%0 Journal Article
%~ Pubmed
%A Whiteman, Ineka T
%A Minamide, Laurie S
%A Goh, De Lian
%A Bamburg, James R
%A Goldsbury, Claire
%T Rapid Changes in Phospho-MAP/Tau Epitopes during Neuronal Stress: Cofilin-Actin Rods Primarily Recruit Microtubule Binding Domain Epitopes.
%B 
%D 2011
%V 6
%N 6
%P e20878
%@ 1932-6203
%X Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies - AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422 - raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.
%Z FOR Codes: 60105



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Baxa, Ulrich
%A Simon, Martha N
%A Steven, Alasdair C
%A Engel, Andreas
%A Wall, Joseph S
%A Aebi, Ueli
%A M?ller, Shirley A
%T Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy.
%B Journal of structural biology
%D 2011
%V 
%N 
%P 
%@ 1095-8657
%X Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).
%Z FOR Codes: 60112



%0 Journal Article
%~ Pubmed
%A Bamburg, J R
%A Bernstein, B W
%A Davis, R C
%A Flynn, K C
%A Goldsbury, C
%A Jensen, J R
%A Maloney, M T
%A Marsden, I T
%A Minamide, L S
%A Pak, C W
%A Shaw, A E
%A Whiteman, I
%A Wiggan, O
%T ADF/Cofilin-actin rods in neurodegenerative diseases.
%B 
%D 2010
%V 7
%N 3
%P 241-50
%@ 1875-5828
%X Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of beta-amyloid peptide (Abeta). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in "striated neuropil threads," characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Abeta. Thus rods may mediate the loss of synapses, production of excess Abeta, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases.
%Z FOR Codes: 110904



%0 Journal Article
%~ Pubmed
%A Whiteman, Ineka T
%A Gervasio, Othon L
%A Cullen, Karen M
%A Guillemin, Gilles J
%A Jeong, Erica V
%A Witting, Paul K
%A Antao, Shane T
%A Minamide, Laurie S
%A Bamburg, James R
%A Goldsbury, Claire
%T Activated actin-depolymerizing factor/cofilin sequesters phosphorylated microtubule-associated protein during the assembly of alzheimer-like neuritic cytoskeletal striations.
%B The Journal of Neuroscience
%D 2009
%V 29
%N 41
%P 12994-3005
%@ 1529-2401
%X In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.
%Z FOR Codes: 110999



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Whiteman, Ineka T
%A Jeong, Erica
%A Lim, Yun-An
%T Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons.
%B Aging cell
%D 2008
%V 7
%N 5
%P 771-5
%@ 1474-9726
%X Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.
%Z FOR Codes: 111601



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Thies, Edda
%A Konzack, Sven
%A Mandelkow, Eva-Maria
%T Quantification of amyloid precursor protein and tau for the study of axonal traffic pathways.
%B The Journal of Neuroscience
%D 2007
%V 27
%N 13
%P 3357-63
%@ 1529-2401
%X 
%Z FOR Codes: 1109



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Mocanu, Maria-Magdalena
%A Thies, Edda
%A Kaether, Christoph
%A Haass, Christian
%A Keller, Patrick
%A Biernat, Jacek
%A Mandelkow, Eckhard
%A Mandelkow, Eva-Maria
%T Inhibition of APP trafficking by tau protein does not increase the generation of amyloid-beta peptides.
%B Traffic (Copenhagen, Denmark)
%D 2006
%V 7
%N 7
%P 873-88
%@ 1398-9219
%X Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.
%Z FOR Codes: 110106



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Green, Janelle
%T Time-lapse atomic force microscopy in the characterization of amyloid-like fibril assembly and oligomeric intermediates.
%B Methods in molecular biology (Clifton, N.J.)
%D 2005
%V 299
%N 
%P 103-28
%@ 1064-3745
%X The atomic force microscope (AFM) images the topography of biological structures adsorbed to surfaces with nanometer to angstrom scale resolution. Amyloid-like fibrils and oligomers can be imaged in buffer solutions, allowing the samples to retain physiological-like properties while temporal changes in structure are monitored, e.g., the elongation of fibrils or the growth of single oligomers. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, i.e., electron microscopy (EM). However, AFM is limited in that the specimen must be firmly attached to a solid support for measurement and that time-lapse imaging of individual assemblies can thus only be achieved for fibrils and oligomers growing on this support. Nevertheless, AFM has provided several insights into the in vitro assembly mechanism and structures of amyloid-like fibrils. The first section of this chapter provides a methodological introduction to AFM, whilst the second details the application of this technique to the investigation of amyloidogenic proteins, specifically amylin and amyloid-beta (Abeta) peptides.
%Z FOR Codes: 60199



