Theresa A. Good

Nanofluidic Biosensing for β-Amyloid Detection Using Surface Enhanced Raman Spectroscopy

Trace detection of the conformational transition of beta-amyloid peptide (Abeta) from a predominantly alpha-helical structure to beta-sheet could have a large impact in understanding and diagnosing Alzheimers disease. We demonstrate how a novel nanofluidic biosensor using a controlled, reproducible surface enhanced Raman spectroscopy active site was developed to observe Abeta in different conformational states during the Abeta self-assembly process as well as to distinguish Abeta from confounder proteins commonly found in cerebral spinal fluid.

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Aβ fibril formation pathway

β‐amyloid peptide (Aβ) is one of the main protein components of senile plaques associated with Alzheimers disease (AD). Aβ readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Aβ aggregates or oligomers is still under investigation. In this article, we show that different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Aβ aggregates faster than Aβ prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Aβ aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Aβ fibrils formed with agitation. In addition, Aβ fibrils formed under quiescent conditions were less toxic to differentiated SH‐SY5Y cells than the Aβ aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Aβ aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Aβ oligomers.

The Role of Prion Peptide Structure and Aggregation in Toxicity and Membrane Binding

Abstract: Prion diseases are neurodegenerative disorders associatedwith a conformational change in the normal cellular isoform of the prionprotein, PrPC, to an abnormal scrapie isoform, PrPSC.Unlike the α‐helical PrPC, the protease‐resistant core ofPrPSC is predominantly β‐sheet and possesses a tendency topolymerize into amyloid fibrils. We performed experiments with two synthetichuman prion peptides, PrP(106‐126) and PrP(127‐147), to determine how peptidestructure affects neurotoxicity and protein‐membrane interactions. Peptidesolutions possessing β‐sheet and amyloid structures were neurotoxic toPC12 cells in vitro and bound with measurable affinities to cholesterol‐richphospholipid membranes at ambient conditions, but peptide solutions lackingstable β‐sheet structures and amyloid content were nontoxic and possessedless than one tenth of the binding affinities of the amyloid‐containingpeptides. Regardless of structure, the peptide binding affinities tocholesterol‐depleted membranes were greatly reduced. These results suggestthat the β‐sheet and amyloid structures of the prion peptides give riseto their toxicity and membrane binding affinities and that membrane bindingaffinity, especially in cholesterol‐rich environments, may be related totoxicity. Our results may have significance in understanding the role of thefibrillogenic cerebral deposits associated with some of the prion diseases inneurodegeneration and may have implications for other amyloidoses.

Use of a mathematical model to estimate stress and strain during elevated pressure induced lamina cribrosa deformation.

Background. High intraocular pressure (IOP), which is generally associated with glaucoma, causes lamina cribrosa retrodisplacement and deformation. Shear stress and strain resulting from lamina cribrosa deformation have been implicated in tissue remodeling, changes in retinal astrocyte function and retinal ganglion cell (RGC) death observed in vivo during glaucoma. Methods. A mathematical model was developed to describe the lamina cribrosa exposed to elevated intraocular pressure (IOP). The model is based on the bending theory of plates, incorporates anatomical properties of the lamina cribrosa, and provides estimates of its biomechanical properties. The model relates IOP, the parameter normally correlated with glaucoma, and lamina cribrosa retrodisplacement to stress and strain experienced by cells, parameters that may be more closely associated with cell injury. Results. We estimate that shear strains of 0.05 occur at the edge of a 200 µm thick lamina cribrosa at an IOP of 25 mm Hg. We estimate greater lamina cribrosa deformation and higher shear stress and strain for thinner lamina cribrosa and lamina cribrosa of larger radii. Conclusion. These results may provide better estimates of the stress and strain experienced by cells in the lamina cribrosa and may further our understanding of the forces that contribute to optic nerve degeneration during glaucoma.

Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimers disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimers disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.

Hsp20, a novel α-crystallin, prevents Aβ fibril formation and toxicity

β‐Amyloid (Aβ) is a major protein component of senile plaques in Alzheimers disease, and is neurotoxic when aggregated. The size of aggregated Aβ responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Aβ aggregation still holds promise as a means to reduce Aβ neurotoxicity. In research presented here, we show that Hsp20, a novel α‐crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Aβ amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Aβ of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Aβ fibril formation. Hsp20 was also able to significantly reduce Aβ toxicity to both SH‐SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Aβ toxicity.

Pulsatile shear stress leads to DNA fragmentation in human SH‐SY5Y neuroblastoma cell line

1 Using an in vitro model of shear stress‐induced cell injury we demonstrate that application of shear to differentiated human SH‐SY5Y cells leads to cell death characterized by DNA fragmentation. Controlled shear stress was applied to cells via a modified cone and plate viscometer. 2 We show that pulsatile shear stress leads to DNA fragmentation, as determined via flow cytometry of fluorescein‐12‐dUTP nick‐end labelled cells, in 45 ± 4% of cells. No lactate dehydrogenase (LDH) release was observed immediately after injury; however, 24 h after injury significant LDH release was observed. 3 Nitric oxide production by cells subjected to pulsatile shear increased two‐ to threefold over that in unsheared control cells. 4 Inhibition of protein synthesis, nitric oxide production, Ca2+ entry into cells, and pertussis toxin‐sensitive G protein activation attenuated the shear stress‐induced cell injury. 5 Our results show for the first time that application of pulsatile shear stress to a neuron‐like cell in vitro leads to nitric oxide‐dependent cell death.

