Project Summary: Engineered environments offer the …biomechanics.stanford.edu/paper/BioX.docx · Web viewSoichi Wakatsuki Structural Biology and Photon Science School of Medicine/SLAC

Bio-X IIP 2016 Molecular Mechanisms of Chronic Traumatic Encephalopathy

1. PROJECT TITLEMolecular Mechanisms of Chronic Traumatic Encephalopathy

2. INVESTIGATORSEllen Kuhl Mechanical Engineering and Bioengineering School of EngineeringSoichi Wakatsuki Structural Biology and Photon Science School of Medicine/SLAC

3. BACKGROUND AND SIGNIFICANCEChronic traumatic encephalopathy is a progressive degenerative disease that affects people who have experienced one or more mechanical insults to the head. To date, the only method to reliably diagnose chronic traumatic encephalopathy is post mortem histopathology, where it manifests itself through extensive damage of axons in the white matter tissue. In the healthy state, the axonal cytoskeleton is made up of microtubules, neurofilaments, and microfila-ments. Microtubules are hollow tubes of α- and -tubulin heterodimers composed of thirteen laterally joined protofilaments. Axonal microtubules are assembled into well-organized, evenly spaced bundles by tau dimers. Recent studies suggest that tau acts as a molecular switch between three major mechanisms of axonal failure: microtubule sliding, detachment, and rupture, Fig. 1. However, the dynamic interactions of the tau-microtubule complex remain poorly understood. This project seeks to provide fundamental insight into the molecular mechanisms of axonal failure in chronic traumatic encephalopathy using cryo-electron microscopy, small angle X-ray scattering, axonal stretching, molecular dynamics simulations, kino-geometric sampling, and multiscale modeling with the ultimate goal to understand, diagnose, slow down, block, or reverse progressive neurodegeneration.

4. OBJECTIVE AND SPECIFIC AIMSTraumatic brain injury is a nondegenerative, noncongenital insult to the brain that affects more than one million people annually in the United States alone. Recent studies suggest that even mild concussions—if repetitive—can trigger progressive neurological degeneration, a condition that is increasingly recognized as chronic traumatic encephalopathy. Progressive axonal failure and structural degradation are classic hallmarks of chronic traumatic encephalopathy. Strikingly, these symptoms appear to be shared, at least in part, by a number of other neurodegenerative diseases including Alzheimer’s disease and Parkinsonism. However, the molecular mechanisms of neurodegeneration remain poorly understood. Our long-term goal is to establish a mechanistic, physico-biochemical model of axonal dynamics to predict the effects of mechanical stimuli on normal and abnormal axon physiology. The overall objective of this proposal is to probe, model, and simulate the tau-microtubule complex to reveal the molecular failure mechanisms of chronic traumatic encephalopathy. Our central hypothesis is that axonal failure is an emergent property of the dynamic interplay of axonal microtubules and cross-linking tau proteins. The rationale for creating a multiscale model of axonal failure is that it will allow us to virtually probe landscapes of parameters across multiple intrinsic spatial and

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Figure 1. Tau protein acts as molecular switch between the three mechanisms associated with axonal failure: microtubule sliding, detachment, and rupture. van den Bedem & Kuhl, Biophysical Journal, 2015;109:2215-2217.


temporal scales and identify common degenerative pathways shared by several neurodegenerative diseases. Ultimately, our macroscopic-exposure/microscopic-response rela-

Figure 2. Close interplay of experiment and computation to characterize structure, function, and failure of the tau-microtubule complex.

tionship can help reduce chronic traumatic encephalopathy by establishing safe limits for a variety of activities. Towards this goal, we will pursue three specific aims, Fig 2.:Aim 1. Characterize tau-microtubule structure and interaction using cryo-electron microscopy, and predict molecular-scale failure mechanisms using molecular dynamics simulations. Aim 2. Identify the molecular failure mechanisms of the tau-microtubule complex using small angle X-ray scattering and interpret conformational ensembles using kino-geometric sampling. Aim 3. Identify the cellular failure mechanisms of an axon using axonal stretching and multiscale, multiphysics models derived from physico-biochemical principles.

