Akira Chiba - US grants

This proposal tests the "chemoaffinity" theory with single cell resolution in vivo. The mechanisms of synaptic target recognition is a major issue in developmental neurobiology. The "chemoaffinity" hypothesis postulates that each neuronal growth cone has an ability to respond to unique molecular labels of its target cells. Many diffusible and cell surface molecules have been proposed as candidate target recognition molecules. However, in most cases it is technically difficult to test their specific roles at the level of individual cells in vivo. The embryonic Drosophila neuromuscular system provides an ideal model system, being amenable to high resolution experimentation. This simple system consists of uniquely identified motoneurons and muscle cells. Connectivity is precise to the level of individual cells. Powerful genetics, allowing precise in vivo manipulations of a molecule under focus, and accessibility to high resolution cell biological assays facilitate direct tests of the candidate molecules at the level of single neurons and single genes. Already more than a dozen candidate target recognition molecules are identified in this system, based on their unique expression patterns and molecular characterization. They include diverse cell surface molecules such as Fasciclin III (Ig-CAM) and Toll (leucine-rich repeat containing cell surface molecule). Most recently, Chiba et al. (1995) have shown that Fasciclin III can function as a specific and "attractive" target recognition molecule. The proposed research will take the advantage of the Drosophila system. Rather than screening for additional candidate target recognition molecules, the study will instead dissect into the molecular nature of synaptic target selection by using both Fasciclin llI and Toll as a molecular model. The identified motoneuron growth cones will be challenged with their microenvironment being variously manipulated for either Fasciclin III or Toll expression (Specific Aims l & 2). This is achieved by combined use of misexpression through genomic transformation and loss- of-function mutations. The results will be assessed at the single cell level, through intracellular dye injection and immunocytochemistry, as well as functional and ultrastructural analyses of the identified synapses. In addition, live visualization of the identified growth cones in both undissected embryos and in vivo/vitro hybrid culture system will be performed. All together they provide comprehensive assessment of the molecular events during synaptic target recognition. Most of the methodology to be employed is already very familiar to the investigators. However, several new assays will be developed and/or fine-tuned to improve resolution of analysis further (Specific Aim 3). The results are anticipated to uncover how synaptic target recognition molecules work in vivo. They will begin to define aspects of these molecules that are crucial for their functions. Also, whether or not members of multiple gene families have evolved to serve functionally redundant roles will be tested. Ultimately, the results from the proposed research are hoped to help identifying synaptic target recognition molecules and understanding their molecular mechanisms in the human and other vertebrate brains.

High density lipoprotein (HDL) has been proposed to be a major mediator of the transport of excess cholesterol from peripheral cells to the liver for clearance from the body. Recent reports have indicated that some subspecies of HDL may have an enhanced ability to perform this function. The most common apolipoprotein in HDL, apolipoprotein (apo) Al, appears to modulate the structure of HDL as well as its interaction with various plasma factors. However, the structure of the apo Al is not yet known in detail. The proposed research will utilize specific apoAl variants produced by site-directed and deletional mutagenesis. The abilities of these variants to self-associate, bind lipid, and form reconstituted HDL particles will be determined. The structure of the variants in both the lipid-free and -bound forms will be probed by circular dichroism and fluorescence techniques. The results from these studies will provide information on the relative abilities of the various domains in apoAl to bind lipid and to interact with other apolipoproteins. In addition, specific information will be obtained on the chemical environments of individual tryptophan residues in the N-terminal region of apoAl.

