-
[show abstract]
[hide abstract]
ABSTRACT: This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid-β peptide (Aβ). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer's disease (AD). Past decades of AD research highlight the dependence of Aβ42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aβ42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aβ. To develop homogenous Aβ42 assemblies, we initially monomerize the peptide, erasing any "structural history" that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aβ42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aβ42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aβ42. We also present a protocol for fluorescently labeling oligomeric Aβ42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections.
Methods in molecular biology (Clifton, N.J.) 01/2011; 670:13-32.
-
01/2008: pages 211 - 243; , ISBN: 9783527619344
-
[show abstract]
[hide abstract]
ABSTRACT: Amyloid-beta (Abeta) is causally implicated in Alzheimer's disease and neuroplasticity failure has acquired validity as a possible mechanism of early AD pathogenesis. We have previously demonstrated that oligomeric Abeta(1-42) inhibits LTP in the dentate gyrus of rat hippocampal slices. We now show, using whole cell recordings in hippocampal granule cells, that oligomeric Abeta(1-42) decreases neuronal excitability. In particular, Abeta(1-42) application was associated with a decrease in the number of action potentials fired in response to current injection, and with an increase in the amplitude of the afterhyperpolarization. Reduced excitability may underlie the Abeta-mediated impairment in neuroplasticity, and ultimately may contribute to the memory loss in Alzheimer disease.
Neuroscience Letters 08/2006; 403(1-2):162-5. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent studies have shown that the lipidation and assembly state of apolipoprotein E (apoE) determine receptor recognition and amyloid-beta peptide (Abeta) binding. We previously demonstrated that apoE secreted by HEK cells stably expressing apoE3 or apoE4 (HEK-apoE) binds Abeta and inhibits Abeta-induced neurotoxicity by an isoform-specific process that requires apoE receptors. Here we characterized the structure of HEK-apoE assemblies and determined their receptor binding specificity. By chromatography, HEK-apoE elutes in high molecular mass fractions and is the size of plasma HDL, consistent with a multiprotein assembly. No lipid was associated with these apoE assemblies. Several methods for analyzing receptor binding indicate that HEK-apoE is a ligand for low-density lipoprotein (LDL) receptor-related protein (LRP) but not the LDL receptor. This suggests that self-assembly of apoE may induce a functional conformation necessary for binding to LRP. Our results indicate that, in addition to lipid content, the assembly state of apoE influences Abeta binding and receptor recognition.
Biochemistry 02/2006; 45(2):381-90. · 3.42 Impact Factor
-
Desiree Watson,
Eduardo Castaño,
Tyler A Kokjohn,
Yu-Min Kuo,
Yuri Lyubchenko,
David Pinsky,
E Sander Connolly,
Chera Esh,
Dean C Luehrs, W Blaine Stine,
Linda M Rowse,
Mark R Emmerling,
Alex E Roher
[show abstract]
[hide abstract]
ABSTRACT: Extracellular fibrillar amyloid deposits are prominent and universal Alzheimer's disease (AD) features, but senile plaque abundance does not always correlate directly with the degree of dementia exhibited by AD patients. The mechanism(s) and dynamics of Abeta fibril genesis and deposition remain obscure. Enhanced Abeta synthesis rates coupled with decreased degradative enzyme production and accumulating physical modifications that dampen proteolysis may all enhance amyloid deposit formation. Amyloid accumulation may indirectly exert the greatest pathologic effect on the brain vasculature by destroying smooth muscle cells and creating a cascade of negative impacts on cerebral blood flow. The most visible manifestation of amyloid dis-equilibrium could actually be a defense mechanism employed to avoid serious vascular wall degradation while the major toxic effects to the gray and white matter neurons are mediated by soluble oligomeric Abeta peptides with high beta-sheet content. The recognition that dynamic soluble oligomeric Abeta pools exist in AD and are correlated to disease severity led to neurotoxicity and physical conformation studies. It is now recognized that the most basic soluble Abeta peptides are stable dimers with hydrophobic regions sequestered from the aqueous environment and are capable of higher order aggregations. Time course experiments employing a modified ELISA method able to detect Abeta oligomers revealed dynamic intermolecular interactions and additional experiments physically confirmed the presence of stable amyloid multimers. Amyloid peptides that are rich in beta-sheet structure are capable of creating toxic membrane ion channels and a capacity to self-assemble as annular structures was confirmed in vitro using atomic force microscopy. Biochemical studies have established that soluble Abeta peptides perturb metabolic processes, provoke release of deleterious reactive compounds, reduce blood flow, induce mitochondrial apoptotic toxicity and inhibit angiogenesis. While there is no question that gross amyloid deposition does contribute to AD pathology, the destructive potential now associated with soluble Abeta suggests that treatment strategies that target these molecules may be efficacious in preventing some of the devastating effects of AD.