5. OUTLINE OF PROPOSED METHODS

Aim 1. Characterize tau-microtubule structure and interaction using cryo-electron microscopy, and predict molecular-scale failure mechanisms using molecular dynamics simulations. While structures of tubulin heterodimers are available from cryo-electron microscopy, tau is an intrinsically disordered protein for which a structure has remained elusive. By binding at the interface between tubulin heterodimers and forming partially ordered dimers, tau modulates the arrangement of individual microtubules into well-organized, evenly spaced bundles. However, the precise structural and dynamic interactions between tau and microtubules are unknown. The objective of this aim is to structurally and dynamically characterize the tau-microtubule complex. Our working hypothesis is that tau adopts partial order in the complex, enabling structure determination with cryo-electron microscopy. Our approach is to create a structural model of the complex for different isoforms of tau and validate the model with cryo-electron microscopy. Cryo-electron microscopy will be performed on the Titan Krios, a first of several state-of-the-art electron microscopes to be operational at SLAC starting December 2016. Our tau-microtubule model will inform non-equilibrium, coarse-grained molecular dynamics simulations to probe molecular failure mechanisms. Our rationale is that the structure-informed simulations will report the response of each molecular interface, tau-tau and tau-microtubule, under different strain and strain rates. Our expectation is that we obtain a high-resolution tau-microtubule structure supplemented by simulated failure modes of tau-microtubule complexes.

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Aim 2. Identify the molecular failure mechanisms of the tau-microtubule complex using small angle X-ray scattering and interpret conformational ensembles using kino-geometric sampling.The objective of this aim is to quantify the forces between microtubules coated with tau using synchrotron small-angle X-ray scattering of reconstituted tau-microtubule complexes under varying osmotic pressures. Our working hypothesis is that the distance between two neighboring microtubules decreases with increasing osmotic pressure and that this effect is more pronounced for tau isoforms with long projection domains. To test this hypothesis, we will use a hybrid experimental-computational approach based on small angle X-ray scattering and kino- geometric sampling. The rationale for perturbing the equilibrium state of the tau-microtubule

Figure 3. Small angle X-ray scattering experiments characterize the molecular mechanisms of tau-microtubule interaction: At low osmotic pressures, the negatively charged projection domains of neighboring tau proteins generate a repulsive force; at high osmotic pressures, the negatively and positively charged projection domains generate an attractive force and form an electrostatic zipper to stabilize microtubule conformations. Adapted from Chung et al., PNAS, 2015;E6416-E3625.

complex through a systematic variation of the osmotic pressure and interpreting the data using our customized kino-geometric sampling algorithms is that this will allow us to directly quantify the physical forces between neighboring microtubules to characterize tau-microtubule dynamics: At low pressures, the negatively charged projection domains of tau will generate repulsion; at high osmotic pressures, the polyampholytic nature of tau will generate attraction, Fig. 3. It is our expectation that this aim will elucidate the fundamental, pressure- and isoform-dependent role of tau as a molecular switch between different failure mechanisms in chronic traumatic encephalopathy, with important implications for other tauopathies including Alzheimer’s disease, frontal dementia, and Parkinsonism.

Aim 3. Identify the cellular failure mechanisms of an axon using axon elongation experiments and multiscale, multiphysics models derived from physico-biochemical principles.

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Figure 4. Axonal stretching reveals the progressive loss of microtubules. When rapidly stretched, control axons gradually lose microtubules; taxol-stabilized axons maintain microtubules; nocoda-zole-destabilized axons rapidly lose microtubules. Tang et al., FASEB Journal, 2010;24:1401-1410.

Diffuse axonal injury has been studied extensively at different scales; yet, to date, there is no single integrative concept to understand how axonal failure propagates across the scales. The objective of this aim is to synthesize the structural and functional information about the tau-microtubule complex and create a multiscale, multi-physics model of the axon to predict cellular failure mechanisms from physico-biochemical principles. Towards this objective, we will test our working hypothesis that extreme stretch and stretch rates induce a break-down of the tau-microtubule complex on the molecular scale, which triggers progressive microtubule depolymerization on the cellular scale, and ultimately results in in axonal failure, Fig. 4. We will test this hypothesis by adopting a hybrid experimental-computational approach using our novel axonal model, informed by controlled axonal stretch experiments. We will micropattern untreated, taxol-stabilized, and nocodazole-destabilized neuronal cell cultures on silicone membranes and subject them to systematically varying stretch and stretch rates to identify cellular level rate constants, Fig. 4. The rationale for combining the entropic elasticity of biomolecules with the binding kinetics of protein bonds in a unified cellular model for axonal failure is that this will allow us, for the first time, to probe the landscapes of interacting intracellular failure mechanisms: At low strain rates, our axon model will be highly compliant and easily deformable; at high strain rates, our axon model will be stiff and vulnerable to mechanical loading. Once calibrated across the scales, it is our expectation that our axon model can reproduce the failure mechanisms of neurodegeneration and predict the timeline of progression in chronic traumatic encephalopathy.6. PREVIOUS INVESTIGATIONS AND PERTINENT WORK BY INVESTIGATORS

