Stuart C Feinstein

Publications of Stuart C Feinstein

• Amyloid beta-mediated cell death of cultured hippocampal neurons reveals extensive Tau fragmentation without increased full-length tau phosphorylation.

  Authors: Jack Reifert, DeeAnn Hartung-Cranston, Stuart C Feinstein

  The Journal of biological chemistry. 06/2011; 286(23):20797-811.

  A variety of genetic and biochemical evidence suggests that amyloid β (Aβ) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer andA variety of genetic and biochemical evidence suggests that amyloid β (Aβ) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer and related dementias. To better understand molecular mechanisms involved in Aβ-mediated neuronal cell death, we have treated primary rat hippocampal cultures with Aβ oligomers and examined the resulting cellular changes occurring before and during the induction of cell death with a focus on altered Tau biochemistry. The most rapid neuronal responses upon Aβ administration are activation of caspase 3/7 and calpain proteases. Aβ also appears to reduce Akt and Erk1/2 kinase activities while increasing GSK3β and Cdk5 activities. Shortly thereafter, substantial Tau degradation begins, generating relatively stable Tau fragments. Only a very small fraction of full-length Tau remains intact after 4 h of Aβ treatment. In conflict with expectations based on suggested increases of GSK3β and Cdk5 activities, Aβ does not cause any major increases in phosphorylation of full-length Tau as assayed by immunoblotting one-dimensional gels with 11 independent site- and phospho-specific anti-Tau antibodies as well as by immunoblotting two-dimensional gels probed with a pan-Tau antibody. There are, however, subtle and transient increases in Tau phosphorylation at 3-4 specific sites before its degradation. Taken together, these data are consistent with the notion that Aβ-mediated neuronal cell death involves the loss of full-length Tau and/or the generation of toxic fragments but does not involve or require hyperphosphorylation of full-length Tau.

• Nanoscale assembly in biological systems: from neuronal cytoskeletal proteins to curvature stabilizing lipids.

  Authors: Cyrus R Safinya, Uri Raviv, Daniel J Needleman, Alexandra Zidovska, Myung Chul Choi, Miguel A Ojeda-Lopez, Kai K Ewert, Youli Li, Herbert P Miller, Joel Quispe, Bridget Carragher, Clinton S Potter, Mahn Won Kim, Stuart C Feinstein, Leslie Wilson

  Advanced materials (Deerfield Beach, Fla.). 05/2011; 23(20):2260-70.

  The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of theThe review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous-actin in neurons where the nature of the interactions, structures, and structure-function correlations remain poorly understood. We describe how three-dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter-ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT-associated-protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio- nanotechnology. In another set of experiments we describe lipid-protein-nanotubes, and lipid nano-tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio-nanotechnology.

• Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts.

  Authors: Erkan Kiris, Donovan Ventimiglia, Mehmet E Sargin, Michelle R Gaylord, Alphan Altinok, Kenneth Rose, B S Manjunath, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 02/2011; 286(16):14257-70.

  Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias.Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

• Tau isoform-specific modulation of kinesin-driven microtubule gliding rates and trajectories as determined with tau-stabilized microtubules.

  Authors: Austin Peck, M Emre Sargin, Nichole E LaPointe, Kenneth Rose, B S Manjunath, Stuart C Feinstein, Leslie Wilson

  Cytoskeleton (Hoboken, N.J.). 01/2011; 68(1):44-55.

