstrate that curcumin reduces toxicity by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure. Further, our fluorescence and two-dimensional nuclear magnetic resonance (2D-NMR) data indicate that curcumin does not bind to monomeric α-Syn but binds specifically to oligomeric intermediates. The degree of curcumin binding correlates with the extent of α-Syn oligomerization, suggesting that the ordered structure of protein is required for effective curcumin binding. The acceleration of aggregation by curcumin may decrease the population of toxic oligomeric intermediates of α-Syn. Collectively; our results suggest that curcumin and related polyphenolic compounds can be pursued as candidate drug targets for treatment of PD and other neurological diseases.