Figure 5. Kino-geometric sampling and subse-quent molecular dynamics simulations identified a higher energy substate of non-coding RNA HIV-1 TAR from sparse data. van den Bedem & Fraser, Nature Methods, 2015;12:307-318.

Cryo-electron microscopy. Through our leadership role in the Biosciences Division at SLAC, our group is actively involved in a number of new electron and optical microscopy initiatives including the installation of the FEI Titan Krios, Stanford’s new 300 kV cryo-electron microscope instrument that will be operational at SLAC starting December 2016. Cryo-electron microscopy has previously been used to characterize structures of tubulin heterodimers, but not of tau.

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Here, we will create a structural model of the tau-microtubule complex, and validate the model using cryo-electron microscopy. For sample preparation, we work closely with the new Macromolecular Structure Knowledge Center, a joint initiative between Stanford ChEM-H, SLAC, and the School of Engineering that helps engineers characterize macromolecules. Characterizing the structure of the tau-microtubule complex is the experimental objective of Aim 1 of this proposal.

Molecular dynamics. Our group has extensive experience in using molecular dynamics simulations to interpret structure and function of large biomolecular complexes. Informed by cryo-electron microscopy data, molecular dynamics simulations allow us to computationally perturb the equilibrium state of the tau-microtubule complex and generate hypotheses to explain its failure mechanisms. We have successfully applied molecular dynamics simulations—both in isolation and in combination with kino-geometric sampling—to analyze the activation pathways of G-protein coupled receptors, reveal previously hidden substates of HIV-1 TAR, and characterize the structure and dynamics of phosphorylation mediated UBAN domain binding to ubiquitin, Fig 5. Our group routinely uses OpenMM and NAMD, two popular open source molecular dynamics codes for high-performance simulation of large biomolecular systems. Harnessing molecular dynamics simulations to identify molecular failure mechanism of the tau-microtubule complex is the simulation objective of Aim 1 of this proposal.

Small angle X-ray scattering. Our group routinely investigates atomic, molecular, cellular interactions of proteins and their complexes using small angle X-ray scattering at the Stanford Synchrotron Radiation Lightsource. For this project, we will use Beamline 4–2 to acquire scattering data with a 2D Area Detector, with a sample-to-detector distance of 3.5m as previously shown. We prepare tau-microtubule samples with an osmotic depletant, polyethylene oxide, following established protocols. Varying the osmotic pressure to characterize forces within the tau-microtubule complex by small angle X-ray scattering line shape analysis is the experimental objective of Aim 2 of this proposal.

Kino-geometric sampling. Our group has developed the widely used, open source kino-geometric sample-and-select tools qFit, CONTACT, and KGS that report on ensembles of biomolecular conformations orders of magnitude faster than traditional molecular dynamics simulations. The size of the tau-microtubule complexes and the time scales at which conformational changes occur hinder adequate sampling of the conformational landscape with molecular dynamics simulations. We have pioneered robotics-inspired conformational sampling procedures that probe the conformational landscape of large biomolecular complexes to exploit their structure-dynamics-function relationship in protein engineering and design. Identifying the molecular failure mechanisms of the tau-microtubule complex using kino-geometric sampling is the simulation objective of Aim 2 of this proposal.

Axonal stretching. Our group has the expertise and knowledge to probe living cells using physical forces. Here, we will apply this concept to probe individual axons using calibrated glass microneedles and quantify forces using bending mechanics. The fabrication, calibration, and use of microneedles to stretch axons is well-established and well-documented. Manipulating the mechanical environment via microneedles allows us to directly stimulate and regulate axonal stretch while monitoring axonal deformation and force at multiple time points. We have successfully adopted this approach to calibrate the rate constants of our model using chicken

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dorsal root ganglion cells at slow stretch rates, Fig. 6. Screening the landscape of varying stretches and stretch rates is the experimental objective of Aim 3 of this proposal.