DESCRIPTION (Adapted from the Applicant's Abstract): The goal of this project is to test the validity of two general hypotheses: (1) the growth cone reprogramming hypothesis, and (2) the unique dendritic guidance hypothesis. According to the first hypothesis, axon guidance occurs through an interactive reprogramming model, in which a growth cone must encounter specific molecular cues early during its navigation so that it can undergo a reprogramming process that enables it to properly respond to cues presented subsequently. The project adopts in vivo single cell analysis, while taking the CNS midline as one example of early guidance cues that may be responsible for growth cone reprogramming. The second hypothesis is a model to explain why dendrites and axons grow differently. According to this hypothesis, axons and dendrites grow differently because they use a different set of growth cone receptor/signaling molecules. A neuron is capable of targeting molecules differentially to its axon or dendrites, thereby allowing the differential expression of receptors at each site. Additional possibilities are that dendritic growth may depend on axonal guidance, or that dendritic growth cones may be more susceptible to neuronal activity. This project will begin to tease apart the unique dendritic guidance hypothesis by using in vivo single cell analysis of dendritic development as compared to axonal development. It is anticipated that the present study will provide a better understanding of operational principles of growth cone guidance.

Many molecules have been described that stimulate or guide axons, but less is known about the molecules that cause axons to stop and form specialized synaptic contacts. This project focuses on a postsynaptic molecule, commissureless (comm), that has such a function and is required for synapse formation at the neuromuscular junction of Drosophila. The proposed experiments will determine whether comm works autonomously and whether it acts by regulating the endocytic resurfacing of postsynaptic cells. Since comm appears to be one of the major coordinators of neuromuscular junction, further characterization of its mechanism of action will enhance our knowledge of synaptogenesis.

DESCRIPTION (Verbatim from the Applicant's Abstract): This project investigates the roles of integrin during axon navigation in vivo. A major challenge in neuroscience today is to explain the genetic porgrams that direct development of the brain. The brain is composed of multi-layered networks of neurons that self-organize specific connections. How does a neuronal growth cone communicate with other cells while choosing specific pathways? What molecules are there to mediate such cell recognition and migration? The prevailing view is that accurate axon guidance relies on the activation of cell surface receptors that translate extrinsic cues into directed cytoskeletal rearrangement within a growth cone. Integrin is a family of multi-functional cell surface receptor/adhesion molecules, enriched in axonal growth cones. Although it has been proposed to play unique and important roles during migration and network formation of neurons, the main functions of integrin during axon navigation are not well understood in vivo. Drosophila offers both genetic and cell biological advantages. We apply our lab's expertise and examine the axon navigation defects in integrin knock out mutants, determine the cell autonomous requirement of integrin in axons and begin characterizing how integrin works inside a growth cone in situ. Specific hypotheses will be evaluated in real life contexts (in situ). Aim 1 evaluates both "speed controller" and "decision mediator" models for integrin's roles during axon navigation. It also tests the idea that much of axon defects in knock out mutants reflect integrin's "cell autonomy" inside neurons that express integrin. Aim 2 looks inside axons and begins to evaluate the idea that integrin works through forming "focal adhesions" and/or "filopodia enrichment." It also examined the differences between alphaPS1 and alphaPS2 knock out phenotypes and ask whether the differences owe to their "expression-based distinctions" or "structure-based distinctions." Whereas Aim 1 establishes cellular contexts in which Integrin's in vivo roles can be studied during axon navigation, Aim 2 explores into the molecule-level experimentation in the same in vivo contexts. Thus, the project "bridges" the gap between the wealth of in vitro (molecule-level) knowledge and the absence of in vivo (cell-level) analysis of integrin. Through this project, we move from the question of "what is integrin capable of doing?" to that of "what is integrin really doing" in real life contexts in situ.

DESCRIPTION (provided by applicant): The mechanisms by which the wiring of the central nervous system is established during development are an important topic of basic neuroscience. Although much attention has been paid to the mechanisms of axon guidance, there are major gaps in our understanding of the processes used by dendrites to establish specific synaptic connections. This project uses the tractable Drosophila embryonic nervous system to tease apart the mechanisms of dendritic guidance. The previous and preliminary results show unexpected and novel aspects of this process that are very likely to have an important impact on how we think about molecules involved in pathfinding. Although previously identified molecules are being studied, the evidence so far obtained makes a clear case that the same molecules work in different ways in specific neurites within the same neuron, and that different identified neurons may use the same set of molecules in different ways. A set of integrated hypotheses, each of which can be adapted to general situations, will be tested by this project. If successful, outcome of this project will provide a much-needed background to understanding the cause of diseases that affect brain connectivity.