  We have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MTWe have utilized tau-assembled and tau-stabilized microtubules (MTs), in the absence of taxol, to investigate the effects of tau isoforms with three and four MT binding repeats upon kinesin-driven MT gliding. MTs were assembled in the presence of either 3-repeat tau (3R tau) or 4-repeat tau (4R tau) at tau:tubulin dimer molar ratios that approximate those found in neurons. MTs assembled with 3R tau glided at 31.1 μm/min versus 25.8 μm/min for 4R tau, a statistically significant 17% difference. Importantly, the gliding rates for either isoform did not change over a fourfold range of tau concentrations. Further, tau-assembled MTs underwent minimal dynamic instability behavior while gliding and moved with linear trajectories. In contrast, MTs assembled with taxol in the absence of tau displayed curved gliding trajectories. Interestingly, addition of 4R tau to taxol-stabilized MTs restored linear gliding, while addition of 3R tau did not. The data are consistent with the ideas that (i) 3R and 4R tau-assembled MTs possess at least some isoform-specific features that impact upon kinesin translocation, (ii) tau-assembled MTs possess different structural features than do taxol-assembled MTs, and (iii) some features of tau-assembled MTs can be masked by prior assembly by taxol. The differences in kinesin-driven gliding between 3R and 4R tau suggest important features of tau function related to the normal shift in tau isoform composition that occurs during neural development as well as in neurodegeneration caused by altered expression ratios of otherwise normal tau isoforms.

• Quantitative analysis of MAP-mediated regulation of microtubule dynamic instability in vitro focus on Tau.

  Authors: Erkan Kiris, Donovan Ventimiglia, Stuart C Feinstein

  Methods in cell biology. 01/2010; 95:481-503.

  The regulation of microtubule growing and shortening dynamics is essential for proper cell function and viability, and microtubule-associated proteins (MAPs) such as the neural protein tau areThe regulation of microtubule growing and shortening dynamics is essential for proper cell function and viability, and microtubule-associated proteins (MAPs) such as the neural protein tau are critical regulators of these dynamic processes. Further, we and our colleagues have proposed that misregulation of microtubule dynamics may contribute to tau-mediated neuronal cell death and dementia in Alzheimer's and related diseases. In the first part of this chapter, we present a general background on microtubule dynamics and then focus in on tau. We review the literature on the roles of tau in normal neuronal cell biology, the tau structure-function relationship, regulatory mechanisms influencing tau action, and pathological tau action, including normal and aberrant regulation of microtubule dynamics. In the second part of this chapter, we present detailed protocols for various in vitro procedures often used in studying tau-mediated regulation of microtubule dynamics, including purification and characterization of necessary reagents, microtubule assembly assays, and microtubule dynamics assays. Importantly, these assays are readily adaptable to examine other regulators of microtubule dynamics besides tau. In the final analysis, in vitro analyses of MAP-mediated regulation of microtubule dynamics will provide extremely valuable insights into our understanding of normal and pathological cell biology.

• The neuroprotective peptide NAP does not directly affect polymerization or dynamics of reconstituted neural microtubules.

  Authors: Mythili Yenjerla, Nichole E LaPointe, Manu Lopus, Corey Cox, Mary Ann Jordan, Stuart C Feinstein, Leslie Wilson

  Journal of Alzheimer's disease : JAD. 01/2010; 19(4):1377-86.

  NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAPNAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is a neuroprotective peptide that shows cognitive protection in patients with amnestic mild cognitive impairment, a precursor to Alzheimer's disease. NAP exhibits potent neuroprotective properties in several in vivo and cellular models of neural injury. While NAP has been found in many studies to affect microtubule assembly and/or stability in neuronal and glial cells at fM concentrations, it has remained unclear whether NAP acts directly or indirectly on tubulin or microtubules. We analyzed the effects of NAP (1 fM-1 microM) on the assembly of reconstituted bovine brain microtubules in vitro and found that it did not significantly (p< 0.05) alter polymerization of either purified tubulin or of a mixture of tubulin and unfractionated microtubule-associated proteins. NAP also had no significant effect (p < 0.05) on the growing and shortening dynamics of steady-state microtubules at their plus ends, nor did it alter the polymerization or dynamics of microtubules assembled in the presence of 3-repeat or 4-repeat tau. Thus, the neuroprotective activity of NAP does not appear to involve a direct action on the polymerization or dynamics of purified tubulin or microtubules.

• Natural product derivative Bis(4-fluorobenzyl)trisulfide inhibits tumor growth by modification of {beta}-tubulin at Cys 12 and suppression of microtubule dynamics.