Figure 6. Axon model (left) and kymographs of axonal stretch experiment (top right) and of our simulation (bottom right) highlight how we use the experimental data to calibrate our model.

Multiscale modeling. Our group has created the first multiphysics model of an axon that enables polymerization and depolymerization of individual microtubules, dynamic cross-linking of neighboring microtubule via tau protein, lateral pressure via a surrounding actin meshwork, and overall elongation and failure in response to physical forces. We have demonstrated that our axon model reproduces axonal stretching on the phenomenological level, Fig. 6; yet, the model has never been calibrated mechanistically with temporal and spatial data from molecular failure experiments. Synthesizing our molecular and cellular level information towards a fully calibrated multiscale axon model is the simulation objective of Aim 3 of this proposal.

7. INTERDISCIPLINARY AND INTERACTIVE NATURE OF THE PROPOSALIn a new collaboration between SLAC and Stanford, this research will cross the boundaries between Photon Science, Structural Biology, Mechanical Engineering, and Bioengineering to create new insights into neurodegeneration in chronic traumatic encephalopathy. We have built a strong research team of leading experts in diverse fields with the immediate goal to reinterpret neurodegeneration correlating nano-scale tau-microtubule breakdown to macro-scale events. Through joint group meetings, mentoring of joint postdoctoral researchers, and interdisciplinary training of graduate students, we will establish a close collaboration between our groups.

8. NEW COLLABORATION THAT LEVERAGES EXPERTISE OF EACH MEMBERThis project is an entirely new collaboration of investigators at Stanford and SLAC who have never collaborated, published together, or served as collaborators on a joint grant. Dr. Kuhl was the PI on a previous Bio-X IIP seed grant on simulations of human brain development in 2014; this seed grant has, to date, generated a News & Views article in Nature Biophysics, 13 peer-reviewed journal articles, 29 conference presentations, and the educational outreach video ‘Unfolding the Brain’ (http://www.phdcomics.com/tv/?v=x9IXtTbt2f0). This 2016 molecular-level project has no overlap with the 2014 organ-level project. Dr. Wakatsuki has never been a PI on a Bio-X IIP seed grant before. In preparation for this project, our team has met regularly over the period of several months—without support—to initiate our collaboration with the goal to improve the fundamental understanding of chronic traumatic encephalopathy and other neurodegenerative diseases. Our new collaboration leverages Dr. Kuhl’s expertise in multiscale modeling and Dr. Wakatsuki’s expertise in structural biology and photon science with first-hand access to the high performance cluster Sherlock at Stanford and to the unique biosciences facilities at SLAC.

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9. POTENTIAL IMPACTS AND OUTCOMESThis project will provide fundamental insight into the common molecular mechanisms shared by chronic traumatic encephalopathy and other tauopathies including Alzheimer’s disease, frontal dementia, and Parkinsonism. It will provide widely applicable new technologies including: (i) a structural characterization of tau-microtubule binding mechanisms; (ii) fundamentally new insight into the molecular failure mechanisms of the tau-microtubule complex ; (iii) insight into the cellular failure mechanisms of individual axons ; (iv) functional relations between protein structure, protein dynamics, and physical forces; and (v) predictive mathematical models to explain how physical forces translate into cellular and molecular failure. Ultimately, a better mechanistic understanding of the tau-microtubule complex will help identify potential drug targets and design inhibitors to slow down, block, or reverse progressive neurodegeneration.

10. POTENTIAL FOR FUTURE FUNDINGThis project will allow us to formally strengthen the collaboration between SLAC and Stanford, collect preliminary data, and support our efforts to apply for external funding at NSF and NIH.

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SUPPORTING DOCUMENTS

1. BIOSKETCHES OF INVESTIGATORS AND ROLES IN THE PROJECT

Our research has the potential to radically change our understanding of chronic traumatic encephalopathy, a progressive neurodegenerative disease that cannot be fully understood by one single discipline alone. This project is an entirely new collaboration of investigators who have never collaborated, published together, or served as collaborators on a joint grant. Yet, in preparation for this project, our team has met regularly over the period of one year—without support—to discuss pathways of future collaboration with the common goal to improve the fundamental understanding of chronic traumatic encephalopathy and other neurodegenerative diseases. To address this challenge, we have assembled a strong and diverse research team of leading experts in complementary fields to integrate the experimental and computational expertise across Stanford and SLAC:

Ellen Kuhl, Principal Investigator, Professor of Mechanical Engineering and Bioengineering, is an expert in the computational modeling of living systems. As the Chair of the Mechanics and Computation Group at Stanford, Dr. Kuhl will oversee the axonal stretch and shear experiments in Aim 3 and the molecular and cellular level failure simulations in Aims 1 and 3. She will closely mentor the Postdoctoral Researcher Dr. Weickenmeier, and provide feedback and support for the modeling aspects of this project.