DESCRIPTION (provided by applicant): This project develops molecular Bioprobes that can be introduced into Drosophila neurons. These Bioprobes are designed to reveal dynamic signal integrations within an individual neuron in situ. During the initial grant period, the project develops new FRET (fluorescent resonance energy transfer)-based nano-scale Bioprobes to study Cdc42 signaling at the onset of dendrogenesis. The molecular mechanisms of dendrogenesis are not well understood. The complexity among cells and molecules in the CNS requires an approach that supports high-resolution analysis. Recent advances in genetically encoded Bioprobes allow an unprecedented opportunity to launch this new study. Preliminary observations lead to the hypothesis that spatiotemporal integration of Cdc42 activation and effector availability accounts for the precise space-time at which dendrogenesis initiates in neuron in vivo. Therefore, the goal for the initial grant period is two-fold: (1) to examine the role of Cdc42 signaling at the onset of dendrogenesis, and (2) to demonstrates the power of a Bioprobe-assisted in vivo approach. The Bioprobe-assisted analysis is readily applicable to studies on various properties of a neuron and its compartments.

DESCRIPTION (provided by applicant): This project aims at causing a major paradigm-shift in regenerative medicine. In the past, attempts to re-establish synaptic connectivity following injury have met great difficulties. A new work yielded evidence that mechanical force, applied either naturally or artificially at individual nascent synapses, initiates and enhances their connectivity. This FORCE (force orchestrated retrograde synaptic enhancement) mechanism is likely based on a property of cells conserved through evolution and, thus, has the potential to apply widely including regenerative medicine. In this project, we seek to establish the basis for novel strategies that make synaptic restoration possible. Genetics, bioengineering, computational bioinformatics and nanotechnology will be combined to test the hypothesis: mechanical force not only initiates and enhances but also restores neural connectivity. If successful, this project will shift the focus in regenerative medicine from moleculo-centric to mechano-centric approaches. PUBLIC HEALTH RELEVANCE: This project aims to introduce an important new concept to regenerative neuromedicine. Regeneration of central nervous system neurons after injury faces daunting challenges. We propose to explore the potential of mechanical force being an integral part of initiation, enhancement and restoration of synapses in vivo and, thereby, seek to establish a foundation for novel neural restoration strategies.

Towards a neuro-mechanical memory element

Abstract

The objective of this project is to explore whether a neuro-mechanical synapse can be formed by interfacing a neuron cell with a silicon substrate. It is based on our recent finding that mechanical force applied at a neuromuscular synapse of Drosophila (fruit fly) embryos produces neuronal memory. This mechano-sensing ability of neurons is likely rooted in evolutionarily conserved properties of cells. It has thus the potential to apply to neuro-silico interfaces. The approach to this goal are: (1) investigate whether mechanically applied tension on a neuron using a silicon probe can be transduced into information that can be captured, e.g., as an image; (2) functionalize the probe to explore whether synapse can be engineered, and (3) explore whether engineered synapse can attain usage dependent force sensitive memory.

Intellectual merit: The study will shed light, for the first time, on whether (and how) nature employs mechanical tension to store and process information in an analogue fashion. It then attempts to translate the knowledge to engineer a neuro-mechanical synapse. If this possibility is realized, it will be a breakthrough in the engineering of learning and memory into synthetic systems.

Broader impact: This study will lay the foundation for creating networks of synthetic cells that could learn and remember patterns and share that knowledge with devices and sensors. The knowledge will be integrated with education through (1) development of a new course on neuro-mechanics (2) student seminars, (3) hands on teaching modules at the local Children?s Science Museum, and (4) web page development.