  Authors: Wanhong Xu, Biao Xi, Jieying Wu, Haoyun An, Jenny Zhu, Yama Abassi, Stuart C Feinstein, Michelle Gaylord, Baoqin Geng, Huifang Yan, Weimin Fan, Meihua Sui, Xiaobo Wang, Xiao Xu

  Molecular cancer therapeutics. 12/2009;

  Bis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potentBis(4-fluorobenzyl)trisulfide (BFBTS) is a synthetic molecule derived from a bioactive natural product, dibenzyltrisulfide, found in a subtropical shrub, Petiveria allieacea. BFBTS has potent anticancer activities to a broad spectrum of tumor cell lines with IC(50) values from high nanomolar to low micromolar and showed equal anticancer potency between tumor cell lines overexpressing multidrug-resistant gene, MDR1 (MCF7/adr line and KBv200 line), and their parental MCF7 line and KB lines. BFBTS inhibited microtubule polymerization dynamics in MCF7 cells, at a low nanomolar concentration of 54 nmol/L, while disrupting microtubule filaments in cells at low micromolar concentration of 1 mumol/L. Tumor cells treated with BFBTS were arrested at G(2)-M phase, conceivably resulting from BFBTS-mediated antimicrotubule activities. Mass spectrometry studies revealed that BFBTS bound and modified beta-tubulin at residue Cys12, forming beta-tubulin-SS-fluorobenzyl. The binding site differs from known antimicrotubule agents, suggesting that BFBTS functions as a novel antimicrotubule agent. BFBTS at a dose of 25 mg/kg inhibited tumor growth with relative tumor growth rates of 19.91%, 18.5%, and 23.42% in A549 lung cancer, Bcap-37 breast cancer, and SKOV3 ovarian cancer xenografts, respectively. Notably, BFBTS was more potent against MDR1-overexpressing MCF7/adr breast cancer xenografts with a relative tumor growth rate of 12.3% than paclitaxel with a rate of 43.0%. BFBTS displays a novel antimicrotubule agent with potentials for cancer therapeutics. [Mol Cancer Ther 2009;8(12):3318-30].

• Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects.

  Authors: Yama A Abassi, Biao Xi, Wenfu Zhang, Peifang Ye, Shelli L Kirstein, Michelle R Gaylord, Stuart C Feinstein, Xiaobo Wang, Xiao Xu

  Chemistry & biology. 08/2009; 16(7):712-23.

  We describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This noninvasive readout allows continuous sampling of cellularWe describe a cell-based kinetic profiling approach using impedance readout for monitoring the effect of small molecule compounds. This noninvasive readout allows continuous sampling of cellular responses to biologically active compounds and the ensuing kinetic profile provides information regarding the temporal interaction of compounds with cells. The utility of this approach was tested by screening a library containing FDA approved drugs, experimental compounds, and nature compounds. Compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRPs). The compounds were clustered based on TCRP similarity. We identified novel mechanisms for existing drugs, confirmed previously reported calcium modulating activity for COX-2 inhibitor celecoxib, and identified an additional mechanism for the experimental compound monastrol. We also identified and characterized a new antimitotic agent. Our findings indicate that the TCRP approach provides predictive mechanistic information for small molecule compounds.

• FTDP-17 mutations in tau alter the regulation of microtubule dynamics - an "Alternative Core" model for normal and pathological tau action.

  Authors: Adria C Leboeuf, Sasha F Levy, Michelle Gaylord, Arnab Bhattacharya, Ambuj K. Singh, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 11/2008;

  Mutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleteriousMutations affecting either the structure or regulation of the microtubule associated protein tau cause neuronal cell death and dementia. However, the molecular mechanisms mediating these deleterious effects remain unclear. Among tau's most characterized activities is the ability to regulate microtubule dynamics, known to be essential for proper cell function and viability. Here, we have tested the hypothesis that tau mutations causing neurodegeneration also alter the ability of tau to regulate the dynamic instability behaviors of microtubules. Using in vitro microtubule dynamics assays to assess average microtubule growth rates, microtubule growth rate distributions and catastrophe frequencies, we found that all tested mutants possessing amino acid substitutions or deletions mapping to either the repeat or inter-repeat regions of tau do indeed compromise its ability to regulate microtubule dynamics. Further mutational analyses suggest a novel mechanism of tau regulatory action based on an "alternative core" of microtubule binding and regulatory activities composed of two repeats and the inter-repeat between them. In this model, the inter-repeat serves as the primary regulator of microtubule dynamics while the flanking repeats serve as tethers to properly position the inter-repeat on the microtubule. Importantly, since there are multiple inter-repeats on each tau molecule, there are also multiple cores on each tau molecule, each with distinct mechanistic capabilities, thereby providing significant regulatory potential. Taken together, the data are consistent with a microtubule misregulation mechanism for tau mediated neuronal cell death and provide a novel mechanistic model for normal and pathological tau action.