Neurological Research 01/2006; 27(8):869-81. · 1.52 Impact Factor
-
Desiree Watson,
Eduardo Castao,
Tyler Kokjohn,
Yu-Min Kuo,
Yuri Lyubchenko,
David Pinsky,
E. Sander Connolly,
Chera Esh,
Dean Luehrs, W. Blaine Stine,
Linda Rowse,
Mark Emmerling,
Alex Roher
[show abstract]
[hide abstract]
ABSTRACT: Extracellular fibrillar amyloid deposits are prominent and universal Alzheimer's disease (AD) features, but senile plaque abundance does not always correlate directly with the degree of dementia exhibited by AD patients. The mechanism(s) and dynamics of A? fibril genesis and deposition remain obscure. Enhanced A? synthesis rates coupled with decreased degradative enzyme production and accumulating physical modifications that dampen proteolysis may all enhance amyloid deposit formation. Amyloid accumulation may indirectly exert the greatest pathologic effect on the brain vasculature by destroying smooth muscle cells and creating a cascade of negative impacts on cerebral blood flow. The most visible manifestation of amyloid dis-equilibrium could actually be a defense mechanism employed to avoid serious vascular wall degradation while the major toxic effects to the gray and white matter neurons are mediated by soluble oligomeric A? peptides with high ?-sheet content. The recognition that dynamic soluble oligomeric A? pools exist in AD and are correlated to disease severity led to neurotoxicity and physical conformation studies. It is now recognized that the most basic soluble A? peptides are stable dimers with hydrophobic regions sequestered from the aqueous environment and are capable of higher order aggregations. Time course experiments employing a modified ELISA method able to detect A? oligomers revealed dynamic intermolecular interactions and additional experiments physically confirmed the presence of stable amyloid multimers. Amyloid peptides that are rich in ?-sheet structure are capable of creating toxic membrane ion channels and a capacity to self-assemble as annular structures was confirmed in vitro using atomic force microscopy. Biochemical studies have established that soluble A? peptides perturb metabolic processes, provoke release of deleterious reactive compounds, reduce blood flow, induce mitochondrial apoptotic toxicity and inhibit angiogenesis. While there is no question that gross amyloid deposition does contribute to AD pathology, the destructive potential now associated with soluble A? suggests that treatment strategies that target these molecules may be efficacious in preventing some of the devastating effects of AD.
Neurological Research 11/2005; 27(8):869-881. · 1.52 Impact Factor
-
Michael O Chaney, W Blaine Stine,
Tyler A Kokjohn,
Yu-Min Kuo,
Chera Esh,
Afroza Rahman,
Dean C Luehrs,
Ann Marie Schmidt,
David Stern,
Shi Du Yan,
Alex E Roher
[show abstract]
[hide abstract]
ABSTRACT: In the AD brain, there are elevated amounts of soluble and insoluble Abeta peptides which enhance the expression of membrane bound and soluble receptor for advanced glycation end products (RAGE). The binding of soluble Abeta to soluble RAGE inhibits further aggregation of Abeta peptides, while membrane bound RAGE-Abeta interactions elicit activation of the NF-kappaB transcription factor promoting sustained chronic neuroinflammation. Atomic force microscopy observations demonstrated that the N-terminal domain of RAGE, by interacting with Abeta, is a powerful inhibitor of Abeta polymerization even at prolonged periods of incubation. Hence, the potential RAGE-Abeta structural interactions were further explored utilizing a series of computational chemistry algorithms. Our modeling suggests that a soluble dimeric RAGE assembly creates a positively charged well into which the negative charges of the N-terminal domain of dimeric Abeta dock.