Attenuation of β-amyloid induced toxicity by sialic acid-conjugated dendrimers: role of sialic acid attachment

beta-Amyloid (Abeta) is the primary protein component of senile plaques in Alzheimers disease and is believed to be associated with neurotoxicity in the disease. We and others have shown that Abeta binds with relatively high affinity to clustered sialic acid residues on cell surfaces and that removal of cell surface sialic acids attenuates Abeta toxicity. We have also shown that sialic acid functionalized dendrimeric polymers can act as mimics of cell surface sialic acid clusters and attenuate Abeta-induced neurotoxicity. In the current study, we prepared sialic-acid-conjugated dendrimers using a physiologically relevant attachment of the sialic acid to the dendrimeric termini, and evaluated the Abeta toxicity attenuation properties of the dendrimers. We compared performance of sialic-acid-conjugated dendrimeric polymers in which the sialic acid moieties were attached to dendrimeric termini via the anomeric hydroxyl group of the sialic acid, a physiological attachment, to polymers in which the attachment was made via the carboxylic acid group on the sialic acid, a non-physiological attachment. This work enhances our understanding of Abeta-cell surface binding and is a step towards the development of new classes of sequestering agents as therapeutics for the prevention of Abeta toxicity in AD.

Hsp20, a novel alpha-crystallin, prevents Abeta fibril formation and toxicity.

β‐Amyloid (Aβ) is a major protein component of senile plaques in Alzheimers disease, and is neurotoxic when aggregated. The size of aggregated Aβ responsible for the observed neurotoxicity and the mechanism of aggregation are still under investigation; however, prevention of Aβ aggregation still holds promise as a means to reduce Aβ neurotoxicity. In research presented here, we show that Hsp20, a novel α‐crystallin isolated from the bovine erythrocyte parasite Babesia bovis, was able to prevent aggregation of denatured alcohol dehydrogenase when the two proteins are present at near equimolar levels. We then examined the ability of Hsp20 produced as two different fusion proteins to prevent Aβ amyloid formation as indicated by Congo Red binding; we found that not only was Hsp20 able to dramatically reduce Congo Red binding, but it was able to do so at molar ratios of Hsp20 to Aβ of 1 to 1000. Electron microscopy confirmed that Hsp20 does prevent Aβ fibril formation. Hsp20 was also able to significantly reduce Aβ toxicity to both SH‐SY5Y and PC12 neuronal cells at similar molar ratios. At high concentrations of Hsp20, the protein no longer displays its aggregation inhibition and toxicity attenuation properties. Size exclusion chromatography indicated that Hsp20 was active at low concentrations in which dimer was present. Loss of activity at high concentrations was associated with the presence of higher oligomers of Hsp20. This work could contribute to the development of a novel aggregation inhibitor for prevention of Aβ toxicity.

Two disaccharides and trimethylamine N-oxide affect Aβ aggregation differently, but all attenuate oligomer-induced membrane permeability

Interaction between aggregates of amyloid beta protein (Abeta) and membranes has been hypothesized by many to be a key event in the mechanism of neurotoxicity associated with Alzheimers disease (AD). Proposed membrane-related mechanisms of neurotoxicity include ion channel formation, membrane disruption, changes in membrane capacitance, and lipid membrane oxidation. Recently, osmolytes such as trehalose have been found to delay Abeta aggregation in vitro and reduce neurotoxicity. However, no direct measurements have separated the effects of osmolytes on Abeta aggregation versus membrane interactions. In this article, we tested the influence of trehalose, sucrose and trimethylamine-N-oxide (TMAO) on Abeta aggregation and fluorescent dye leakage induced by Abeta aggregates from liposomes. In the absence of lipid vesicles, trehalose and sucrose, but not TMAO, were found to delay Abeta aggregation. In contrast, all of the osmolytes significantly attenuated dye leakage. Dissolution of preformed Abeta aggregates was excluded as a possible mechanism of dye leakage attenuation by measurements of Congo red binding as well as hydrogen-deuterium exchange detected by mass spectrometry (HX-MS). However, the accelerated conversion of high order oligomers to fibril caused by vesicles did not take place if any of the three osmolytes presented. Instead, in the case of disaccharide, osmolytes were found to form adducts with Abeta, and change the dissociation dynamics of soluble oligomeric species. Both effects may have contributed to the observed osmolyte attenuation of dye leakage. These results suggest that disaccharides and TMAO may have very different effects on Abeta aggregation because of the different tendencies of the osmolytes to interact with the peptide backbone. However, the effects on Abeta membrane interaction may be due to much more general phenomena associated with osmolyte enhancement of Abeta oligomer stability and/or direct interaction of osmolyte with the membrane surface.