• Complementary dimerization of microtubule-associated tau protein: Implications for microtubule bundling and tau-mediated pathogenesis.

  Authors: Kenneth J Rosenberg, Jennifer L Ross, H Eric Feinstein, Stuart C Feinstein, Jacob Israelachvili

  Proceedings of the National Academy of Sciences of the United States of America. 06/2008; 105(21):7445-50.

  Tau is an intrinsically unstructured microtubule (MT)-associated protein capable of binding to and organizing MTs into evenly spaced parallel assemblies known as "MT bundles." How tau achieves MTTau is an intrinsically unstructured microtubule (MT)-associated protein capable of binding to and organizing MTs into evenly spaced parallel assemblies known as "MT bundles." How tau achieves MT bundling is enigmatic because each tau molecule possesses only one MT-binding region. To dissect this complex behavior, we have used a surface forces apparatus to measure the interaction forces of the six CNS tau isoforms when bound to mica substrates in vitro. Two types of measurements were performed for each isoform: symmetric configuration experiments measured the interactions between two tau-coated mica surfaces, whereas "asymmetric" experiments examined tau-coated surfaces interacting with a smooth bare mica surface. Depending on the configuration (of which there were 12), the forces were weakly adhesive, strongly adhesive, or purely repulsive. The equilibrium spacing was determined mainly by the length of the tau projection domain, in contrast to the adhesion force/energy, which was determined by the number of repeats in the MT-binding region. Taken together, the data are incompatible with tau acting as a monomer; rather, they indicate that two tau molecules associate in an antiparallel configuration held together by an electrostatic "zipper" of complementary salt bridges composed of the N-terminal and central regions of each tau monomer, with the C-terminal MT-binding regions extending outward from each end of the dimeric backbone. This tau dimer determines the length and strength of the linker holding two MTs together and could be the fundamental structural unit of tau, underlying both its normal and pathological action.

• A general modeling and visualization tool for comparing different members of a group: application to studying tau-mediated regulation of microtubule dynamics.

  Authors: Arnab Bhattacharya, Sasha Levy, Adria LeBoeuf, Michelle Gaylord, Leslie Wilson, Ambuj K. Singh, Stuart C Feinstein

  BMC bioinformatics. 02/2008; 9:339.

  ABSTRACT: BACKGROUND: Innumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost allABSTRACT: BACKGROUND: Innumerable biological investigations require comparing collections of molecules, cells or organisms to one another with respect to one or more of their properties. Almost all of these comparisons are performed manually, which can be susceptible to inadvertent bias as well as miss subtle effects. The development and application of computer-assisted analytical and interpretive tools could help address these issues and thereby dramatically improve these investigations. RESULTS: We have developed novel computer-assisted analytical and interpretive tools and applied them to recent studies examining the ability of 3-repeat and 4-repeat tau to regulate the dynamic behavior of microtubules in vitro. More specifically, we have developed an automated and objective method to define growth, shortening and attenuation events from real time videos of dynamic microtubules, and demonstrated its validity by comparing it to manually assessed data. Additionally, we have used the same data to develop a general strategy of building different models of interest, computing appropriate dissimilarity functions to compare them, and embedding them on a two-dimensional plot for visualization and easy comparison. Application of these methods to assess microtubule growth rates and growth rate distributions established the validity of the embedding procedure and revealed non-linearity in the relationship between the tau:tubulin molar ratio and growth rate distribution. CONCLUSION: This work addresses the need of the biological community for rigorously quantitative and generally applicable computational tools for comparative studies. The two-dimensional embedding method retains the inherent structure of the data, and yet markedly simplifies comparison between models and parameters of different samples. Most notably, even in cases where numerous parameters exist by which to compare the different samples, our embedding procedure provides a generally applicable computational strategy to detect subtle relationships between different molecules or conditions that might otherwise escape manual analyses.