Understanding force-induced learning and memory
(NSF CMMI 0800870)
PI: Taher Saif, University of Illinois at Urbana-Champaign


Recent in vivo experiments using Drosophila (fruit fly) embryos in the PI's lab reveal that mechanical force applied at an individual neuromuscular junction (synapse) produces neuronal memory. Under normal conditions, axons of neuron cells actively maintain a resting tension of about 1 nN. Increased tension, applied artificially, causes increased accumulation of neurotransmitters at the neuro muscular junction. Reduction of tension causes a decrease in neurotransmitters. This force-mediated control of neurotransmitter accumulation appears to be essential for memory formation. It is envisioned that a specific set of molecules serves as a "force sensor" that senses mechanical force at the synapse to induce the accumulation. Furthermore, this mechano-sensing ability of neurons is likely rooted in evolutionarily conserved properties of cells. This project will investigate the underlying cellular and molecular mechanisms of force sensing and force-induced neuronal memory in vivo in Drosophila embryos using advanced nano-mechanical force/stretch sensors, and new molecular FRET (fluorescence resonance energy transfer) based biosensors. The quest for the force sensor will be based on the hypothesis: localization of neurotransmitters is mediated by an appropriate level of intracellular calcium. The latter is induced by force/stretch.

The study will shed light, for the first time, on whether (and if so, how) nature employs mechanical tension to store and process information in an analogue fashion. It will thus reveal a new mechanism of memory formation, in contrast to the conventional view that neurotransmission is entirely a result of electro-chemical signaling process. The study will offer significant fundamental knowledge in the field neuroscience, and has the potential of laying the foundation of the new field of neuro-mechanics. The study will also offer clues for new engineering treatment protocols for various neuronal disorders such as Alzheimer?s, and Parkinson?s diseases, possibly involving mechanical stimuli.

DESCRIPTION (provided by applicant): A team of University of Miami investigators requests $4,970,522 to undertake a readily deployable collaborative project that will redefine the proteomics as a context-rich molecular bioinformatics. Proteomics has been hailed as 'the next big thing'after genomics. It has progressed from cataloging the whole complement of proteins, or proteome, to charting their interactions, or interactome. Yet the major predicament in proteomics today is its paucity of in situ contexts. The team proposes to launch an imaging-based survey of protein-protein interaction networks within neurons. Its ultimate goal is reconstruction of genome-wide protein- protein interaction networks within each and every subcellular compartment of neurons at progressive steps of their development. This project will be the first systematic inspection of when and where each protein-protein interaction takes place in vivo. The investigators bring their expertise in neuronal imaging, Drosophila genetics, and computational analysis. The project will isolate GFP (green fluorescent protein) protein-trap lines for endogenous proteins present in well-studied model CNS and PNS neurons, and create inducible transgenic lines for neurologically 'relevant'proteins tagged with either GFP or monomeric RFP (red fluorescent protein). Using crosses to combine the green and red tagged proteins in a single fly, the project will both determine the localization of each protein and reveal the dynamic interactions between proteins in cell body, axon, dendrite and/or synapse of intact neurons within a whole organism during development. To achieve high-content screens within two years, the project takes advantage of a robotic imaging system as well as a FLIM (fluorescent lifetime microscopy) cluster. The latter quantifies GFP-to-RFP FRET (Forster resonance energy transfer), an indicator of direct association, between each of approximately 100,000 protein protein pairs. Over one million 3D images of model neurons will be analyzed to construct proteomic maps specific for different neuronal types, developmental stages and subcellular compartments. This image library will then undergo cross-correlation analysis to arrive at a model of the dynamics of the molecular networks of wild type neurons. At its completion, the project not only delivers the first context-rich proteomics resource, but also offers a new intellectual infrastructure for determining the molecular circuitries affected by neurological disorders, aging or drug addiction and designing strategies to repair and/or protect neurons. PUBLIC HEALTH RELEVANCE: Proteomics remains virtually context-less. The proposed project will show context-specific proteomic maps of wild type neurons in the intact brain, furnishing a much-needed intellectual infrastructure to transform definitions of the molecular circuitries affected by neurological disorders as well as strategies to repair/protect neurons.