Soichi Wakatsuki, co-Investigator, Professor of Structural Biology at Stanford and of Photon Science at SLAC, is a leading expert in protein crystallography and small angle X-ray scattering. As the Director of the Biosciences Division at SLAC with a Laboratory in the Clark Center, Dr. Wakatsuki will oversee the sample preparation, cryo-electron microscopy, and small angle X-ray scattering in Aims 1 and 2. He will closely mentor the second postdoctoral researcher, and provide feedback and support for the experimental aspects of this project.

Henry van den Bedem, Ph.D., Senior Staff Scientist in the Biosciences Division at SLAC , is an expert in computational biodynamics and crystallography. He designed and developed the open source kino-geometric sampling algorithms qFit (https://simtk.org/projects/qfit), CONTACT, and KGS (https://simtk.org/projects/kgs) to interpret high-resolution X-ray data with multi-conformer models, to automatically identify contact networks of conformationally heterogeneous residues in high-resolution X-ray crystallography data, and to report on ensembles of RNA molecular conformations orders of magnitude faster than ordinary molecular dynamics simulations. He will contribute to the experimental design and data analysis in Aims 1 and 2 of this project. Johannes Weickenmeier, Ph.D., Postdoctoral Researcher in the Department of Mechanical Engineering is an expert in computational modeling of biological systems. He will be responsible for the axon stretch and shear experiments and for the multiscale modeling in Aim 3. He will be mentored by Dr. Kuhl and will collaborate closely with Dr. Wakatsuki and Dr. van den Bedem to coordinate data acquisition and data analysis in Aims 1 and 2 to calibrate the axonal model across the scales. He will oversee the modeling aspects of the project and support the second postdoc’s molecular dynamics simulations and Rijk de Rooij’s axonal failure simulations.

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TBD, Postdoctoral Researcher in the Department of Structural Biology. The postdoctoral researcher will be responsible for sample preparation, data collection, and data analysis of cryo-electron microscopy and small angle X-ray scattering in Aims 1 and 2. He/she will be mentored by Dr. Wakatsuki and Dr. van den Bedem to perform kino-geometric sampling and coarse-grained molecular dynamics simulations. He/she will collaborate closely with Dr. Kuhl and Dr. Weickenmeier to coordinate data analysis and model calibration as molecular level input for the cell level model in Aim 3.

Rijk de Rooij, M.Sc., Ph.D. student in the Department of Mechanical Engineering is an expert in multiscale, multiphysics modeling who has implemented the first computational model for axons that includes dynamic microtubule polymerization, dynamic microtubule cross-linking via cross linking proteins, and physical forces. He is a post-masters, 4 th year PhD student who is fully supported through the Stanford Graduate Fellowship. Rijk de Rooij will contribute to the axon model development in Aim 3, informed by the parameters from Aims 1 and 2.

Maria Holland, M.Sc., Ph.D. student in the Department of Mechanical Engineering is an expert in multiscale modeling of the brain. She has created and implemented acute and chronic axon elongation models and calibrated these models with short-term and long-term axonal stretch experiments to characterize the time scales of axon elongation. She is a post-masters, 5th year PhD student who is fully supported through a Stanford DARE fellowship. Maria Holland will contribute to the calibration of the axon model in Aim 3.

Caitlin Ploch, M.Sc., Ph.D. student in the Department of Mechanical Engineering, is an expert in the mechanical characterization of brain tissue. She has tested the macroscopic properties of mammalian brain using uniaxial testing and created neurosurgical models of the human brain. She is a 3rd year PhD student who is fully supported through the Stanford Graduate Fellowship and the National Science Foundation Graduate Research Fellowship. Caitlin Ploch will contribute to the sample preparation in Aims 1 and 2 and to the experimental testing in Aim 3.

