A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux.

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

Z-scan analysis and theoretical studies of dopamine under physiological conditions.

Dopamine (DA) is a widely researched catecholamine best known for its role in motor, motivation, addiction, and reward. Disruption in dopamine homeostasis and signaling within the central nervous system (CNS) can lead to disorders such as attention deficit hyperactivity disorder (ADHD), schizophrenia, Parkinson's disease, and obsessive-compulsive disorder. In the periphery, circulating DA is stored in blood platelets, and its disruption correlates with pathological conditions such as head and neck paragangliomas, Huntington's chorea, and schizophrenia. Various methods to sensitively and selectively detect dopamine have been reported, but sparse attempts have been made to exploit its intrinsic properties. Previously, we have harnessed dopamine's natural mid-ultraviolet auto-fluorescence to carry out its label-free imaging in live brain tissues. Recently, we used the closed-aperture (CA) Z-scan method to provide the first line of evidence on the existence of dopamine nonlinearity. Here, we utilized this simple, sensitive, and straightforward CA Z-scan technique and coupled this with theoretical simulations to further investigate the nonlinear photophysical properties of DA under physiological conditions. Our combined approach revealed that the nonlinear property of dopamine is governed by the thermo-optical effects, and the CA Z-scan profiles can be modulated by parameters such as phase-shift, orders of absorption, and time dependency. Simple and physiologically relevant systems, such as the platelets, are amenable to Z-scan analysis, thereby empowering us to scrutinize in the future if nonlinearity and its alterations, if any, have a direct bearing on DA homeostasis and associated diseases.

Norepinephrine exhibits thermo-optical nonlinearity under physiological conditions.

Norepinephrine (NE), a crucial modulatory neurotransmitter, plays a significant role in human physiology. Here, we use the Z-scan technique to investigate the nonlinear properties of NE at physiological conditions. Results reveal that NE exhibits thermo-optical nonlinearity. Outcomes can be utilized to investigate noradrenergic processes in correlation with various diseases.

A simulation study on the influence of energy migration and relative interaction strengths of homo- and hetero-FRET on the net FRET efficiency.

Förster resonance energy transfer (FRET) is a powerful method for probing biomolecular conformations and dynamics in bulk as well as at a single-molecule level. FRET utilizes non-radiative mechanisms to transfer energy between fluorophores, donor and acceptor when placed in close proximity. The FRET efficiency has a strong distance dependence and serves as a direct read-out for molecular interaction. In case of a significant overlap of donor emission and absorption spectra, the excited state energy can be exchanged between the identical donors in close proximity, which eventually migrates back and forth until it gets dissipated. This form of energy transfer is called energy migration or homo-FRET. Here, we have simulated FRET efficiency by considering the donor-donor interaction strength (ξDD) and donor-acceptor interaction strength (ξDA) under conditions of non-uniform distribution of molecules. Our earlier studies indicate that energy migration modulate the FRET efficiency for various values of ξDD and ξDA. We, therefore, determined the limiting values of acceptor concentration (CLA) that will allow the determination of FRET efficiency in the absence and presence of energy migration. Taken together, our study optimizes the conditions for meaningful FRET efficiency for a given FRET pair for better reporting of molecular interactions.

Probing third-order nonlinearity in serotonin: A Z-scan study.

Serotonin (5-hydroxytryptamine, 5-HT) is a crucial endogenous monoamine neurotransmitter that modulates neurotransmission, gastrointestinal motility, hemostasis, and cardiovascular integrity. There have been numerous attempts to study the biochemical and photophysical properties of serotonin to carry out its molecular imaging and quantitative estimation. Here, we investigate the properties of serotonin at physiological concentration and pH using a continuous wave (CW) laser excitation closed-aperture (CA) Z-scan technique. Serotonin is packaged at high concentration inside the acidic environment of vesicles, and upon release gets diluted at the release sites in a neutral pH environment. Our solution-based measurements indicate that serotonin showed negative refractive nonlinearity and positive absorptive nonlinearity at a neutral pH. However, in the acidic medium, it showed negative refractive nonlinearity and mostly negative absorptive nonlinearity. The effect of excitation laser power on the observed nonlinearity is also verified. We attribute the origin of the nonlinearity in serotonin to the thermal lensing effect. Our robust and straightforward strategy to probe the monoamine neurotransmitter properties will provide new avenues to investigate serotonergic processes.

Ordered and Disordered Segments of Amyloid-ß Drive Sequential Steps of the Toxic Pathway.

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aß). Aß oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aß fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aß10-40 or Aß1-9, is toxic by itself. The ordered part (Aß10-40) is the major determinant of how Aß attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aß enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aß to the toxic pathway. Our findings correlate well with Aß sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.

Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP-hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP-hDAT co-exist and that higher-order molecular complexes of mGFP-hDAT are absent at the plasma membrane. The mGFP-hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP-hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP2) stabilized the oligomers, the stability of mGFP-hDAT dimers was PIP2 independent.

Development of a Next-Generation Fluorescent Turn-On Sensor to Simultaneously Detect and Detoxify Mercury in Living Samples.

