David Meaney, Ph.D. - US grants

DESCRIPTION (Investigator's Abstract): The broad, long-term objective of the proposed research is to provide more consistent and comparable models of traumatic brain injury (TBI) in the rat and mouse. Currently, it is difficult to precisely compare animal models across laboratories and optimize the settings in these models to study the distribution and severity of human closed head injury lesions. In this application, we use modeling and experimental tools to better quantitatively define and improve the biomechanics of small animal TBI models. The specific aims of the research are: Aim A: To determine the brain tissue deformations caused by three common rodent models of traumatic brain injury-weight - weight drop, controlled cortical impact, modified weight. Aim B: To determine the in vivo axonal, neuronal, and vascular tissue thresholds for mechanical injury in TBI models. Aim C: To rigorously determine the in vivo brain tissue deformation caused by the fluid percussion technique (lateral and midline) in the rat, developing biomedical measures that best predict mechanical injury. Aim D: To standardize a cortical impact model in the mouse that applies for a variety of mouse strains. By accomplishing the aims of the research plan, we expect to predict the mechanically-mediated damage caused by rat and mouse models of traumatic brain injury and establish precise parametric relationships to compare these models across laboratories. In addition, we expect to minimize the variability in new TBI models targeted to study traumatic brain injury sequelae in transgenically altered animals. Once accomplished, the research will significantly enhance existing models to understand the sequelae and treatment of human closed head injury.

9733928 Meaney Cells in the human body are aware constantly of their outside environment and often respond to even small changes in surrounding mechanical forces. However, the exact nature and course of these mechanically initiated changes remains largely unexplained. If better understood, the adaptation of cells to mechanical stress could be better controlled to understand and direct cell growth, maintenance, and the restoration of cell function. The underlying premise of the research and educational activities of this CAREER award is that critical new advances in the control of cell function are accomplished by combining molecular based control of biological systems with an engineered modulation of the external mechanical forces sensed by the cell. An experimental approach is used to understand how mechanical forces are sensed, or transduced, by neuronal cells of the brain and spinal cord. The focus of research in this CAREER award is on understanding how changes to the structural architecture of the neuron can affect the sensing mechanisms for the cell. By developing structure-function relationships for normal neurons, we seek to extend these studies and accomplish our long term objective of using these sensing mechanisms to assist in the repair or recovery of the diseased or injured neuron. The central aim of the educational plan is to bring elements of cellular bioengineering research to the undergraduate level and form a stronger interface with the clinical sciences in the bioengineering undergraduate program. These two elements as critical for bioengineering undergraduates to experience, since they both bring undergraduates closer to the process of creating new knowledge through basic research and viewing the practical applications of basic bioengineering research in medicine. It is expected that the exposure to research based laboratories and modern tools in biomedicine at the undergraduate level will help prepare students for careers where research is a more pervasive element and is constantly translated into improvements in health care technology. ***

Funds are requested for the purchase of a multi-photon microscope to be conveniently located near two of the major Penn Institutes whose faculty will share the equipment. Some 14 users from the School of Engineering and Applied Science, the School of Medicine, and several campus institutes will use the equipment for biomedical and biomaterial research investigations. The new equipment offers significant advantages over existing equipment in imaging penetration depth, sensitivity, photodamage, and the ability to observe multiple fluorophores simultaneously. Specific studies include the effect of environmental stimuli on cells, the microscopic basis of biomaterial behavior, polymer behavior at interfaces, etc. The facility will be made available to researchers at nearby Universities.

Description: The proposed project will bring together a broad team of bioengineers, neuroscientists, molecular biologists, bioinformaticists and clinical scientists to examine the molecular etiology of traumatic brain injury (TBI). The focus will be to study the genomic and protein expression of force transmission in the CNS, with the long term goal of treating and preventing neuronal necrosis and apoptosis in gray matter contusions, the most common form of damage in brain injured patients.

