James R. Bamburg - US grants

Using an assay for G-actin based on its ability to inhibit the hydrolytic activity of DNAase, we have isolated a protein from brain which actively promotes the depolymerization of F-actin in solution. This protein will now be further characterized as to its molecular weight, isoelectric point, amino acid composition and its ability to interact with F-actin alone and in the presence of associated proteins such as tropomyosin. A modification of the DNAase inhibition assay has been developed which permits the assay of G-actin in the presence of F-actin under conditions where no exchange between the two actin pools occurs. This assay will be applied to the measurement of G and F-actin concentrations in synchronized CHO cells during the cell cycle.

J.R. Bamburg will use the epi-fluorescence microscope with stage incubator for the microinjection of fluorescently labelled proteins into living cells. The image intensification and analysis system will be utilized to follow the distribution of fluorescently labelled cytoskeletal proteins into their cellular components. The dynamics of actin and actin depolymerizing factor in the neuronal growth cone will be observed during the transition from active neurite outgrowth to the quiescent state. Subcellular distribution of calcium ion will be measured to correlate changes in calcium with alterations seen in the organization and assembly of actin. The linear photodiode array camera and intensity stabilized light table will be used to identify, by two-dimensional (2-D) gel analysis, proteins whose interaction with the growth cone cytoskeleton is modulated by agents which alter growth. J.S. Bedford will use the epi-fluorescence microscope to measure the proliferative status of irradiated cells by a new immunofluorescent assay for DNA polymerase alpha. The image analysis system coupled to the fluorescence microscope will be used to analyze interphase chromosomal damage and the early stages of chromosomal aberration formation following radiation exposure. The 2-D gel analysis system is required for examining the differences which exist in the proteins synthesized in thermotolerantless mutants of CHO cells which have been isolated. R.J. Gillies will use the instrumentation to discern the spatial and temporal subcellular distribution of both hydronium and calcium ions in mouse fibroblasts. These ions will be monitored during the cells proliferative response to serum or growth factors. The effects of these agents on the assembly of actin in these cells will also be monitored by cytochemistry of fluorescent analogs. The 2-D gel analysis system will be used to identify proteins that have been synthesized in response to mitogenic signals. M.R. Paule will use the microscope and microinjection system for transformation of Acanthamoeba. The image analysis system will be used for identification of modified RNA polymerase subunits on 2-D gels, for quantitative transcription assays and footprinting, and for DNA sequence analysis using the molecular biology workstation.

This award will support collaborative research between Dr. James Bamburg, Colorado State University, and Dr. Dennis Bray, Cell Biophysics Unit, Medical Research Coucil, London. In this project Dr. Bamburg and Dr. Bray will investigate the sites of assembly of actin into the neuronal cortex using fluorescence and electron microscopy. Actin filaments are the major cytoskeletal element of the neuronal growth cone and comprise the core structure of microspikes, pseudopodia which extend from the growth cone and which generate the tension required for directed neurite growth. Drs. Bamburg and Bray plan to identify putative complexes between actin and other proteins in transit to the axonal cortex. The proposed experiments should determine if actin subunits exchange between the filament population in the axon during transport, and if the cortical actin layer of the axon arises from deposition at the growth cone or the shaft. During the course of the project, Dr. Bamburg will prepare proteins for microinjection; Dr. Bray will prepare antibodies against the fluorescent labels. The microinjections studies will be carried out in both laboratories. The proposed studies are well suited for collaboration between these two laboratories. Dr. Bray has been a leader in the development of neuronal culture methods, production of antibodies to neuronal cell proteins, and the application of immunocytochemical methods for other localization of proteins within neurons. Dr. Bamburg has been active in isolation and characterization in vitro of proteins ________ involved in cytoskeletal structures and in regulating assembly of cytoskeletal components.

biomedical equipment resource; biomedical equipment purchase;

Determination of the sequence of amino acids is the first step toward the study of cloning and structure-function relationships of proteins. DNA of known base sequence can be synthesized if the amino acid sequence of a protein is known. Genes for several proteins are proposed to be cloned in the Biochemistry Department; these include actin depolymerization factor and an rRNA transcription initiation factor. Amino acid sequence is also essential to find the site of proteins damaged by oxyradicals and hydrogen peroxide; for the region of neurotoxin-acetylcholine receptor binding; to elucidate the active center of snake hemorrhagic toxins; and to determine the regulatory site of phosphorylation in actin depolymerizing factor. The active sites of the RNA polymerases from E. coli and bacteriophage T7 are also proposed to be studied. Unfortunately, there is no protein sequencer at Colorado State University. At the moment we are relying on a facility at a neighboring state university. Our ability to do protein research is impaired by the lack of more direct and timely access to this instrumentation. Acquisition of a sequenator will enhance the efficiency and quality of protein research at Colorado State University.