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2. ABSTRACT DESCRIBING THE WORK AND HOW IT RELATES TO THE MISSION OF BIO-X

Traumatic brain injury is a mechanical insult to the brain that affects more than one million people in the United States every year. We now know that even mild concussions can trigger progressive neurological degeneration, a condition that is increasingly recognized as chronic traumatic encephalopathy. There is currently no way to diagnose chronic traumatic encephalopathy during life; it can only be diagnosed after death by histopathological analysis. In the brain of a healthy person, nerve cells transmit electrical signals through thin, long cables called axons. These axons are made up of microtubules that are assembled into well-organized, evenly spaced bundles by tau proteins. Progressive failure of the tau-microtubule complex and gradual axonal degradation are classic hallmarks of chronic traumatic encephalopathy. Strikingly, these symptoms appear to be shared by a number of other neurodegenerative diseases including Alzheimer’s disease and Parkinsonism. However, to date, the molecular mechanisms of neurodegeneration remain poorly understood. The objective of this research is to probe, model, and simulate tau-microtubule interaction to provide fundamental insight into the molecular and cellular mechanisms of axonal failure during chronic traumatic encephalopathy. In a new collaboration between SLAC National Accelerator Laboratory and Stanford University, this research will cross the boundaries between Photon Science, Structural Biology, Mechanical Engineering, and Bioengineering and combine interdisciplinary solutions including cryo-electron microscopy, small angle X-ray scattering, kino-geometric sampling, molecular dynamics simulations, and multiscale computational modeling to predict the effects of mechanical stimuli on normal and abnormal axonal behavior. For cryo-electron microscopy experiments, we will capitalize on the Titan Krios, a first of several state-of-the-art electron microscopes that will be operational at SLAC starting December 2016. Taken together, this study will allow us to probe landscapes of interacting intracellular events at their intrinsic spatial and temporal scales and predict the timeline of neurodegenerative disease. A macro-exposure/micro-response relationship can help prevent chronic traumatic encephalopathy by establishing safe limits for a variety of activities. Ultimately, a better mechanistic understanding of the tau-microtubule complex could radically change our understanding of neurodegeneration and help identify potential drug targets and inhibitors to slow down, block, or reverse chronic traumatic encephalopathy.

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3. BUDGET

We request a total budget of $200,000 for a period of two years, with an annual budget of $100,000 for each year. The budget will support two postdoctoral researchers, sample preparation for cryo-electron microscopy and small angle X-ray scattering, mechanical testing, and support of conference travel.

Year 1

A. Personnel Postdoctoral Researcher, TBD, Molecular Experiment/Simulation $40,000Postdoctoral Researcher, Johannes Weickenmeier, Cell Experiment/Simulation $40,000

C. Supplies Aim 1. Protein Structure: Sample Preparation for Cryo-EM $ 5,000Aim 2. Protein Function: Sample Preparation for SAXS/Rheology $ 5,000Aim 3. Mechanical Testing: Testing Device and Sample Preparation $ 5,000Data Storage, Analysis Software, and Documentation $ 2,000

E. Travel Support for Conference Presentations: $1,500 per Postdoctoral Researcher $ 3,000

Total Year 1 $100,000

Year 2

A. Personnel Postdoctoral Researcher, TBD, Molecular Experiment & Simulation $40,000Postdoctoral Researcher, Johannes Weickenmeier, Cell Experiment & Simulation $40,000

C. Supplies Aim 1. Protein Structure: Sample Preparation for Cryo-EM $ 6,250Aim 2. Protein Function: Sample Preparation for SAXS/Rheology $ 6,250Aim 3. Mechanical Testing: Sample Preparation for Axonal Stretch and Shear $ 2,500Data Storage, Analysis Software, and Documentation $ 2,000

E. Travel Support for Conference Presentations: $1,500 per Postdoctoral Researcher $ 3,000

Total Year 2 $100,000

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BUDGET JUSTIFICATION

We request a total budget of $200,000 for the time period of two years as detailed below.

A. Personnel. This research will provide a holistic multiscale understanding of chronic traumatic encephalopathy, a progressive neurodegenerative disease that cannot be fully understood by a single discipline alone. To address this challenge, we have assembled a strong and diverse research team of leading experts in complementary fields to integrate the experimental and computational expertise across Stanford and SLAC.

