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Featured researches published by Jeff Kuret.


Journal of Neurochemistry | 2002

Differential Assembly of Human Tau Isoforms in the Presence of Arachidonic Acid

Michelle E. King; T. Chris Gamblin; Jeff Kuret; Lester I. Binder

Abstract: Six tau isoforms arise from the alternative splicing of a single gene in humans. Insoluble, filamentous deposits of tau protein occur in a number of neuro‐degenerative diseases, and in some of these diseases, the deposition of polymers enriched in certain tau isoforms has been documented. Because of these findings, we have undertaken studies on the efficacy of fatty acid‐induced polymerization of the individual tau isoforms found in the adult human CNS. The polymerization of each tau isoform in the presence of two concentrations of arachidonic acid indicated that isoforms lacking N‐terminal exons e2 and e3 formed small, globular oligomers that did not go on to elongate into straight (SF) or paired helical (PHF) filaments under our buffer conditions. The polymerization of all isoforms containing e2 or e2 and e3 occurred readily at a high arachidonic acid concentration. Conversely, at a lower arachidonic acid concentration, only tau isoforms containing four microtubule binding repeats assembled well. Under all buffer conditions employed, filaments formed from three of the isoforms containing e2 and e3 resembled SFs in morphology but began to form PHF‐like structures following extended incubation at 37°C. These results indicate that polymerization of the intact tau molecule may be facilitated by e2 and e3. Moreover, tau isoforms containing three versus four microtubule binding repeats display different assembly properties depending on the solvent conditions employed.


American Journal of Pathology | 1999

A New Molecular Link between the Fibrillar and Granulovacuolar Lesions of Alzheimer's Disease

Nupur Ghoshal; John F. Smiley; Anthony J. Demaggio; Merl F. Hoekstra; Elizabeth J. Cochran; Lester I. Binder; Jeff Kuret

Alzheimers Disease (AD) is a progressive neurodegenerative disorder involving select neurons of the hippocampus, neocortex, and other regions of the brain. Markers of end stage disease include fibrillar lesions, which accumulate hyperphosphorylated tau protein polymerized into filaments, and granulovacuolar lesions, which appear primarily within the hippocampus. The mechanism by which only select populations of neurons develop these lesions as well as the relationship between them is unknown. To address these questions, we have turned to AD tissue to search for enzymes specifically involved in tau hyperphosphorylation. Recently, we showed that the principal phosphotransferases associated with AD brain-derived tau filaments are members of the casein kinase-1 (CK1) family of protein kinases. Here we report the distribution of three CK1 isoforms (Ckialpha, Ckidelta, and Ckiepsilon) in AD and control brains using immunohistochemistry and Western analysis. In addition to colocalizing with elements of the fibrillar pathology, CK1 is found within the matrix of granulovacuolar degeneration bodies. Furthermore, levels of all CK1 isoforms are elevated in the CA1 region of AD hippocampus relative to controls, with one isoform, Ckidelta, being elevated >30-fold. We propose that overexpression of this protein kinase family plays a key role in the hyperphosphorylation of tau and in the formation of AD-related pathology.


Journal of Biological Chemistry | 2004

Pseudophosphorylation and Glycation of Tau Protein Enhance but Do Not Trigger Fibrillization in Vitro

Mihaela Necula; Jeff Kuret

Alzheimers disease is defined in part by the intraneuronal aggregation of tau protein into filamentous lesions. The pathway is accompanied by posttranslational modifications including phosphorylation and glycation, each of which has been shown to promote tau fibrillization in vitro when present at high stoichiometry. To clarify the site-specific impact of posttranslational modification on tau fibrillization, the ability of recombinant full-length four repeat tau protein (htau40) and 11 pseudophosphorylation mutants to fibrillize in the presence of anionic inducer was assayed in vitro using transmission electron microscopy and laser light scattering assays. Tau glycated with d-glucose was examined as well. Both glycated tau and pseudophosphorylation mutants S199E, T212E, S214E, double mutant T212E/S214E, and triple mutant S199E/S202E/T205E yielded increased filament mass at equilibrium relative to wild-type tau. Increases in filament mass correlated strongly with decreases in critical concentration, indicating that both pseudophosphorylation and glycation promoted fibrillization by shifting equilibrium toward the fibrillized state. Analysis of reaction time courses further revealed that increases in filament mass were not associated with reduced lag times, indicating that these posttranslational modifications did not promote filament nucleation. The results suggest that site-specific posttranslational modifications can stabilize filaments once they nucleate, and thereby support their accumulation at low intracellular tau concentrations.


