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ABSTRACT: We developed atomic force microscope (AFM) based protocols that enable isolation and characterization of antibody based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single chain antibody fragments (scFvs) that specifically recognize anoligomeric amyloid-beta (Aβ) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We employedseveral subtractive panning steps to remove all phagebinding non-desired antigens and then employed a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones,again using minimal material, selecting the C6 scFv based on expression levels. We show that C6selectively bindscell and brain derived oligomeric Aβ. The protocols described are readily adapted to isolating antibody based reagents against other antigenic targets with limited availability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
Biotechnology Progress 01/2013; · 2.34 Impact Factor
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ABSTRACT: Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5'-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (>4 nm) oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates) exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD.
PLoS ONE 01/2013; 8(2):e55886. · 4.09 Impact Factor
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ABSTRACT: While accumulation and deposition of beta amyloid (Aβ) is a primary pathological feature of Alzheimer's disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aβ as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aβ have potential diagnostic and therapeutic value. Using a novel biopanning technique that combines phage display technology and atomic force microscopy, we isolated the nanobody E1 against oligomeric Aβ. Here we show that E1 specifically recognizes a small oligomeric Aβ aggregate species distinct from the species recognized by the A4 nanobody previously reported by our group. While E1, like A4, blocks assembly of Aβ into larger oligomeric and fibrillar forms and prevents any Aβ induced toxicity toward neuronal cells, it does so by binding a small Aβ oligomeric species, directing its assembly toward a stable nontoxic conformation. The E1 nanobody selectively recognizes naturally occurring Aβ aggregates produced in human AD brain tissue indicating that a variety of morphologically distinct Aβ aggregate forms occur naturally and that a stable low-n nontoxic Aβ form exists that does not readily aggregate into larger forms. Because E1 catalyses the formation of a stable nontoxic low-n Aβ species it has potential value as a therapeutic reagent for AD which can be used in combination with other therapeutic approaches.
Neurobiology of aging 11/2010; 33(7):1320-8. · 5.94 Impact Factor
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ABSTRACT: 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.
BMC Neuroscience. 01/2010;
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ABSTRACT: 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.
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.
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.
BMC Neuroscience 01/2010; 11:57. · 3.04 Impact Factor
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ABSTRACT: Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of alpha-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of alpha-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of alpha-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 alpha-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 alpha-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of alpha-synuclein in vitro, blocks extracellular alpha-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.
Journal of Biological Chemistry 02/2009; 284(17):11048-58. · 4.77 Impact Factor
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ABSTRACT: 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.
Langmuir 01/2009; 25(2):912-8. · 4.19 Impact Factor
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ABSTRACT: The Amyloid-beta (Abeta) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Abeta are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Abeta using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Abeta, we identified an scFv that bound oligomeric Abeta specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Abeta aggregation and toxicity, and reduces the toxicity of preformed oligomeric Abeta towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Abeta scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Abeta in AD brains.
Journal of Molecular Biology 11/2008; 384(4):917-28. · 4.00 Impact Factor
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ABSTRACT: Protein misfolding and aggregation are a common thread in numerous diseases including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, diabetes, and prion-related diseases. Elucidation of the role played by the various protein forms in these diseases requires reagents that can target specific protein forms. Here we present a method to isolate antibodies that bind to a specific protein form. We combined the imaging and nanomanipulation capabilities of atomic force microscopy (AFM) with the protein diversity of phage display antibody libraries to develop a technology that allows us to recover a single antibody molecule that is bound to a single protein molecular target. The target protein-antibody complex is first imaged by AFM, the AFM tip is then manipulated by nanolithography over the target antibody to recover the associated phage, and the antibody gene is recovered from the single phage particle by polymerase chain reaction.
Nanomedicine: nanotechnology, biology, and medicine 10/2007; 3(3):192-7. · 5.44 Impact Factor
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ABSTRACT: Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-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 alpha-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 alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.
