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Abstract Background Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinson's disease (PD) and other synucleinopathies. αS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results Here we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra- or extracellulary. Conclusions Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.

Many polymer-stabilized silver nanoparticles (AgNPs) with enhanced antibacterial properties have been synthesized, but very little is known about the fate of these materials and their interactions with microbes in physiological solutions. In this study, we evaluated the role of cellulose nanofibrils (CNFs) in stabilizing AgNPs (CNF-AgNPs) in a bacterial growth medi µm, determined the antibacterial properties of CNF-AgNPs and assessed the CNF and CNF-AgNP interactions with bacteria. The attachment of AgNPs to CNFs significantly improved the stability and dispersibility of AgNPs in the bacterial growth medium compared to colloidal AgNPs. The CNF-AgNPs exerted a concentration-dependent growth-inhibitory effect on E. coli. An extracellular polysaccharide (EPS)-like structure was formed around the E. coli bacterium when incubated with a sub-lethal CNF-AgNP concentration, which led to the clustering of neighboring bacteria. No EPS-like structure was observed around the bacterium incubated with high concentration of CNF-AgNPs. Overall, this study demonstrated that CNFs significantly improved the stability and dispersibility of AgNPs in physiological medi µm, validated the antimicrobial potential of CNF-AgNPs, and provided an insight into the physio-chemical interactions between bacteria and CNF- AgNPs in a physiological growth medium. [ABSTRACT FROM AUTHOR]

Background: Overexpression and abnormal accumulation of aggregated a-synuclein (aS) have been linked to Parkinson's disease (PD) and other synucleinopathies. aS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase aS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results: Here we show that curcumin can alleviate aS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of aS and extracellular addition of oligomeric aS increase ROS which induces apoptosis, suggesting that aggregated aS may induce similar toxic effects whether it is generated intra- or extracellulary. Conclusions: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Abstract: The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimer’s (AD) and Niemann-Pick type C (NPC) diseases. NFTs have been correlated with elevated cholesterol levels and a cholesterol-scavenging compound, cyclodextrin, effectively modulates and traffics cholesterol from cell bodies in NPC disease models. Cyclodextrins are also used as drug carriers to the blood–brain barrier (BBB) and other tissues. While cyclodextrins have potential value in treating brain diseases, it is important to determine how cyclodextrins affect natively unfolded proteins such as beta-amyloid (Aβ) whose aggregation has been correlated with AD. We show that cyclodextrins drastically alter Aβ aggregation kinetics and induce morphological changes to Aβ that can enhance toxicity towards SH-SY5Y human neuroblastoma cells. These results suggest that care must be taken when using cyclodextrins for BBB delivery or for treatment of brain disease because cyclodextrins can promote toxic aggregation of Aβ. [Copyright &y& Elsevier]

Background: Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely a-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes. Methodology/Principal Findings: Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of α-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains. Conclusions/Significance: These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases. [ABSTRACT FROM AUTHOR]

Abstract: The distal to proximal degeneration of axons, or “dying back” is a common pattern of neuropathology in many diseases of the PNS and CNS. A long-standing debate has centered on whether this pattern of neurodegeneration is due to an insult to the cell body or to the axon itself, although it is likely that mechanisms are different for specific disease entities. We have addressed this question in a model system of vincristine-induced axonal degeneration. Here, we created a novel experimental apparatus combining a microfluidic divider with a multielectrode array substrate to allow for independent monitoring of injury-induced electrical activity from dorsal root ganglion (DRG) cell bodies and axons while isolating them into their own culture microenvironments. At specified doses, exposure of the cell body to vincristine caused neither morphological neurodegeneration nor persistent hyperexcitablility. In comparison, exposure of the distal axon to the same dose of vincristine first caused a decrease in the excitability of the axon and then axonal degeneration in a dying back pattern. Additionally, exposure of axons to vincristine caused an initial period of hyperexcitability in the cell bodies, suggesting that a signal is transmitted from the distal axon to the soma during the progression of vincristine-induced axonal degeneration. These data support the proposition that vincristine has a direct neurotoxic effect on the axon. [Copyright &y& Elsevier]

Abstract: This paper describes a microfabricated compartmented culture system (μ-CCS) for studying the effects of drugs on cultured neurons. We describe the fabrication of the microsystem and show the ability to culture DRG neurons in the microsystem. Furthermore, we demonstrate the ability to culture neurons with extensions growing into adjoining compartments while maintaining fluid isolation between compartments. The axonal growth pattern was controlled along the surface of the glass microelectrode substrate using a micropatterned collagen film. The ability to isolate fluids to selected compartments while simultaneously allowing intercompartmental growth of the axons enables various studies in which selected segments of neurons or populations of neurons can be selectively exposed to biochemical treatment. The neurotoxin vincristine was used as the test vehicle to demonstrate the functionality of the μ-CCS. Vincristine was applied to the axonal compartment to show that the interaction of drugs with DRG neurons progresses in a way similar to neurons cultured/exposed using conventional techniques. [Copyright &y& Elsevier]