This proposal is directed toward elucidating the mechanisms which regulate the spatial and temporal changes in actin filament organization that accompany growth cone movement during neuronal pathfinding. Actin filaments, the core structure of filopodia, are the major cytoskeletal component in nerve terminals. In order to understand actin filament dynamics in growth cones we must know: 1) in what form the actin arrives at the growing tip of the neuron; 2) where the actin monomer is incorporated into the filament; and, 3) what factors regulate its assembly and organization. We will utilize cultured neurons in an active state of growth to address these questions. Classical pulse-chase experiments followed by differential extraction to separate the soluble actin pool from the cytoskeletal actin will be combined with video imaging and fluorescence energy transfer experiments to examine the forms of actin and the regions within the neuron where the actin assembles. Studies on the regulation of actin assembly and organization will focus on two proteins already demonstrated in other cell types to be of significance in actin assembly and membrane association. One of these proteins, actin depolymerizing factor (ADF), is regulated in other cells by phosphorylation and we have identified a phosphorylated form of ADF in growth cone particles. Since ADF is abundant in neurons (20% to 40% of the level of actin on a molar basis), and since ADF binds actin available for assembly will depend directly on the inactivation of ADF. We will use isolated growth cone particles to identify the nature of the membrane signal transduction mechanisms involved. A second protein, alpha-actinin, identified in neuronal growth cones, is a alpha-actinin in growth cone adhesion. Defined surface adhesion and cell adhesion molecules covalently attached to glass will be used to form adhesion sites on growth cones. The adhesion plaque- associated molecules will be removed attached to the glass and the cytoplasmic proteins will help us to understand how encounters between the growth cone and substratum affect the membrane attachment of actin and translate the environmental cues into an organized growth response.

Actin depolymerizing factor (ADF) is a 19kDa protein which can sever actin filaments and sequester actin monomers. It is widely distributed among different tissues of the embryonic and adult chicken, and immunologically cross-reactive proteins of identical molecular mass occur in mammalian cells. ADF levels are high in embryonic tissue (as much as 0.6 mole ADF per mole of actin) but disappear from some adult tissues such as muscle, and decline 50% in others such as nerve. The ADF level does not decline during in vitro myogenesis, indicating one or more factors which regulate ADF synthesis are lacking. One of these factors is likely to be thyroid hormone which appears to regulate ADF expression in developing brain. ADF activity can also be controlled by posttranslational modifications. Several isoforms of ADF, both active and inactive, have been isolated from tissues and from cultured cells. Some cells have multiple active isoforms. These isoforms of ADF will be fully characterized with respect to activity, primary structure and posttranslational modifications, as well as to determine certain structure/function relationships within a single isoform of the molecule by site-directed mutagenesis of our cloned full length cDNA. This characterization will include a genomic analysis for the number and structure of the ADF gene(s), and the mechanisms by which the multiple mRNAs for ADF arise. The nature of the posttranslational regulatory system which controls ADF activity in vivo will be identified. The cellular function of ADF will be investigated by microinjection of ADF and activity- inhibiting antibodies. Specific antibody probes will be prepared which will allow us to analyze the temporal and spatial relationships between the inactivation of ADF and the assembly of actin in developing muscle. These studies will utilize chimeric myotubes formed from myocytes of mice carrying a dysgenic mutation and rescued by the insertion of a normal nucleus. The level at which ADF expression is regulated in myocytes and the effect of thyroid hormone on ADF expression will be determined. Successful completion of these studies will provide the means to determine the structure of actin in a filament and the role that the control of ADF expression and activity have in regulating actin assembly in many cell types.