%0 Journal Article
%~ Pubmed
%A Goldsbury, Claire
%A Frey, Peter
%A Olivieri, Vesna
%A Aebi, Ueli
%A M?ller, Shirley A
%T Multiple assembly pathways underlie amyloid-beta fibril polymorphisms.
%B Journal of Molecular Biology
%D 2005
%V 352
%N 2
%P 282-98
%@ 0022-2836
%X The amyloid beta-protein transiently forms low and high molecular mass oligomers and protofibrils in vitro, and after longer incubation times assembles into polymorphic mature fibrils. The precursor-to-product relationship of these species remains to be understood. Protofibrils are up to approximately 600 nm in length and have mass-per-lengths of 19(+/-2) kDa/nm measured by scanning transmission electron microscopy. Two predominant mature fibril types, several microns in length and with mass-per-lengths of 18(+/-3) and 27(+/-3) kDa/nm, are identified after longer incubation times. The difference of approximately 9 kDa/nm between the two fibril types indicates a bona fide elementary protofilament subunit of this mass-per-length. Fibrils in the 18(+/-3) kDa/nm group often exhibited distinct coiling with axial cross-over spacings of approximately 25 nm. Although strikingly different in morphology, the mass-per-length (MPL) of these coiled fibrils is equivalent to that measured for protofibrils. They could therefore arise from a conformational change in the protofibril concurrent with coiling and rapid elongation. Alternatively, we cannot rule out an assembly pathway not directly related to protofibrils. In contrast, the 27(+/-3) kDa/nm fibrils correspond to a MPL of approximately 1.5 x the protofibril and thus can neither arise from a simple conformational transition nor from lateral association of 19 kDa/nm protofibril precursors. Twisted ribbons with axial periodicities ranging from approximately 80 nm to 130 nm were prominent in the 27(+/-3) kDa/nm group as well as more tightly coiled fibrils. Individual fibril ribbons had elongation rates of 20(+/-12) nm/min when imaged by time-lapse atomic force microscopy. Protofibrils exhibited growth rates approximately 15 x slower at 1.3(+/-0.5) nm/min. The data support a model where concurrent multiple assembly pathways give rise to the various polymorphic fibril types.
%Z FOR Codes: 60199



%0 Journal Article
%~ Pubmed
%A Green, Janelle D
%A Goldsbury, Claire
%A Kistler, Joerg
%A Cooper, Garth J S
%A Aebi, Ueli
%T Human amylin oligomer growth and fibril elongation define two distinct phases in amyloid formation.
%B Journal of Biological Chemistry
%D 2004
%V 279
%N 13
%P 12206-12
%@ 0021-9258
%X Human amylin (hA), a 37-amino-acid polypeptide, is one of a number of peptides with the ability to form amyloid fibrils and cause disease. It is the main constituent of the pancreatic amyloid deposits associated with type 2 diabetes. Increasing interest in early assembly intermediates rather than the mature fibrils as the cytotoxic agent has led to this study in which the smallest hA oligomers have been captured by atomic force microscopy. These are 2.3 +/- 1.9 nm in height, 23 +/- 14 nm in length, and consist of an estimated 16 hA molecules. Oligomers first grow to a height of about 6 nm before they begin to significantly elongate into fibrils. Congo red inhibits elongation but not the growth in height of hA oligomers. Two distinct phases have thus been identified in hA fibrillogenesis: lateral growth of oligomers followed by longitudinal growth into mature fibrils. These observations suggest that mature fibrils are assembled directly via longitudinal growth of full-width oligomers, making assembly by lateral association of protofibrils appear less likely.
%Z FOR Codes: 60199



%0 Journal Article
%~ Pubmed
%A Green, J D
%A Kreplak, L
%A Goldsbury, C
%A Li Blatter, X
%A Stolz, M
%A Cooper, G S
%A Seelig, A
%A Kistler, J
%A Aebi, U
%T Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide.
%B Journal of Molecular Biology
%D 2004
%V 342
%N 3
%P 877-87
%@ 0022-2836
%X To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.
%Z FOR Codes: 60199