Abstract: A simple method to generate cell microarrays with high-percentage well occupancy and well-defined cell confinement is presented. This method uses a synergistic combination of vacuum degassing and coverslip sweeping. The vacuum degassing step dislodges air bubbles from the microwells, which in turn enables the cells to enter the microwells, while the physical sweeping step using a glass coverslip removes the excess cells outside the microwells. This low-cost preparation method provides a simple solution to generating cell microarrays that can be performed in basic research laboratories and point-of-care settings for routine cell-based screening assays. [Copyright &y& Elsevier]

Background: Overexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models.Results: Here we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary.Conclusions: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SYSY), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue. [ABSTRACT FROM AUTHOR]

Abstract: Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of α-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson''s Disease (PD). A natively unfolded protein, α-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric α-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of α-synuclein. The scFv described here binds only to an oligomeric form of α-synuclein and inhibits both aggregation and toxicity of α-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of α-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody. [Copyright &y& Elsevier]

A highly ordered array of T7 bacteriophages was created by the electrophoretic capture of phages onto a nanostructured array with wells that accommodated the phages. Electrophoresis of bacteriophages was achieved by applying a positive potential on an indium tin oxide electrode at the bottom of the nanowells. Nanoscale arrays of phages with different surface densities were obtained by changing the electric field applied to the bottom of the nanowells. The applied voltage was shown to be the critical factor in generating a well-ordered phage array. The number of wells occupied by a phage, and hence the concentration of phages in a sample solution, could be quantified by using a DNA intercalating dye that rapidly stains the T7 phage. The fluorescence signal was enhanced by the intrinsic photonic effect made available by the geometry of the platform. It was shown that the quantification of phages on the array was 6 orders of magnitude better than could be obtained with a fluorescent plate reader. The device opens up the possibility that phages can be detected directly without enrichment or culturing, and by detecting phages that specifically infect bacteria of interest, rapid pathogen detection becomes possible.

It has been reported that the oxidation of phosphatidylcholine (PC) is necessary for C-reactive protein (CRP) to bind to lipid membranes, but it remains elusive why CRP only binds oxidized membranes. Here we offer a new perspective on the role of membrane curvature and CRP binding using engineered lipoprotein particle (LPP) mimics. We show that CRP binds preferentially to LPP mimics with diameters of ≤ 28 nm, and binding of CRP to these mimics leads to the dissociation of native CRP into monomeric CRP, exposing CRP neo-epitopes that bind C1q. We also show that the smaller LPP mimics compete for CRP binding to oxidized low density lipoproteins (oxLDLs), suggesting that these mimics expose the same PC epitopes as those found on oxLDLs. Results from this study suggest that membrane curvature could be an additional factor influencing CRP binding of damaged membranes distinct from the oxidation of PC lipids.

Lipoprotein particles (LPPs) are biological nanoparticles whose physiological roles are greatly influenced by their sizes. The four major classes of LP are: very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein (LDL) and high density lipoprotein. Since the predominance of small, dense LDLs is associated with increased risk of coronary artery disease (CAD) and diabetes mellitus, LPP profiling can be used to predict metabolic risk factors. Highly tunable LPP mimics can be synthesized using nanoparticles to carefully control for size, lipid composition and surface charge to facilitate the study LPPs in CAD. Here, we engineered LPP mimics using gold nanoparticles between 10-50 nm in diameters. We measured the mobility and zeta potential of these LPP mimics and showed that each mimics have distinct electrokinetic properties and are electrostatically stable.

Protein misfolding and aggregation can lead to several neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD). While the respective proteins involved in each disease differ in their pathological effects and amino acid sequences, the aggregated forms all share a common cross beta-sheet conformation. Substantial controversy exists over the roles of the different aggregate morphologies in disease onset and progression, and analytical tools such as morphology specific antibodies are needed to distinguish between the different protein morphologies in situ. Here we utilize atomic force microscopy (AFM) to characterize the binding of three single chain antibody fragments (scFvs) to different morphologies of alpha-synuclein (alphaS). From the topographic images generated using the AFM, we were able to show that one scFv bound all morphologies of alphaS, a second bound only oligomeric alphaS, and a third bound only fibrillar alphaS by comparing the height distribution of the different alphaS morphologies with and without addition of the different scFvs. These results demonstrate the versatility of the AFM-based technique as an easy tool to characterize specific antigen-antibody binding and the potential applications of scFvs as promising immunodiagnostics for protein misfolding diseases.

This paper describes the design, fabrication, and characterization of a microfabricated compartmented culture system (micro-CCS) useful for electrophysiological signaling studies in cultured neurons. The focus of the paper is the process of interfacing the micro-CCS with cultured neurons and to demonstrate the applicability of the system for biochemical-mediated electrophysiological studies. Moreover, we show that we can record action potentials from cultured neurons through the extracellular compartmented application of elevated levels of K(+) ions. Finally, we show that we can isolate the electrophysiological effects of the sodium channel blocker tetrodotoxin in one of the compartments of a two compartment culture while recording electrophysiological data from both compartments.