Journal of Biological Chemistry | 2007

Tau aggregation and toxicity in a cell culture model of tauopathy.

Bhaswati Bandyopadhyay; Guibin Li; Haishan Yin; Jeff Kuret

Intracellular aggregation of the microtubule-associated protein tau into filamentous inclusions is a defining characteristic of Alzheimer disease. Because appearance of tau-aggregate bearing lesions correlates with both cognitive decline and neurodegeneration, it has been hypothesized that tau aggregation may be directly toxic to cells that harbor them. Testing this hypothesis in cell culture has been complicated by the resistance of full-length tau isoforms to aggregation over experimentally tractable time periods. To overcome this limitation, a small-molecule agonist of the tau aggregation reaction, Congo red, was used to drive aggregation within HEK-293 cells expressing full-length tau isoform htau40. Formation of detergent-insoluble aggregates was both time and agonist concentration dependent. At 10 μm Congo red, detergent-insoluble aggregates appeared with pseudo-first order kinetics and a half-life of approximately 5 days. By 7 days in culture, total tau levels increased 2-fold, with ∼30% of total tau converted into detergent-insoluble aggregates. Agonist addition also led to rapid losses in the tubulin binding activity of tau, although tau was not hyperphosphorylated as judged by occupancy of phosphorylation sites Ser396/Ser404. Tau aggregation was associated with decreased viability as detected by ToPro-3 uptake. The results, which establish a new approach for analysis of tau aggregation in cells independent of tau hyperphosphorylation, suggest that conformational changes associated with aggregation are incompatible with microtubule binding, and that toxicity associated with intracellular tau aggregation is not acute but develops over a period of days.


Journal of Neurochemistry | 2001

Tau‐66: evidence for a novel tau conformation in Alzheimer's disease

Nupur Ghoshal; Francisco García-Sierra; Yifan Fu; Laurel A. Beckett; T. Elliott J. Mufson; Jeff Kuret; Robert W. Berry; Lester I. Binder

We have characterized a novel monoclonal antibody, Tau‐66, raised against recombinant human tau. Immunohistochemistry using Tau‐66 reveals a somatic‐neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimers disease (AD) brain than in normal brain. In hippocampus, Tau‐66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau‐66 stains plaques lacking obvious dystrophic neurites (termed herein ‘diffuse reticulated plaques’) in STG and the hippocampus. Enzyme‐linked immunosorbent assay (ELISA) analysis reveals that Tau‐66 is specific for tau, as there is no cross‐reactivity with MAP2, tubulin, Aβ1−40, or Aβ1−42, although Tau‐66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau‐66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155–244 and 305–314. Tau‐66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau‐66 epitope is conformation‐dependent, perhaps involving a close interaction of the proline‐rich and the third microtubule‐binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.


Brain Research | 2000

Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain.

Koji Yasojima; Jeff Kuret; Anthony J. Demaggio; Edith G. McGeer; Patrick L. McGeer

The casein kinase-1 (Ck1) family are serine/threonine specific protein kinases. They are highly associated with Alzheimer disease (AD) brain-derived tau filaments and granulovacuolar bodies. Recently we have demonstrated that one family member, Ckidelta, colocalizes with tau containing neurofibrillary tangles (NFTs) and other tau deposits in a number of neurodegenerative diseases. Here we show that the association in AD is accompanied by a sharp upregulation of Ckidelta mRNA in brain but not in peripheral organs. The degree of upregulation in AD brain is correlated with the degree of regional pathology. There was a 24.4-fold increase of Ckidelta mRNA in AD hippocampus compared with control, 8.04-fold in the amygdala, 7.45 in the entorhinal cortex and 7.30-fold in the midtemporal gyrus. These are areas with a high burden of NFTs, neuropil threads and dystrophic neurites. In areas almost devoid of this tau pathology, such as the caudate nucleus, occipital cortex and cerebellum, the increases in AD compared to control brain were only 2.21-, 1.89- and 1.87-fold, respectively. Western blot analysis showed that the upregulation of Ckidelta mRNA was paralleled by an upregulation of Ckidelta protein. These data establish that the association of Ckidelta with the tau pathology of AD is reflective of an increase in gene transcription. Since Alzheimer-like phosphoepitopes of tau can be generated by Ck1, the Ckidelta isoform may play an important role in this fundamental aspect of AD pathology.