Actin depolymerizing factor (ADF) is a 19kDa protein which can sever actin filaments and sequester actin monomers. It is widely distributed among different tissues of the embryonic and adult chicken, and immunologically cross-reactive proteins of identical molecular mass occur in mammalian cells. ADF levels are high in embryonic tissue (as much as 0.6 mole ADF per mole of actin) but disappear from some adult tissues such as muscle, and decline 50% in others such as nerve. The ADF level does not decline during in vitro myogenesis, indicating one or more factors which regulate ADF synthesis are lacking. One of these factors is likely to be thyroid hormone which appears to regulate ADF expression in developing brain. ADF activity can also be controlled by posttranslational modifications. Several isoforms of ADF, both active and inactive, have been isolated from tissues and from cultured cells. Some cells have multiple active isoforms. These isoforms of ADF will be fully characterized with respect to activity, primary structure and posttranslational modifications, as well as to determine certain structure/function relationships within a single isoform of the molecule by site-directed mutagenesis of our cloned full length cDNA. This characterization will include a genomic analysis for the number and structure of the ADF gene(s), and the mechanisms by which the multiple mRNAs for ADF arise. The nature of the posttranslational regulatory system which controls ADF activity in vivo will be identified. The cellular function of ADF will be investigated by microinjection of ADF and activity- inhibiting antibodies. Specific antibody probes will be prepared which will allow us to analyze the temporal and spatial relationships between the inactivation of ADF and the assembly of actin in developing muscle. These studies will utilize chimeric myotubes formed from myocytes of mice carrying a dysgenic mutation and rescued by the insertion of a normal nucleus. The level at which ADF expression is regulated in myocytes and the effect of thyroid hormone on ADF expression will be determined. Successful completion of these studies will provide the means to determine the structure of actin in a filament and the role that the control of ADF expression and activity have in regulating actin assembly in many cell types.

Because many critical vents in organismal development require the proper functioning of the actin cytoskeleton, functional mutations in proteins required for these processes are lethal. However, many disorders of early development might arise through abnormal modulation of signal transduction pathways which alter the temporal or spatial regulation of these events, leading to less severe phenotypic variants which survive. Many different actin binding proteins are involved in the organization of the actin cytoskeleton and its dynamic reorganization in response to environmental cues. Among these are the ubiquitous F-actin binding/severing and monomer sequestering proteins of the ADF/cofilin family. These proteins are enriched in ruffling membranes and neuronal growth cones, regions of high actin assembly dynamics. Functional mutations in the ADF/cofilin gene are lethal in yeast, fruit fly and worm. Interactions of ADF/cofilin with actin are modulated by phosphatidylinositides, pH and direct phosphorylation of a single serine residue. ADF cofilin can limit the amount of cytoplasmic actin available for assembly by transporting it to the nucleus. Many intracellular events coupled to cytoskeletal reorganization are triggered by transmembrane signals involving pH, phosphatidylinositol (PI) metabolism, or cAMP production. A direct activation (dephosphorylation) of ADF/cofilin by a cAMP-dependent mechanism occurs rapidly in many cell types undergoing a change in morphology. Thus, the ADF/cofilin family could be a common target of all these pathways and link transmembrane signalling to enhanced actin dynamics. Studies are proposed here to determine how the diverse modes of ADF/cofilin regulation are utilized by cells to alter their behavior. Using cultured animal cells, each cell type selected to best answer important questions about ADF regulation and function in differentiated cells, we propose: 1) to study the behavior of cells expressing a non- phosphorylatable site-directed mutant of ADF, and to determine the effect of its expression upon actin synthesis; 2) to elucidate the signal transduction pathway(s) leading to ADF phosphorylation/dephosphorylation by studying the dynamics of ADF, and to determine the effect of its expression upon actin synthesis; 2) to elucidate the signal transduction pathway(s) leading to ADF phosphorylation/dephosphorylation by studying the dynamics of ADF phosphorylation in cells treated with activators and inhibitors of membrane ruffling; 3) to investigate the role of intracellular pH in regulating ADF activity; 4) to quantify ADF/cofilin binding to PIs in cell membranes and the effect of binding on PI turnover or ADF release following activation of PI-3-kinase and PLCgamma; and 5) to determine the mechanism by which ADF expression is down regulated in cells expressing a mutant form of actin. Because actin has such a vast number of interdependent roles in normal cell function, it is not surprising that multiple signal transduction pathways can impact its organization. It would make sense for a regulator of actin assembly to be a common target for many, if not all, of these pathways.