• Model based dynamics analysis in live cell microtubule images.

  Authors: Alphan Altinok, Erkan Kiris, Austin J Peck, Stuart C Feinstein, Leslie Wilson, B S Manjunath, Kenneth Rose

  BMC cell biology. 02/2007; 8 Suppl 1:S4.

  BACKGROUND: The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubuleBACKGROUND: The dynamic growing and shortening behaviors of microtubules are central to the fundamental roles played by microtubules in essentially all eukaryotic cells. Traditionally, microtubule behavior is quantified by manually tracking individual microtubules in time-lapse images under various experimental conditions. Manual analysis is laborious, approximate, and often offers limited analytical capability in extracting potentially valuable information from the data. RESULTS: In this work, we present computer vision and machine-learning based methods for extracting novel dynamics information from time-lapse images. Using actual microtubule data, we estimate statistical models of microtubule behavior that are highly effective in identifying common and distinct characteristics of microtubule dynamic behavior. CONCLUSION: Computational methods provide powerful analytical capabilities in addition to traditional analysis methods for studying microtubule dynamic behavior. Novel capabilities, such as building and querying microtubule image databases, are introduced to quantify and analyze microtubule dynamic behavior.

• Tracing Microtubules in Live Cell Images.

  Authors: Mehmet Emre Sargin, Alphan Altinok, Erlam Kiris, Stuart C. Feinstein, Leslie Wilson, Kenneth Rose, B. S. Manjunath

  Proceedings of the 2007 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, Washington, DC, USA, April 12-16, 2007; 01/2007

• FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells.

  Authors: Janis M Bunker, Kathy Kamath, Leslie Wilson, Mary Ann Jordan, Stuart C Feinstein

  The Journal of biological chemistry. 05/2006; 281(17):11856-63.

  The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function ofThe neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.

• Automated tracking and modeling of microtubule dynamics.

  Authors: Motaz A. El Saban, Alphan Altinok, Austin J. Peck, Charles S. Kenney, Stuart C. Feinstein, Leslie Wilson, Kenneth Rose, B. S. Manjunath

  Proceedings of the 2006 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, Arlington, VA, USA, 6-9 April 2006; 01/2006

• Activity Analysis in Microtubule Videos by Mixture of Hidden Markov Models.

  Authors: Alphan Altinok, Motaz A. El Saban, Austin J. Peck, Leslie Wilson, Stuart C. Feinstein, B. S. Manjunath, Kenneth Rose

  2006 IEEE Computer Society Conference on Computer Vision and Pattern Recognition (CVPR 2006), 17-22 June 2006, New York, NY, USA; 01/2006

• Three- and four-repeat tau regulate the dynamic instability of two distinct microtubule subpopulations in qualitatively different manners. Implications for neurodegeneration.

  Authors: Sasha F Levy, Adria C Leboeuf, Michelle R Massie, Mary Ann Jordan, Leslie Wilson, Stuart C Feinstein

  The Journal of biological chemistry. 05/2005; 280(14):13520-8.

  The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), inThe microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type approximately 50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

• SNP detection using peptide nucleic acid probes and conjugated polymers: applications in neurodegenerative disease identification.

  Authors: Brent S Gaylord, Michelle R Massie, Stuart C Feinstein, Guillermo C Bazan

  Proceedings of the National Academy of Sciences of the United States of America. 02/2005; 102(1):34-9.