Biochimica et Biophysica Acta 07/2005; 1741(1-2):199-205. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Amyloid-beta1-42 (Abeta1-42) is crucial to Alzheimer disease (AD) pathogenesis but the conformation of the toxic Abeta species remains uncertain. AD risk is increased by apolipoprotein E4 (apoE4) and decreased by apoE2 compared with the apoE3 isoform, but whether inheritance of apoE4 represents a gain of negative or a loss of protective function is also unresolved. Using hippocampal slices from apoE knockout (apoE-KO) and human apoE2, E3, and E4 targeted replacement (apoE-TR) mice, we found that oligomeric Abeta1-42 inhibited long-term potentiation (LTP) with a hierarchy of susceptibility mirroring clinical AD risk (apoE4-TR > apoE3-TR = apoE-KO > apoE2-TR), and that comparable doses of unaggregated Abeta1-42 did not affect LTP. These data provide a novel link among apoE isoform, Abeta1-42, and a functional cellular model of memory. In this model, apoE4 confers a gain of negative function synergistic with Abeta1-42, apoE2 is protective, and the apoE-Abeta interaction is specific to oligomeric Abeta1-42.
Neurobiology of Disease 02/2005; 18(1):75-82. · 5.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Abnormalities in the processing of amyloid precursor protein to amyloid-beta (Abeta) are causal factors, and the presence of the epsilon4 allele of apolipoprotein E (apoE) is the primary risk factor for Alzheimer's disease (AD). Based, at least in part, on these genetics, the potential structural and functional interactions between these two proteins are the focus of our research. To understand the nature of the physical interactions between apoE and Abeta, we initially utilized gel-shift assays to demonstrate that native apoE2 and E3 (associated with lipid particles) form an SDS-stable complex with Abeta that is more abundant than the apoE4:Abeta complex. We further demonstrated that exogenous apoE3 but not E4 prevents Abeta-induced neurotoxicity by a process that requires apoE receptors. In addition, both exogenous apoE3 and E4 prevent Abeta-induced, glial-mediated inflammation, also via a process that requires apoE receptors. These functional effects all occur at a molar ratio of apoE to Abeta of 1:30. Because the biological activities for both apoE and Abeta are profoundly influenced by their isoform and conformation, respectively, we further investigated the idea that apoE3 and E4 differentially interact with particular aggregation species of Abeta1-42. Our overall hypothesis is that apoE has two general functions in relation to Abeta. First, apoE interacts with oligomeric Abeta via an apoE receptor-mediated process to inhibit neurotoxicity and neuroinflammation (apoE3 > apoE4) a process possibly related to binding and clearance of apoE3:oligomer complexes. Second, apoE facilitates the deposition of Abeta as amyloid (apoE4 > apoE3). We will continue to investigate the effect of apoE isoform and Abeta conformation on the structural and functional interactions between these two proteins in relation to the pathogenesis of AD.
Journal of Molecular Neuroscience 01/2004; 23(3):235-46. · 2.50 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Extensive research causally links amyloid-beta peptide (A beta) to Alzheimer's disease, although the pathologically relevant A beta conformation remains unclear. A beta spontaneously aggregates into the fibrils that deposit in senile plaques. However, recent in vivo and in vitro reports describe a potent biological activity for oligomeric assemblies of A beta. To consistently prepare in vitro oligomeric and fibrillar forms of A beta 1-42, a detailed knowledge of how solution parameters influence structure is required. This manuscript represents the first study using a single chemically and structurally homogeneous unaggregated starting material to demonstrate that the formation of oligomers, fibrils, and fibrillar aggregates is determined by time, concentration, temperature, pH, ionic strength, and A beta species. We recently reported that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta 1-42-induced neurotoxicity significant at 10 nm. In addition, we were able to differentiate by structure and neurotoxic activity wild-type A beta1-42 from isoforms containing familial mutations (Dahlgren, K. N., Manelli, A. M., Stine, W. B., Jr., Baker, L. K., Krafft, G. A., and LaDu, M. J. (2002) J. Biol. Chem. 277, 32046-32053). Understanding the biological role of specific A beta conformations may define the link between A beta and Alzheimer's disease, re-focusing therapeutic approaches by identifying the pernicious species of A beta ultimately responsible for the cognitive dysfunction that defines the disease.