This award will support the participation of undergraduate students in the Research Experiences for Undergraduates program offered by the Colorado State University in Biochemistry and Molecular Biology. Biochemistry/molecular biology employs the methods of chemistry, physics, cellular biology, microbiology and genetic manipulation to study the structure and function of complex biomolecules, how these molecules generate the phenomenon we call life, and how alterations in these molecules perturb the living organism. Because of its breadth, biochemistry/molecular biology serves to integrate didactic and experimental material learned in a multitude of basic science courses. This REU site program in Molecular Biosciences will enable qualified undergraduates to do independent research projects in a range of areas and using a variety of experimental approaches. The program is designed to (a) offer the participants a wide variety of research areas from which to choose an independent studies project; (b) offer project areas that are meaningful and exciting to the student; (c) teach the student how to formulate and test hypotheses; (d) help develop the ability to trouble-shoot and problem solve when confronted with the unexpected; (e) instruct the participant in state-of-the-art techniques and instrumentation of biochemistry/molecular biology; (f) develop the ability to communicate with one's peers concerning ongoing research efforts as well as more formal presentations of completed research projects; (g) provide, in our faculty and in outside scientists invited to speak to the REU students, appropriate role models that will reinforce the intellectual excitement of a scientific career; (h) emphasize to all participants gender and diversity issues that impact research careers in science; and (I) render assistance to participants in applying for graduate studies and fellowships. This award will foster the continued education and training of individuals who will be part of the next generation of basic researchers in the diverse aspects of biochemistry and molecular biology.

DESCRIPTION: ADF was discovered by Bamburg at the start of his career and he has focused on it, almost exclusively. ADF is a small monomer that typifies a class of actin-binding proteins by having a curious mixture of actin-binding activities including severing actin filaments and binding actin monomers. These proteins are found in all eukaryotes and are essential in all of several studied cases, including yeast and Dictyostelium. Cells contain a large amount of them, at a molar ratio of about 1:10 relative to actin. They are regulated by phosphoinositides, pH and phosphorylation. Most notably, Bamburg has found that phosphorylation regulates the actin-binding activity of ADF in vitro, and that phosphorylation occurs in developing muscle cells in a manner predicted from the actin assembly that occurs there. ADF is therefore the best case of regulation of a protein that regulates actin assembly. Bamburg has identified the phosphorylation site as a Serine and prepared substitution mutants that constitutively mimic the on and off states (Glu and Ala, respectively). In preliminary work for this application, Bamburg has isolated and analyzed cDNA's and Ab's for Xenopus ADF along with preparing recombinant ADF that is fully functional in vitro. He finds developmental changes in the phosphorylation that controls actin-binding activity. Ab localization studies show ADF in the same places as filamentous actin. Aim 1 is to determine whether ADF is important for actin reorganization during oogenesis. Aim 2 is to determine the nature of the phosphatase that activates ADF by dephosphorylating it after fertilization. The strategy will be to increase the concentration of various second messengers that lie upstream of individual phosphatases. Also, the phosphatase will be purified biochemically from fertilized eggs. Aim 3 is to determine if ADF is important in actin reorganization events that follow fertilization, using injection of inhibitory Abs, and active or inactive ADF mutants. Aim 4 is to determine how ADF influences actin filaments during cytokinesis, which has been found in new preliminary data. The approach will be similar to that in Aim 3. Aim 5 is to determine if the small G-proteins rho, rac1 and cdc42 are important for cortical rotation and cytokinesis and if so, if their effects are mediated through ADF. Again, dominant negative and constitutively active forms will be microinjected, with observation of changes in the biological phenomena. Effects on phosphorylation of ADF will be examined, along with the ability of inhibitory anti-ADF Abs to block these effects.

