Bidyut Sarkar

Nature of the Amyloid-β Monomer and the Monomer-Oligomer Equilibrium

The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-β (Aβ) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both Aβ40 and Aβ42 remain predominantly monomeric up to 3 μm, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If Aβ oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

Significant Structural Differences between Transient Amyloid‐β Oligomers and Less‐Toxic Fibrils in Regions Known To Harbor Familial Alzheimer′s Mutations

Small oligomers of the amyloid β (Aβ) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimers disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aβ40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aβ mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Curcumin Alters the Salt Bridge-containing Turn Region in Amyloid β(1–42) Aggregates

Background: Curcumin reduces the risk of Alzheimer disease via an unknown mechanism. Results: Curcumin-incubated Aβ42 aggregates retain the hairpin architecture but have disruptions in the turn region (surprising similarity with Zn2+ incubation). Conclusion: Salt bridge-containing turn region is a major determinant of morphology and toxicity. Significance: Identification of crucial structural changes provides a checkpoint for developing effective AD therapeutics. Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn2+ ions, another agent known to disrupt the fibrils and alter Aβ42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ42, which is stabilized both by curcumin and by Zn2+.

Thermodynamically stable amyloid-β monomers have much lower membrane affinity than the small oligomers

Amyloid beta (Aβ) is an extracellular 39–43 residue long peptide present in the mammalian cerebrospinal fluid, whose aggregation is associated with Alzheimers disease (AD). Small oligomers of Aβ are currently thought to be the key to toxicity. However, it is not clear why the monomers of Aβ are non-toxic, and at what stage of aggregation toxicity emerges. Interactions of Aβ with cell membranes is thought to be the initiator of toxicity, but membrane binding studies with different preparations of monomers and oligomers have not settled this issue. We have earlier found that thermodynamically stable Aβ monomers emerge spontaneously from oligomeric mixtures upon long term incubation in physiological solutions (Nag et al., 2011). Here we show that the membrane-affinity of these stable Aβ monomers is much lower than that of a mixture of monomers and small oligomers (containing dimers to decamers), providing a clue to the emergence of toxicity. Fluorescently labeled Aβ40 monomers show negligible binding to cell membranes of a neuronal cell line (RN46A) at physiological concentrations (250 nM), while oligomers at the same concentrations show strong binding within 30 min of incubation. The increased affinity most likely does not require any specific neuronal receptor, since this difference in membrane-affinity was also observed in a somatic cell-line (HEK 293T). Similar results are also obtained for Aβ42 monomers and oligomers. Minimal amount of cell death is observed at these concentrations even after 36 h of incubation. It is likely that membrane binding precedes subsequent slower toxic events induced by Aβ. Our results (a) provide an explanation for the non-toxic nature of Aβ monomers, (b) suggest that Aβ toxicity emerges at the initial oligomeric phase, and (c) provide a quick assay for monitoring the benign-to-toxic transformation of Aβ.

The Dynamics of Somatic Exocytosis in Monoaminergic Neurons

Some monoaminergic neurons can release neurotransmitters by exocytosis from their cell bodies. The amount of monoamine released by somatic exocytosis can be comparable to that released by synaptic exocytosis, though neither the underlying mechanisms nor the functional significance of somatic exocytosis are well understood. A detailed examination of these characteristics may provide new routes for therapeutic intervention in mood disorders, substance addiction, and neurodegenerative diseases. The relatively large size of the cell body provides a unique opportunity to understand the mechanism of this mode of neuronal exocytosis in microscopic detail. Here we used three photon and total internal reflection fluorescence microscopy to focus on the dynamics of the pre-exocytotic events and explore the nature of somatic vesicle storage, transport, and docking at the membrane of serotonergic neurons from raphe nuclei of the rat brain. We find that the vesicles (or unresolved vesicular clusters) are quiescent (mean square displacement, MSD ∼0.04 μm2/s) before depolarization, and they move minimally (<1 μm) from their locations over a time-scale of minutes. However, within minutes of depolarization, the vesicles become more dynamic (MSD ∼0.3 μm2/s), and display larger range (several μm) motions, though without any clear directionality. Docking and subsequent exocytosis at the membrane happen at a timescale (∼25 ms) that is slower than most synaptic exocytosis processes, but faster than almost all somatic exocytosis processes observed in endocrine cells. We conclude that, (A) depolarization causes de-tethering of the neurotransmitter vesicles from their storage locations, and this constitutes a critical event in somatic exocytosis; (B) their subsequent transport kinetics can be described by a process of constrained diffusion, and (C) the pre-exocytosis kinetics at the membrane is faster than most other somatic exocytosis processes reported so far.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Aβ self-assembles into parallel cross-β fibrillar aggregates, which is associated with Alzheimers disease pathology. A central hairpin turn around residues 23–29 is a defining characteristic of Aβ in its aggregated state. Major biophysical properties of Aβ, including this turn, remain unaltered in the central fragment Aβ18–35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aβ18–35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aβ18–35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe19 and Leu34 residues, observed in full-length Aβ and Aβ18–35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel β-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel β-sheet. We conclude that the self-assembly of Aβ is critically dependent on the hairpin turn and on the contact between the Phe19 and Leu34 regions, making them potentially sensitive targets for Alzheimers therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Label-free dopamine imaging in live rat brain slices.