Journal of Molecular Biology 05/2007; 368(4):1132-44. · 4.00 Impact Factor
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ABSTRACT: Isolation of antibodies to antigens that are either unstable, exist in multiple morphologies or have very limited availability can be prohibitively difficult. Here we describe a novel technique combining the capabilities of phage display antibody technology and atomic force microscopy (AFM) that is used to isolate antibody fragments that bind to a specific morphology of the target antigen, alpha-synuclein. AFM imaging allows us to both visualize the presence and morphology of the target antigen as well as to monitor the efficiency of each step in the bio-panning process. We demonstrate that phage displayed antibodies specific to the target antigen morphology can be isolated after only two rounds of selection. The target antigen, alpha-synuclein, has been correlated with the Parkinson's disease (PD). Accumulation of alpha-synuclein fibrillar aggregates into Lewy body inclusions is a hallmark feature of PD. While alpha-synuclein can form several different aggregate morphologies including oligomers, protofibrils and fibrils, the role of these morphologies in the progression of PD is not known. The successful selection of the recombinant antibody described here can have potential therapeutic value since the single-chain fragment variable (scFv) can be expressed intracellularly to control folding and toxicity of the specific protein aggregates.
Protein Engineering Design and Selection 12/2006; 19(11):497-502. · 2.94 Impact Factor
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ABSTRACT: A key event in Alzheimer's disease (AD) pathogenesis is the conversion of the peptide beta-amyloid (Abeta) from its soluble monomeric form into various aggregated morphologies in the brain. Preventing aggregation of Abeta is being actively pursued as a primary therapeutic strategy for treating AD. Trehalose, a simple disaccharide, has been shown to be effective in preventing the deactivation of numerous proteins and in protecting cells against stress. Here, we show that trehalose is also effective in inhibiting aggregation of Abeta and reducing its cytotoxicity, although it shows differential effects toward Abeta40 and Abeta42. When co-incubated with Abeta40, trehalose inhibits formation of both fibrillar and oligomeric morphologies as determined by fluorescence staining and atomic force microscopy (AFM). However, when co-incubated with Abeta42, trehalose inhibits formation only of the fibrillar morphology, with significant oligomeric formation still present. When aggregated mixtures were incubated with SH-SY5Y cells, trehalose was shown to reduce the toxicity of Abeta40 mixtures, but not Abeta42. These results provide additional evidence that aggregation of Abeta into soluble oligomeric forms is a pathological step in AD and that Abeta42 in particular is more susceptible to forming these toxic oligomers than Abeta40. These results also suggest that the use of trehalose, a highly soluble, low-priced sugar, as part of a potential therapeutic cocktail to control Abeta peptide aggregation and toxicity warrants further study.
Neurobiology of Disease 11/2005; 20(1):74-81. · 5.40 Impact Factor
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ABSTRACT: alpha-Synuclein (alpha-syn) has been identified as the major component of Lewy bodies that characterize neurodegenerative synucleinopathies, including Parkinson's disease. Overexpression of alpha-syn, and prefibrillar alpha-syn oligomers, has been implicated in these pathologies; therefore, prevention of prefibril accumulation, and inhibition of other aberrant effects of overexpressed alpha-syn, could provide novel treatments. Here, we have selected a human single-chan Fv (scFv) antibody, D10, that binds human monomeric wild-type alpha-syn. We demonstrate, by retargeting assays and coimmunoprecipitation, that the D10 scFv is a specific and efficient intracellular antibody (intrabody). By transfecting the D10 scFv gene into an HEK 293 cell line that overexpresses wild-type alpha-syn, we show that the D10 intrabody stabilizes detergent-soluble monomeric alpha-syn and inhibits the formation of detergent-insoluble high-molecular-weight alpha-syn species. In addition, the D10 intrabody ameliorates the decreased cell adhesion that characterizes the alpha-syn-overexpressing cells. Given the important role of alpha-syn pathology, and the facility with which intrabodies can be further engineered in vitro, anti-alpha-syn intrabodies may represent novel molecular therapeutics for synucleinopathies, with implications for other neurodegenerative disorders caused by misfolded accumulated proteins.