DESCRIPTION (VERBATIM FROM APPLICATION): Directed neuronal growth depends upon the dynamics of actin filaments within the highly motile growth cone. Although actin dynamics are regulated by the complex interactions of many actin-binding proteins, the high turnover rate of actin filaments in vivo is dependent upon proteins in the actin depolymerization factor (ADF)/cofilin (AC) family. AC proteins are essential in all eukaryotes and are enriched within neuronal growth cones. Furthermore, overexpression of ADF in neurons leads to an enhanced rate of outgrowth that is sustained for days. AC proteins are phosphorylated on a single regulatory site. Their phosphorylation and dephosphorylation are common targets of signaling pathways linking external signals to actin cytoskeletal reorganization. Enhanced dynamics of this phosphocycle, without a net change in the phosphorylation state of the AC proteins, often accompanies induction of ruffling membrane. Vertebrate AC proteins are phosphorylated by LIM kinases for which they are the only known substrates. Many growth factors and guidance cues signal in part through the rho family GTPases, each of which has specific targets for actin organization, but all of which target at least one member of the LIM kinase family. Hemizygosity of LIM kinase I causes Williams syndrome, a visuo-spatial cognitive defect resulting from aberrant neuronal migration in the human brain. Thus we hypothesize that the response of growth cones to various guidance cues is modulated by the regulation of AC activity, which is likely to be the final integrator of multiple signaling pathways. We propose to use a combination of molecular, cellular, immunological, and ultrastructural approaches to test the hypothesis that phosphorylation regulation of ADF activity is required for initiation of neuritic outgrowth cone pathfinding in response to attractive or repulsive guidance cues. Tropomyosin isoforms, which may compete with AC for actin filament binding, and Arp2/3, which caps pointed ends of actin and may regulate the ability of AC to depolymerize the filaments, will be localized along with AC and phosphorylated AC in growth cones undergoing a turning response. We will also test the hypothesis that guidance cues signal via bifurcating pathways that regulate both AC phosphorylation and dephosphorylation through activation of PI-3 kinase, and we will isolate and characterize the phosphatase involved. Adenoviral mediated gene transfer is a major tool to be used in sorting out the signal transduction pathways in regulating AC. These studies will advance our understanding of nerve growth cone guidance and the design of agents that allow neurons to grow and regenerated in a normally non-permissive environment.

DESCRIPTION (provided by applicant): Microischemia, oxidative stress, and glutamate excitotoxicity are likely initiators of age-related neurodegenerative diseases. Many senile dementias are characterized by a loss of synapses (up to 50 percent loss compared to age related controls) within the hippocampus and cortex, whereas neuronal loss is far less. The mechanism by which some neuronal processes selectively degenerate while associated cell bodies and other processes remain intact is largely unexplained. Treatment of cultured hippocampal and cortical neurons with common mediators of neurodegeneration leads to the formation of axonal and dendritic inclusion bodies in the form of actin depolymerizing factor (ADF)/cofilin-cortactin-actin rods. ADF/cofilins are essential proteins that regulate the turnover of actin in vivo. Through their regulation by phosphorylation on a single serine residue by LIM kinase, ADF/cofilins are a common target of signaling pathways for actin cytoskeletal reorganization via the rho family of GTPases. Cortactin normally associates with the cortical actin cytoskeleton and is regulated by non-receptor tyr kinases. It has domains for binding Arp2/3 complex and Factin and may cross-link filaments in the rods. Treatments that induce rods cause ADF/cofilin dephosphorylation and at least a transient inactivation of mitochondria. Rod-like inclusions containing ADF are found in Alzheimer brain: half of the inclusions occur nearby amyloid plaques, but >98 percent of plaques have neurites nearby that contain inclusions. Normal human brains do not contain similar ADF-inclusions. Growth cones disappear from neurites containing persistent rods but normal growth cones are found on other processes extending from the same soma, confirming that persistent rod formation is accompanied by degeneration of the neurite distal to the inclusion. Rods may provide a mechanism linking mitochondrial dysfunction to the selective pruning of synaptic terminals. Here we propose to determine the role of rods in synapse elimination. Our specific aims are to test the following interrelated hypotheses: Formation of a persistent rod in a neurite process inhibits transport to the distal regions of that neurite. Synaptic function in a process distal to a rod will be impaired. Amyloidpeptides and plaque will induce rods and therefore rods will contribute to the pathology in mice expressing the mutant amyloid precursor protein (APP v717F). Rod formation requires cortactin dephosphorylation and cortactin domains that bind Arp2/3 complex and F-actin.