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.

Polyunsaturated fatty acids induce polarized submembranous F-actin aggregates and kill Entamoeba histolytica.

We have recently identified a novel galacto‐glycerolipid (GGL) from the plant Oxalis corniculata that killed the human pathogen Entamoeba histolytica. In this study, we show that the anti‐amoebic activity of GGL was due to the polyunsaturated fatty acid α‐linolenic acid (C18:3) side chain. Treatment of α‐linolenic acid to E. histolytica trophozoites disrupted the cytoskeletal network and led to polarization of F‐actin at one end of the cells with prominent filopodial extensions. In addition, clustering of surface receptors and signaling molecules was also observed adjacent to the polarized actin similar to concanavalin‐A‐(Con‐A) induced capping. But, in contrast to Con‐A‐induced capping, α‐linolenic acid induced caps were not shed and showed accumulation of long and numerous filopodia at the cap site. We found that α‐linolenic acid disrupts the actin cytoskeletal network, which led to the detachment of plasma membrane from the underlying cytoskeleton. A similar effect was observed with other dietary fatty acids such as linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (C20:5), and docosahexaenoic acid (C22:6). Our findings showed that dietary polyunsaturated fatty acids are powerful anti‐amoebic agents that lead to disruption of the actin cytoskeleton.

Fluorogenic Detection of Monoamine Neurotransmitters in Live Cells

Monoamine neurotransmission is key to neuromodulation, but imaging monoamines in live neurons has remained a challenge. Here we show that externally added ortho-phthalaldehyde (OPA) can permeate live cells and form bright fluorogenic adducts with intracellular monoamines (e.g., serotonin, dopamine, and norepinephrine) and with L-DOPA, which can be imaged sensitively using conventional single-photon excitation in a fluorescence microscope. The peak excitation and emission wavelengths (λex = 401 nm and λem = 490 nm for serotonin; λex = 446 nm and λem = 557 nm for dopamine; and λex = 446 nm and λem = 544 nm for norepinephrine, respectively) are accessible to most modern confocal imaging instruments. The identity of monoamine containing structures (possibly neurotransmitter vesicles) in serotonergic RN46A cells is established by quasi-simultaneous imaging of serotonin using three-photon excitation microscopy. Mass spectrometry of cell extracts and of in vitro solutions helps us identify the chemical nature of the adducts and establishes the reaction mechanisms. Our method has low toxicity, high selectivity, and the ability to directly report the location and concentration of monoamines in live cells.

Multiphoton ultraviolet microscopy images dopamine dynamics in live brain tissue

The selection of NEs based on sensitivity analysis, employing Artificial Neural Network (ANN) using NeuroSolutions V5, was conceded. Ligand selection was then followed by a detailed interaction studies using a more focused fragment-based geometrical optimization (isobolographic analysis) which was further later substantiated through the use of docking studies (Glide 4.0) and design of experiments. Results and discussion: Curcumin and glycosylated nornicotine demonstrated higher sensitivities toward energy minimizations with A P based upon the Mean Square Error and input–output mapping via ANN (Fig. 2). Interestingly, curcumin formed Hbonds with the alanine residues and were capable of binding to the aliphatic amino acids residues (A 12–28; Glide score: −3.79) whereas the glucose side-chain of glycosylated nornicotine exhibited H-bonding with histidine and phenylalanine (AMBER) and with glutamine, phenylalanine and aspartic acid during the docking studies (Glide score: −2.89; Fig. 3). A highly synergistic interaction was observed, using an isobolographic analysis and Loewe additivity relationship (Quantitative parameter ( ): 0.45),displaying a possible reduction in individual effective concentration by a factor of 4 and 5, respectively, without compromising and even substantiating the therapeutic benefit. A neuroprotective combination of three molecules of Curcumin and three molecules of Glyconornicotine was proposed by the DoE model indicating a possible 1:1 combination with maximum of three molecules of each NE per A P oligomer. Conclusion: Our work offers a novel mathematical and in silico approach that constitutes a new frontier in providing neuroscientists with a template for in vitro and in vivo molecular experimentation.