Strategies for simultaneous detection and detoxification of Hg2+ using a single sensor from biological and environmental samples are limited and have not been realized in living organisms so far. We report a highly selective, small molecule "turn-on" fluorescent sensor, PYDMSA, based on the cationic dye Pyronin Y (PY) and chelating agent meso-2,3-dimercaptosuccinic acid (DMSA) for the simultaneous detection and detoxification of inorganic mercury (Hg2+). After Hg2+ detection, concomitant detoxification was carried out with sufficient efficacy in living samples, which makes the sensor unique. PYDMSA exhibits high selectivity for Hg2+ over other competing metal ions with an experimental detection limit of ∼300 pM in aqueous buffer solution. When PYDMSA reacts with Hg2+, the CS-C9 bond in the sensor gets cleaved. This results in the "turn-on" response of the fluorescence probe with a concomitant release of one equivalent of water-soluble Hg2+-DMSA complex which leads to a synchronous detoxifying effect. The sensor by itself is nontoxic to cells in culture and has been used to monitor the real-time uptake of Hg2+ in live cells and zebrafish larvae. Thus, PYDMSA is a unique sensor which can be used to detect and detoxify mercury at the same time in living samples.

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.

Label-Free Ratiometric Imaging of Serotonin in Live Cells.

Ratiometric imaging can quantitatively measure changes in cellular analyte concentrations using specially designed fluorescent labels. We describe a label-free ratiometric imaging technique for direct detection of changes in intravesicular serotonin concentration in live cells. At higher concentrations, serotonin forms transient oligomers whose ultraviolet emission is shifted to longer wavelengths. We access the ultraviolet/blue emission using relatively benign three-photon excitation and split it into two imaging channels, whose ratio reports the concentration. The technique is sensitive at a physiologically relevant concentration range (10-150 mM serotonin). As a proof of principle, we measure the increase of intravesicular serotonin concentration with the addition of external serotonin. In general, since emission spectra of molecules are often sensitive to concentration, our method may be applicable to other natively fluorescent intracellular molecules which are present at high concentrations.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

Effect of amyloids on the vesicular machinery: implications for somatic neurotransmission.

Certain neurodegenerative diseases are thought to be initiated by the aggregation of amyloidogenic proteins. However, the mechanism underlying toxicity remains obscure. Most of the suggested mechanisms are generic in nature and do not directly explain the neuron-type specific lesions observed in many of these diseases. Some recent reports suggest that the toxic aggregates impair the synaptic vesicular machinery. This may lead to an understanding of the neuron-type specificity observed in these diseases. A disruption of the vesicular machinery can also be deleterious for extra-synaptic, especially somatic, neurotransmission (common in serotonergic and dopaminergic systems which are specifically affected in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively), though this relationship has remained unexplored. In this review, we discuss amyloid-induced damage to the neurotransmitter vesicular machinery, with an eye on the possible implications for somatic exocytosis. We argue that the larger size of the system, and the availability of multi-photon microscopy techniques for directly visualizing monoamines, make the somatic exocytosis machinery a more tractable model for understanding the effect of amyloids on all types of vesicular neurotransmission. Indeed, exploring this neglected connection may not just be important, it may be a more fruitful route for understanding AD and PD.

Rapid, cell-free assay for membrane-active forms of amyloid-ß.

Small oligomers of amyloid beta (Aß) are suspected to be the key to Alzheimer's disease (AD). However, identifying these toxic species in the background of other similar but nontoxic Aß aggregates has remained a challenge. Recent studies indicate that Aß undergoes a global structural transition in an early step of aggregation. This transition is marked by a strong increase in its affinity for cell membranes, which suggests that the resultant oligomers could be the key to Aß toxicity. Here we use this increased membrane affinity to develop a rapid, quantitative, cell-free assay for these bioactive oligomers. It uses fluorescence correlation spectroscopy of fluorescently labeled Aß and requires only 30 s of measurement time. We also describe a simpler (though less rapid) assay based on the same principles, which uses a dialysis step followed by conventional fluorescence spectroscopy. Our results potentially provide a much-needed high-throughput assay for AD drug development.

Curcumin alters the salt bridge-containing turn region in amyloid ß(1-42) aggregates.

Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß 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 ß(1-42) (Aß42) 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 Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)

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.

Development of a rhodamine-rhodanine-based fluorescent mercury sensor and its use to monitor real-time uptake and distribution of inorganic mercury in live zebrafish larvae.

We introduce a new rhodamine-rhodanine-based "turn-on" fluorescent sensor (RR1) and describe its application for detection of mercury, including in solution, in live cells, and in a living vertebrate organism. The sensor RR1, which is a one-pot synthesis from rhodamine B, undergoes a rapid and irreversible 1:1 stoichiometric reaction with Hg(2+) in aqueous medium. Using fluorescence correlation spectroscopy (FCS), RR1 was shown to detect the presence of as low as a 0.5 pM concentration of Hg(2+). It may also lend itself to tagging with biomolecules and nanoparticles, leading to the possibility of organelle-specific Hg detection. Results of experiments with mammalian cells and zebrafish show that RR1 is cell and organism permeable and that it responds selectively to mercury ions over other metal ions. In addition, real-time monitoring of inorganic mercury ion uptake by cells and live zebrafish using this chemosensor shows that saturation of mercury ion uptake occurs within 20-30 min in cells and organisms. We also demonstrate the acquisition of high-resolution real-time distribution maps of inorganic mercury (Hg(2+)) in the zebrafish brain by using a simple fluorescence confocal imaging technique.

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 µm(2)/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 µm(2)/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.