DESCRIPTION (provided by applicant) Integrated investigations using methods from biochemistry, molecular biology, cell biology, anatomy and physiology are central to advancing our understanding of synapse formation, organization and function. The purpose of the proposed program is to provide postdoctoral and predoctoral training focused in synaptic neurobiology. Our strategy for training gains from its ability to emphasize a specific set of current issues that require an interdisciplinary approach while still maintaining a breadth of training experience and exposure to a wide variety of intellectual tools. This research is the foundation for understanding - and hopefully alleviating - numerous neurological diseases. The training will be provided by 7 faculty members of the Molecular, Cellular and Integrative Neuroscience (MCIN) Program. The faculty members come from four departments in two colleges. These faculty, along with 14 others, have organized and teach a core neurobiology curriculum offering lecture, literature, laboratory and discussion courses. Within the program are individuals who can apply this fundamental knowledge in actual treatment of neurological disorders, through their joint appointments as clinical scientists at the Colorado State University Veterinary Teaching Hospital. In 2000 - 2001, 15 predoctoral and 20 postdoctoral students received training in the laboratories of the training faculty. These trainees were supported from individual research grants, postdoctoral and graduate fellowships, and by graduate assistantships from the MCIN Program. Most of the predoctoral trainees have degrees in traditional disciplines and were recruited either through participating departments or through the MCIN program. All of the postdoctoral trainees initiated contact with their mentors directly and were not recruited. The program described in this proposal would allow us to recruit 4 postdoctoral trainees and support 2 predoctoral students to participate in cross-disciplinary projects involving more than one laboratory for up to 3 years. These recruits will be important for strengthening the interactions between laboratories and will provide the trainees with a unique opportunity to acquire the multidisciplinary skills needed for success in today's research environment. All trainees will have access to modern cell and molecular biological facilities including computer enhanced light, confocal and electron microscopy (intermediate voltage), combined electrophysiology and light/fluorescence microscopy equipment, macromolecular synthesis and sequencing facilities, gene cloning and cell transfection laboratories, and a high quality lab animal resource facility.

[unreadable] DESCRIPTION (provided by applicant): Within neurites of nearly all cultured hippocampal neurons, transient ATP depletion rapidly induces rod- shaped structures composed primarily of actin and ADF/cofilin (AC). Rod formation, which sequesters a portion of the actin but virtually all of the AC, is transiently beneficial to the stressed neuron because it spares ATP associated with actin turnover. However, rods can completely occlude the neurite, blocking transport and causing distal neurite withering. Rods are prominent features of Alzheimer's disease (AD) brain but not of control human brain lacking amyloid plaques. Similar structures are found in brains of animals with Niemann-Pick disease typed (NPC1) and of transgenic mice (Tg2576) expressing mutant human amyloid precursor protein (APP). In cultured neurons and mouse brain slices, rods are induced by ischemia, peroxide, NO, and excitotoxic glutamate. In up to 20% of hippocampal neurons, whether from region CA1 or CAS, the AD amyloid beta peptide (Ab) also induces rods: induction is dose-dependent, occurring within 6 h after treatment and reaching a plateau 12-24 h later. As little as 10 nM of Ab oligomer has a significant effect compared to the scrambled peptide control. The nature of the sensitivity of only a subset of neurons to Ab will be explored. Rods block vesicular transport of APP. APP-containing vesicles accumulate at the ends and sides of rods. Within these stalled vesicles is beta-secretase cleaved APP, suggesting that these may be sites of Ab production and/or conversion into more damaging conformers. Taken together, these results suggest a model for AD in which neuronal stress, including Ab formed in familial AD, induces rods that stall vesicle transport and increase toxic Ab, thus inducing rods in neighboring cells. Such a model could explain the formation of amyloid plaques, which would enlarge around the initial site of injury. Using cell culture and organotypic brain slices, we will determine: 1) what activities of cofilin are required for rod formation; 2) if mutations in AC can be identified that prevent rod formation; 3) if rods promote the production or oligomerization of Ab; 4) what makes a subset of neurons sensitive to Ab: and 5) how organotypic brain slices can be used as a model to study where rods form and how they disrupt synapses. Relevance to public health: AD dramatically impacts life quality of senior Americans, affecting 25% of those > 85. This proposal tests a new hypothesis for AD progression and identifies possible sites for targeted intervention. [unreadable] [unreadable]