Molecular Therapy 01/2005; 10(6):1023-31. · 6.87 Impact Factor
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ABSTRACT: Beta-amyloid (Abeta) is a major pathological determinant of Alzheimer's disease (AD). Both active and passive immunization studies have shown that antibodies against Abeta are effective in decreasing cerebral Abeta levels, reducing Abeta accumulation, and attenuating cognitive deficits in animal models of AD. However, the therapeutic potential of these antibodies in human AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhaging associated with the treatments. Here we show that single chain variable fragments (scFv's) represent an attractive alternative to more conventional antibody-based therapeutics to reduce Abeta toxicity. The binding affinities and binding epitopes of two different scFv's to Abeta were characterized using a surface plasmon resonance (SPR) biosensor. An scFv binding the 17-28 region of Abeta effectively inhibited in vitro aggregation of Abeta as determined by thioflavin T (ThT) fluorescence staining and atomic force microscopy (AFM) analysis, while an scFv binding the carboxyl-terminal region of Abeta (residues 29-40) did not inhibit aggregation. The scFv to the 17-28 region when co-incubated with Abeta not only decreased aggregation but also eliminated any toxic effects of aggregated Abeta on the human neuroblastoma cell line, SH-SY5Y. The ability of scFv's to inhibit both aggregation and cytotoxicity of Abeta indicates that scFv's have potential therapeutic value for treating AD.
Biochemistry 07/2004; 43(22):6959-67. · 3.42 Impact Factor
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ABSTRACT: β-Amyloid (Aβ) is a major pathological determinant of Alzheimer's disease (AD). Both active and passive immunization studies have shown that antibodies against Aβ are effective in decreasing cerebral Aβ levels, reducing Aβ accumulation, and attenuating cognitive deficits in animal models of AD. However, the therapeutic potential of these antibodies in human AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhaging associated with the treatments. Here we show that single chain variable fragments (scFv's) represent an attractive alternative to more conventional antibody-based therapeutics to reduce Aβ toxicity. The binding affinities and binding epitopes of two different scFv's to Aβ were characterized using a surface plasmon resonance (SPR) biosensor. An scFv binding the 17−28 region of Aβ effectively inhibited in vitro aggregation of Aβ as determined by thioflavin T (ThT) fluorescence staining and atomic force microscopy (AFM) analysis, while an scFv binding the carboxyl-terminal region of Aβ (residues 29−40) did not inhibit aggregation. The scFv to the 17−28 region when co-incubated with Aβ not only decreased aggregation but also eliminated any toxic effects of aggregated Aβ on the human neuroblastoma cell line, SH-SY5Y. The ability of scFv's to inhibit both aggregation and cytotoxicity of Aβ indicates that scFv's have potential therapeutic value for treating AD.
05/2004;
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ABSTRACT: The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from naïve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.
Biochemistry 04/2004; 43(10):2871-8. · 3.42 Impact Factor
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ABSTRACT: The Amyloid-β (Aβ) peptide is a major component of the amyloid plaques associated with Alzheimer's disease (AD). Recent studies suggest that the most toxic forms of Aβ are small, soluble oligomeric aggregates. Here, we report the isolation and characterization of a single-chain variable domain (scFv) antibody isolated against oligomeric Aβ using a protocol developed in our laboratory that combines phage display technology and atomic force microscopy (AFM). Starting with a randomized, single framework phage display library, after three rounds of selection against oligomeric Aβ, we identified an scFv that bound oligomeric Aβ specifically, but not monomeric or fibrillar forms. The anti-oligomeric scFv inhibits Aβ aggregation and toxicity, and reduces the toxicity of preformed oligomeric Aβ towards human neuroblastoma cells. When used to probe samples of human brain tissue, the scFv reacted with AD tissue but not a healthy control or Parkinson's disease brain samples. The anti-oligomeric Aβ scFv therefore has potential therapeutic and diagnostic applications in specifically targeting or identifying the toxic morphologies of Aβ in AD brains.
Journal of Molecular Biology.