Journal of Biological Chemistry | 2008

Nucleation-dependent Tau Filament Formation THE IMPORTANCE OF DIMERIZATION AND AN ESTIMATION OF ELEMENTARY RATE CONSTANTS

Erin E. Congdon; Sohee Kim; Jonathan Bonchak; Tanakorn Songrug; Anastasios Matzavinos; Jeff Kuret

Filamentous inclusions composed of the microtubule-associated protein tau are found in Alzheimer disease and other tauopathic neurodegenerative diseases, but the mechanisms underlying their formation from full-length protein monomer under physiological conditions are unclear. To address this issue, the fibrillization of recombinant full-length four-repeat human tau was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods and a small-molecule inducer of aggregation, thiazine red. Data were then fit to a simple homogeneous nucleation model with rate constant constraints established from filament dissociation rate, critical concentration, and mass-per-unit length measurements. The model was then tested by comparing the predicted time-dependent evolution of length distributions to experimental data. Results indicated that once assembly-competent conformations were attained, the rate-limiting step in the fibrillization pathway was tau dimer formation. Filament elongation then proceeded by addition of tau monomers to nascent filament ends. Filaments isolated at reaction plateau contained ∼2 tau protomers/β-strand spacing on the basis of mass-per-unit length measurements. The model suggests four key steps in the aggregation pathway that must be surmounted for tau filaments to form in disease.


Journal of Neurochemistry | 2002

Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

S. N. Prasanna Murthy; James H. Wilson; Thomas J. Lukas; Jeff Kuret; Laszlo Lorand

Abstract: A portion of the neurofibrillary tangles of Alzheimers disease has the characteristics of cross‐linked protein. Because the principal component of these lesions is the microtubule‐associated protein tau, and because a major source of cross‐linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (τ23 and τ40) capable of participating in human TGase‐mediated isopeptide bond formation. Using dansyl‐labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule‐binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross‐linked during the TGase‐catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.


Neuropathology and Applied Neurobiology | 2011

Granulovacuolar degeneration (GVD) bodies of Alzheimer's disease (AD) resemble late-stage autophagic organelles

Kristen E. Funk; Robert E. Mrak; Jeff Kuret

K. E. Funk, R. E. Mrak and J. Kuret (2011) Neuropathology and Applied Neurobiology37, 295–306
Granulovacuolar degeneration (GVD) bodies of Alzheimers disease (AD) resemble late‐stage autophagic organelles


Journal of Neuroscience Research | 2008

Two motifs within the tau microtubule-binding domain mediate its association with the hsc70 molecular chaperone

Mitul Sarkar; Jeff Kuret; Gloria Lee

Tau, a microtubule‐associated protein with multiple phosphorylation sites, forms aggregates that correlate with neurodegeneration in Alzheimers disease and several other neurodegenerative diseases, termed tauopathies. Hsc70 is a highly expressed constitutive chaperone that can drive conformational change in proteins, prevent the aggregation of its substrates, recognize misfolded substrates, and facilitate their degradation. Here, we show that hsc70 binds to the microtubule‐binding domain of tau in vitro and in vivo, without an absolute requirement for tau phosphorylation. Binding requires a carboxy‐terminal region of hsc70 comprising its peptide‐binding and variable domains. We have identified two hsc70 binding sites on tau and hydrophobic amino acids crucial for hsc70 binding. Interestingly, these hsc70 binding sites correspond to the β‐structure elements that have been previously reported to facilitate tau aggregation. Thus, it is possible that hsc70 binding might directly inhibit tau–tau interactions that precede tau oligomerization and aggregation. Our results provide an important stimulus for research into how the hsc70–tau interaction might affect tau fate in normal cells and in disease.

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Guibin Li

Ohio State University

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