Ellen Kuhl, Professor of Mechanical Engineering and Bioengineering, is an expert in the computational modeling of living systems. As the Chair of the Mechanics and Computation Group at Stanford, Dr. Kuhl will oversee the axonal stretch and shear experiments in Aim 3 and the molecular and cellular level failure simulations in Aims 1 and 3. She will closely mentor the Postdoctoral Researcher Dr. Weickenmeier, and provide feedback and support for the modeling aspects of this project.

Soichi Wakatsuki, Professor of Structural Biology at Stanford and of Photon Science at SLAC , is a leading expert in protein crystallography and small angle X-ray scattering. As the Director of the Biosciences Division at SLAC with a Laboratory in the Clark Center, Dr. Wakatsuki will oversee the sample preparation, cryo-electron microscopy, and small angle X-ray scattering in Aims 1 and 2. He will closely mentor the second postdoctoral researcher, and provide feedback and support for the experimental aspects of this project.

Henry van den Bedem, Ph.D., Senior Staff Scientist in the Biosciences Division at SLAC , is an expert in computational biodynamics and crystallography. He designed and developed the open source kino-geometric sampling algorithms qFit (https://simtk.org/projects/qfit), CONTACT, and KGS (https://simtk.org/projects/kgs) to interpret high-resolution X-ray data with multi-conformer models, to automatically identify contact networks of conformationally heterogeneous residues in high-resolution X-ray crystallography data, and to report on ensembles of RNA molecular conformations orders of magnitude faster than ordinary molecular dynamics simulations. Dr. van den Bedem is supported through SLAC and will contribute to the experimental design and data analysis in Aims 1 and 2 of this project at no additional cost to the BioX IIP program. Johannes Weickenmeier, Ph.D., Postdoctoral Researcher in the Department of Mechanical Engineering, effort 59%. Dr. Weickenmeier will be responsible for the axon stretch and shear experiments and for the multiscale modeling in Aim 3. He will be mentored by Dr. Kuhl and will collaborate closely with Dr. Wakatsuki and Dr. van den Bedem to coordinate data acquisition and data analysis in Aims 1 and 2 to calibrate the axonal model across the scales. Dr. Weickenmeier will oversee the modeling aspects of the project and support the second postdoc’s molecular dynamics simulations and Rijk de Rooij’s axonal failure simulations.

TBD, Postdoctoral Researcher in the Department of Structural Biology, effort 59%. The postdoctoral researcher will be responsible for sample preparation, data collection, and data analysis of cryo-electron microscopy and small angle X-ray scattering in Aims 1 and 2. He/she will be mentored by Dr. Wakatsuki and Dr. van den Bedem to perform kino-geometric sampling and coarse-grained molecular dynamics simulations. He/she will collaborate closely with Dr.

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Kuhl and Dr. Weickenmeier to coordinate data analysis and model calibration as molecular level input for the cell level model in Aim 3.

Rijk de Rooij, M.Sc., Ph.D. student in the Department of Mechanical Engineering, effort 75%. Rijk de Rooij is an expert in multiscale, multiphysics modeling who has implemented the first computational model for axons that includes dynamic microtubule polymerization, dynamic microtubule cross-linking via cross linking proteins, and physical forces. He is a post-masters, 4 th

year PhD student who is fully supported through the Stanford Graduate Fellowship. Rijk de Rooij will contribute to the axon model development in Aim 3, informed by the parameters from Aims 1 and 2, at no extra cost to the BioX IIP program.

Maria Holland, M.Sc., Ph.D. student in the Department of Mechanical Engineering, effort 50%. Maria Holland is an expert in multiscale modeling of the brain. She has created and implemented acute and chronic axon elongation models and calibrated these models with short-term and long-term axonal stretch experiments to characterize the time scales of axon elongation. She is a post-masters, 5th year PhD student who is fully supported through a Stanford DARE fellowship. Maria Holland will contribute to the calibration of the axon model in Aim 3 at no extra cost to the BioX IIP program.

Caitlin Ploch, M.Sc., Ph.D. student in the Department of Mechanical Engineering, effort 50%. Caitlin Ploch is an expert in the mechanical characterization of brain tissue. She has tested the macroscopic properties of mammalian brain using uniaxial testing and created neurosurgical models of the human brain. She is a 3rd year PhD student who is fully supported through the Stanford Graduate Fellowship and the National Science Foundation Graduate Research Fellowship. Caitlin Ploch will contribute to the sample preparation in Aims 1 and 2 and to the experimental testing in Aim 3 at no extra cost to the BioX IIP program.