  A strategy employing a combination of peptide nucleic acid (PNA) probes, an optically amplifying conjugated polymer (CP), and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, andA strategy employing a combination of peptide nucleic acid (PNA) probes, an optically amplifying conjugated polymer (CP), and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, and sensitive manner. The recognition is accomplished by sequence-specific hybridization between the uncharged, fluorescein-labeled PNA probe and the DNA sequence of interest. After subsequent treatment with S1 nuclease, the cationic water soluble CP electrostatically associates with the remaining anionic PNA/DNA complex, leading to sensitized emission of the labeled PNA probe via FRET from the CP. The generation of fluorescent signal is controlled by strand-specific electrostatic interactions and is governed by the complementarity of the probe/target pair. To assess the method, we compared the ability of the sensor system to detect normal, wild-type human DNA sequences, and those sequences containing a single base mutation. Specifically, we examined a PNA probe complementary to a region of the gene encoding the microtubule associated protein tau. The probe sequence covers a known point mutation implicated in a dominant neurodegenerative dementia known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), which has clinical and molecular similarities to Alzheimer's disease. By using an appropriate PNA probe, the conjugated polymer poly[(9,9-bis(6'-N,N,N-trimethylammoniumhexylbromide)fluorene)-co-phenylene] and S1 nuclease, unambiguous FRET signaling is achieved for the wild-type DNA and not the mutant sequence harboring the SNP. Distance relationships in the CP/PNA assay are also discussed to highlight constraints and demonstrate improvements within the system.

• Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death.

  Authors: Stuart C Feinstein, Leslie Wilson

  Biochimica et biophysica acta. 02/2005; 1739(2-3):268-79.

  Interest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerativeInterest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerative diseases. Under normal circumstances, tau performs its functions by binding to microtubules and powerfully regulating their stability and growing and shortening dynamics. On the other hand, genetic analyses have established a clear cause-and-effect relationship between tau dysfunction/mis-regulation and neuronal cell death and dementia in FTDP-17, but the molecular basis of tau's destructive action(s) remains poorly understood. One attractive model suggests that the intracellular accumulation of abnormal tau aggregates causes cell death, i.e., a gain-of-toxic function model. Here, we describe the evidence and arguments for an alternative loss-of-function model in which tau-mediated neuronal cell death is caused by the inability of affected cells to properly regulate their microtubule dynamic due to mis-regulation by tau. In support of this model, our recent data demonstrate that missense FTDP-17 mutations that alter amino acid residues near tau's microtubule binding region strikingly modify the ability of tau to modulate microtubule dynamics. Additional recent data from our labs support the notion that the same dysfunction occurs in the FTDP-17 regulatory mutations that alter tau RNA splicing patterns. Our model posits that the dynamics of microtubules in neuronal cells must be tightly regulated to enable them to carry out their diverse functions, and that microtubules that are either over-stabilized or under-stabilized, that is, outside an acceptable window of dynamic activity, lead to neurodegeneration. An especially attractive aspect of this model is that it readily accommodates both the structural and regulatory classes of FTDP-17 mutations.

• Modulation of microtubule dynamics by tau in living cells: implications for development and neurodegeneration.

  Authors: Janis M Bunker, Leslie Wilson, Mary Ann Jordan, Stuart C Feinstein

  Molecular biology of the cell. 07/2004; 15(6):2720-8.

  The neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observationsThe neural microtubule-associated protein tau binds to and stabilizes microtubules. Because of alternative mRNA splicing, tau is expressed with either 3 or 4 C-terminal repeats. Two observations indicate that differences between these tau isoforms are functionally important. First, the pattern of tau isoform expression is tightly regulated during development. Second, mutation-induced changes in tau RNA splicing cause neuronal cell death and dementia simply by altering the isoform expression ratio. To investigate whether 3- and 4-repeat tau differentially regulate microtubule behavior in cells, we microinjected physiological levels of these two isoforms into EGFP-tubulin-expressing cultured MCF7 cells and measured the effects on the dynamic instability behavior of individual microtubules by time-lapse microscopy. Both isoforms suppressed microtubule dynamics, though to different extents. Specifically, 4-repeat tau reduced the rate and extent of both growing and shortening events. In contrast, 3-repeat tau stabilized most dynamic parameters about threefold less potently than 4-repeat tau and had only a minimal ability to suppress shortening events. These differences provide a mechanistic rationale for the developmental shift in tau isoform expression and are consistent with a loss-of-function model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal cell death and dementia.