Journal of Biological Chemistry 04/2003; 278(13):11612-22. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).
Journal of Biological Chemistry 09/2002; 277(35):32046-53. · 4.77 Impact Factor
-
Hai-Wei Wang,
Joseph F Pasternak,
Helen Kuo,
Helen Ristic,
Mary P Lambert,
Brett Chromy,
Kirsten L Viola,
William L Klein, W Blaine Stine,
Grant A Krafft,
Barbara L Trommer
[show abstract]
[hide abstract]
ABSTRACT: The dementia in Alzheimer disease (AD) is usually attributed to widespread neuronal loss in conjunction with the pathologic hallmarks of intracellular neurofibrillary tangles and extracellular plaques containing amyloid (A beta) in fibrillar form. Recently it has been demonstrated that non-fibrillar assemblies of A beta possess electrophysiologic activity, with the corollary that they may produce dementia by disrupting neuronal signaling prior to cell death. We therefore examined the effects of soluble oligomers of A beta(1-42) on long-term potentiation (LTP) and long-term depression (LTD), two cellular models of memory, in the dentate gyrus of rat hippocampal slices. Compared with vehicle controls, slices pre-incubated 60 min in the presence of A beta-derived diffusible ligands (ADDLs) showed no differences in threshold intensity to evoke a synaptic response, slope of field excitatory post-synaptic potentials (EPSPs), or the input/output function. Tetanus-induced LTP and reversal of LTD were strongly inhibited in ADDLs-treated slices whereas LTD was unaffected. These data suggest that soluble non-fibrillar amyloid may contribute to the pathogenesis of AD both by impairing LTP/memory formation at the cellular level and by creating 'neuroplasticity imbalance' manifested by unopposed LTD in the setting of impaired capacity for neural repair via reversal of LTD or LTP.
Brain Research 02/2002; 924(2):133-40. · 2.73 Impact Factor
-
Alex E. Roher,
Michael O. Chaney,
Yu-Min Kuo,
Scott D. Webster, W. Blaine Stine,
Lanny J. Haverkamp,
Amina S. Woods,
Robert J. Cotter,
James M. Tuohy,
Grant A. Krafft,
Barry S. Bonnell,
Mark R. Emmerling
[show abstract]
[hide abstract]
ABSTRACT: In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified
stable dimeric and trimeric components of Aβ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by
size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of
monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic
Aβ-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry.
Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments,
whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Aβ-(1-40/42)
were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal
killing. It is possible that these potentially pathogenic Aβ-(1-40/42) dimers and trimers from Alzheimer's disease amyloid
represent the soluble oligomers of Aβ recently described in Alzheimer's disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey,
C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).
Journal of Biological Chemistry 08/1996; 271(34):20631-20635. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In this chapter, we attempt to analyze the evolution of the amyloid-L (AL) molecular structure from its inception as part of the AL precursor protein to its release by the secretases and its extrusion from membrane into an aqueous environment. Biophysical studies suggest that the AL peptide sustains a series of transitions from a molecule rich in K-helix to a molecule in which L-strands prevail. It is proposed that initially the extended C-termini of two opposing AL dimers form an antiparallel L-sheet and that the subsequent addition of dimers generates a helical AL protofilament. Two or more protofilaments create a strand in which the hydrophobic core of the L-sheets is shielded from the aqueous environment by the N-terminal polar domains of the AL dimers. Once the nucleation has occurred, the AL filament grows in length by the addition of dimers or tetramers. ß 2000 Elsevier Science B.V. All rights reserved.