C. SuppliesTo characterize the protein structure of the tau-microtubule complex, we will perform cryo- electron microscopy experiments at SLAC for which we budget $5,000 in year 1 and $6,250 in year 2 for sample preparation. To characterize the protein function of the tau-microtubule complex, we will perform rheological small angle X-ray scattering experiments at SLAC for which we budget $5,000 in year 1 and $6,250 in year 2 for sample preparation. To characterize cellular failure of the axon, we will perform axonal stretching and shearing tests for which we budget $5,000 in year 1, including building the testing device, and $6,250 in year 2 for sample preparation. We further budget $2,000 for data storage, analysis software, and documentation in years 1 and 2.

E. TravelTo disseminate our results to the scientific community, we budget support for conference participation of Dr. Johannes Weickenmeier and of the second postdoctoral researcher for $1,500 per year each resulting in $3,000 in years 1 and 2.

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4. OTHER PERTINENT SUPPORT AND POTENTIAL LEVERAGING

OTHER SUPPORT AND POTENTIAL LEVERAGINGKUHL, ELLEN

ACTIVE:

U01 HL119578. Guccione, Kassab, Kuhl (PI) 07/01/2014-06/30/2019National Institutes of Health (NHLBI)“Multi-scale laws of myocardial growth and remodeling”This overall goal of this study is to establish constitutive laws for cardiac growth and remodeling to predict the progression of heart failure using animal models and computational simulation. Role: Principal Investigator (2.27 calendar months)

NSF INSPIRE 1233054. Kuhl (PI) 08/15/2012-08/14/2016“Optogenetic control of the human heart – Turning light into force”This objective of this hybrid experimental/computational project is to create a biological pacemaker from genetically engineered heart cells and establish predictive models towards pacing hearts with light.Role: Principal Investigator (1.0 calendar months)

OVERLAP:

None

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OTHER SUPPORT AND POTENTIAL LEVERAGINGWAKATSUKI, SOICHI

ACTIVE:

R01HD084422-01A1. Mochly-Rosen (PI), Wakatsuki (co-PI) 04/01/2016–03/31/2021National Institutes of Health $250,000“Development of a novel treatment for hyperbilirubinemia-induced kernicterus”The objective of this proposal is to develop a treatment for kernicterus using a totally novel approach, by developing activators of wild-type (Wt) and mutant G6PDs is described.Role: co-PI

MEXT. Wakatsuki (co-PI), Nagano (co-PI), Gojobori (co-PI) 04/01/2012-03/31/2017Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT)“Platform for drug discovery, informatics and structural life science (PDIS)”, The objective of this Japanese 5-year national project to develop and operate infrastructure for structural life science with key applications in drug discovery, food and environment. Role: co-PI; more than 150 researchers participating with an annual budget of about $27M

JSPS. Wakatsuki (PI) 01/01/2011-12/31/2015Japan Society for the Promotion of Science (JSPS) “Development of 2D detectors using DEPFET sensors”The goal of this project is to develop fast pixel array detectors for X-ray crystallography and scattering in collaboration with particle physicists at KEK and DEPFET development consortium headed by Max Planck Institute, Munich

TIP 2014. Mochly-Rosen (PI), Wakatsuki (co-PI) 01/01/2014-12/31/2016Child Health Research Institute Stanford $46,750Transdisciplinary Initiatives Program“Generation of allosteric chaperones for glucose-6-phosphate dehydrogenase (G6PD) deficiency; application for neonatal jaundice-induced kernicterus”Role: co-PI

DOE Mesoscale Pilot Project. Wakatsuki (PI) 01/01/2015-12-31-2017DOE Office of Biological and Environmental Research $300,000 “On ammonia oxidizing archaea”Role: PI

SLAC LDRD 2016 Barger (PI). Wakatsuki (co-PI) 10/01/2015-09/30/2016SLAC Laboratory Directed Research and Development Program $300,000 “Molecular basis of ecosystem N cycling: A strategic SLAC biosciences program”The goal of this project is to develop capabilities to produce functional proteins and their complexes from environmental matagenomes and characterize the structures of these proteins. Role: co-PI

OVERLAP:

None15

Documents

Project Summary: Engineered environments offer the …biomechanics.stanford.edu/paper/BioX.docx · Web viewSoichi Wakatsuki Structural Biology and Photon Science School of Medicine/SLAC