This is a 5-day workshop on the cytoskeleton to be held in Pingree Park, Colorado State University in the summer of 2010, entitled: The Cellular Cytoskeleton: Common Organizing Principles in Mitosis, Motility and Cell Polarization. The main theme of this meeting will be on using quantitative experiments and theory to gain insight into (i) how the behaviors of complex cytoskeletal structure arises from the individual molecules from which they are composed, and (ii) how these dynamic properties of the cytoskeleton are used by cells during mitosis, motility, and cell polarization. The conference will bring together a group of about 60 researchers and graduate students that have an interest, exposure and/or training in interdisciplinary research but who are not necessarily experts in multiples areas or techniques of cytoskeletal dynamics. The workshop will be organized into morning and evening sessions with invited speakers who are experts in cell biology, biophysics, biochemistry, microscopy, genetics and theoretical modeling. The target is more than 30% of women invited speakers and more than 40% women participants. The organizerss will select and provide support for applicants who are minorities and/or persons with disabilities. This conference will fulfill an important goal of broadening graduate student education by facilitating interactions of graduate students from diverse areas with each other. The organizers plan to publish a report summarizing the novel ideas and collaborations that developed during the meeting.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca2+, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation;furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

DESCRIPTION (provided by applicant): Cofilin is a well-documented promoter of actin turnover in all eukaryotic cells. It undergoes dephosphorylation (activation) and oxidation to dimers when neurons are stressed by agents associated with Alzheimer disease (AD), all of which increase reactive oxygen species (ROS). This cofilin oxidation results within neurites in the formation of rod shaped cofilin-saturated actin bundles (rods). Rods immediately after their appearance are energy conserving, but when sustained causes neurite degeneration by blocking transport. Agents can induce rods via mitochondrial inhibition that generates ROS (e.g., excitotoxic glutamate) or via a prion-dependent pathway, probably involving membrane NADPH oxidase (NOX) activation. The latter pathway is activated by the proinflammatory cytokine TNF¿ as well as by soluble SDS-stable dimers/trimers of Amyloid-¿ (A¿d/t), the A¿ form that correlates best with dementia severity. Cofilin-actin rod formation causes the synapse elimination without neuronal cell death that is characteristic of amnestic mild cognitive impairment, an early stage in progression to Alzheimer disease. Our hypothesis is that it is cofilin-actin rods that initiate and exacerbate synaptic dysfunction typical of both sporadic (SAD) and familial AD (FAD). To demonstrate definitively that rod formation per se contributes to cognitive decline associated with AD, we need to develop a mouse model resistant to rod formation. Only a rod resistant mouse will allow us to answer the critical question: Do rods, themselves, cause synaptic loss or is synaptic loss due to stress-induced changes other than rod formation? It should be possible to make such a model since we have characterized a non-rod forming mutant of cofilin (K22Q), which is able to rescue normal behavior of cofilin-silenced cells as well as wild type cofilin. Three strategies are described to make a knock-in mouse in which cofilin K22Q will replace wild type cofilin. Two of these will make a conditional mouse which expresses wild type cofilin until mice are given tamoxifen. In these mice tamoxifen activates expression of Cre recombinase, which will initiate the inactivation of the wild type gene and the activation of the K22Q cofilin gene. The project is high risk since obtaining such a mouse is not guaranteed. Ultimately these mice will be used in behavioral assays to assess their cognitive ability under normal and stress conditions mimicking SAD or FAD. Finding that rods per se are necessary for synapse loss and cognitive impairment would make this project high reward because we already have identified a nutraceutical, the pentacyclic triterpene ursolic acid (UA), that blocks and reverses A¿d/t- and TNF¿-induced rods in cultured neurons and reverses oxidative stress markers and cognitive deficits in a brain oxidative stress mouse model. Thus we will determine if UA functions by reducing cofilin pathology and if it can be a major therapy for reducing cognitive deficits in mouse models of both SAD and FAD.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the only leading cause of death with no effective means of slowing its progression. Varying models predict the involvement of diverse neurodegenerative stresses, including amyloid beta (Aß) peptides, proinflammatory cytokines, oxidative stress, and energetic stress in mediating the pathogenesis of both familial (FAD) and sporadic (SAD) forms of the disease. In spite of decades of active research and a deep molecular understanding of many of the major molecular players in the progression of dementia, there are many areas of confusion. For example, there is good agreement on the fact that excess production of amyloid-ß is a causative or major contributing factor to the initiation of dementia and most mouse models to study AD utilize the expression of human amyloid precursor protein (APP) with early onset AD mutations (usually along with other mutant human proteins) to generate mice that develop cognitive deficits and at least some aspects of human AD pathology. However, there is still much debate over the actual form of the Aß that induces the synaptic deficits. In addition, a plethora of neuronal proteins with totally unrelated functions that interact with human Aß have been identified and elimination of any one of these binding partners alone was sufficient to reduce or eliminate the cognitive deficits when these mice were crossed with an AD mouse model over producing Aß. No current model can explain how single elimination of the different Aß binding partners protects against development of Aß-induced cognitive deficits. We recently showed that very active forms of soluble Aß consisting of dimers and trimers (Aßd/t), as well as proinflammatory cytokines (TNFa, IL-1ß, IL-6) stimulate NADPH oxidase (NOX) and production of reactive oxygen species (ROS) in neurons through a cellular prion protein (PrPC)-dependent pathway. This pathway stimulated the formation of rod-shaped bundles of 1:1 cofilin:actin (rods), which cause synaptic dysfunction. Formation of rods requires activation (dephosphorylation) of the actin binding protein cofilin as well as its oxidation to form intermolecular disulfide bonds. Rods do not form i response to Aß or proinflammatory cytokines in PrPC-null neurons, but surprisingly, over expression of PrPC alone is sufficient to induce rods at much higher levels than are induced by Aß of proinflammatory cytokine treatment. Thus, we have proposed a new model in which multiple receptors can contribute to NOX activation and ROS production through PrPC-interactions in enlarged membrane domains. The triggering of cofilin oxidation to form rods is dependent on achieving a threshold level of ROS and this is why coalescence of many different receptors into signaling complexes contributes to achieving this ROS level. Rods sequester cofilin and can occlude neurites, blocking transport, either of which inhibit normal synaptic function. Using cultured primary neurons and several knock-out or transgenic mouse lines, we propose to determine (1) if the relative rod-inducing activities of different forms of Aß relate t their direct affinity for PrPC, (2) if specific proinflammatory cytokine receptors are required for their rod induction through the PrPC- dependent pathway, (3) if the PrPC-pathway functions in both axons and dendrites and if mislocalization of rod signaling components occurs between compartments, and (4) the role in cofilin activation played by three likely components of the cytoplasmic domain of PrPC-signaling complexes.

Project Summary This proposal seeks funding for a Keyence BZ-X710 all-in-one fluorescence microscope, which provides a wide range of wide-field imaging capabilities in concert with incredible ease-of-use. The unique set of microscopy capabilities provided in this microscope permit high throughput, rapid imaging of a vast array of fixed specimens in various preparations, e.g., multi-well microtiter plates, culture dishes, microscope slides, roller tubes, etc. Further, the BZ-X710 incorporates an optional stage-top incubation chamber to enable imaging of live cells and in vitro tissues. Another advantage of the BZ-X710 that has a transformative impact on live cell imaging is the high speed at which large images can be acquired and stitched into a mosaic, with demonstrated repeatability to well below the spatial resolution of the imaging system. Finally, the nature of the illumination system significantly decreases deleterious effects such as photobleaching and photodamage, and thus both living and fixed specimens can be imaged for longer periods of time than with, for example, a confocal microscope. The structured illumination system and deconvolution algorithms allow for rapid imaging of relatively thick tissue slices (up to 500 ?m) at multiple wavelengths. This combination of unique attributes permits the use of BZ-X710 for long-term fluorescent imaging studies of living cells and tissue slices. Observation of living cells with a large number of exposures is paramount for studying long-term behavior of a specimen, and could greatly aid in elucidating the function of biomolecules within the cell. Here we will exploit the power of the BZ-X710 by applying it to many projects of biomedical significance. These projects ask questions whose answers will improve our understanding of basic cell biological phenomena and apply this knowledge to diverse areas of human health, such as neurodegenerative disorders and cancer. The ease-of-use of the Keyence BZ-X710 microscope allows us to incorporate this technology into a microscopy core facility that will enable its use by many research groups, while maintaining access to the major users who have proposed many exciting questions to be addressed here. Incorporation into a Foundational Core and having oversight, training and data acquisition and analysis provided by a university funded facility director with an applied physics PhD and several years of experience in bioscience applications will ensure proper long-term operation.