DESCRIPTION (provided by applicant): Regenerating axons within the central nervous system (CNS) remains a fundamental challenge in neuroscience. Recently, we have shown that a large number (10/5) of axons integrated with CNS neuronal cultures will grow rapidly (8-10 mm/day) and over long distances ( >5 cm) if the axons are placed under a continuous mechanical tension. We feel the impact of this discovery could be significant. This technique provides a method to culture cell transplants for bridging lesions in the white matter that are centimeters long, distances that are not readily traversed with such a large number of axons using other techniques (e.g., ensheathing cell transplants, directed material scaffolds, controlled release). In addition, this model represents an opportunity to study the mechanisms of accelerated axonal growth in a large population of axons that was previously not possible. However, the technology is at a critical nascent stage with risk - it is not widely used or available to investigators, and we do not know if axonal tracts developed with this technique have viable electrophysiological function. In this proposed, we will build the appropriate technical infrastructure for rapidly culturing a large number of cell transplant constructs using commercially available materials, creating a more generalizable resource for the neuroscience community. Embedded within this re-design of the system is to allow for the measurement of electrophysiological properties of the constructs. Once developed, we use this to propose a series of studies on how a specific cytoskeletal component (neurofilaments) may be a key limiting factor in controlling the growth rate with this technique.

DESCRIPTION (provided by applicant): This is a competing continuation of a Bioengineering Research Partnership (BRP) grant focusing on the molecular mechanisms of traumatic brain injury (TBI). Our work in the last phase points out a potentially critical receptor - the calcium permeable AMPA receptor (CP-AMPAR) - that appears in neurons after mechanical injury and plays a key role in neuronal death. In the final phase of this BRP, we define the mechanisms regulating the appearance of CP-AMPARs (Aim 1), determine when CP-AMPAR activation leads to neuronal death (Aim 2), and develop therapies for reversing neuronal death initiated by CP- AMPARs (Aim 3). Our therapeutic approaches include strategies we can test immediately with available compounds, as well as new therapies developed with unique technologies to target key molecular events that lead to neuronal death. Our overlying hypotheses are (a) CP-AMPARs increase following injury due a change in the translation of AMPAR subunits, a change in the editing of GluR2 mRNA, and an ERK mediated insertion of GluR1 homomeric AMPARs. (b) Immediate or delayed inhibition of calcium permeable AMPARs reduce neuronal death after mechanical injury, and their effect is enhanced restoring G^luR2 editing (calpain inhibition) or inhibiting ERK phosphorylation, (c) Restoring ADAR2 editing activity of GluR2 mRNA, limiting the GluR2 synthesis, and interrupting Elk-1 signaling selectively in dendrites are effective delayed strategies to improve neuronal survival after injury. . We integrate the collective expertise of the BRP labs to test these hypotheses across the subcellular, cellular and organ scale. We evaluate transcription factor (Elk-1) signaling and the synthesis/regulation of CP- AMPAR subunits within individual dendrites after injury, measure changes in RNA editing and transcription within individual neurons and in slice culture, and test newly developed therapies in animal models of TBI. Relevance: This work studies factors that cause cell death after traumatic brain injury. The investigators test treatments to reduce neuronal death using commercially available compounds, and design new molecules that may be even more effective in reducing cell death. Both treatment approaches are tested for their effectiveness if given either immediately or several hours after injury, which is critical to know if these will be used clinically in the future.

Axonal damage is a hallmark of diffuse brain injuries, and is considered by many as a nearly universal consequence of traumatic brain injury. Recent evidence shows that unmyelinated axons are particularly vulnerable to damage in DAI. In this project, we study the mciromechanical aspects of axonal injury to unmyelinated axons. Our long term objective to study when sodium channel proteolysis occurs in axons after mild TBI, determine the mechanism(s) that regulate this channel proteolysis, and to evaluate the effectivenes of different biomarkers to evaluate this form of axonal damage. Our specific aims are to: Aim 1: To measure the strain rate sensitive threshold for sodium channel mechanoactivation following axonal stretch injury in vitro, determining the pathway(s) that contribute to both immediate and sustained stretch-induced increases in axoplasmic calcium, Aim 2: To determine the thresholds and timecourse of sodium channel proteolysis after axonal stretch, assess if this proteolysis is mediated by calpain activation and linked to a specific stretch-induced calcium pathway, and evaluate if the proteolysis can be reduced using delayed treatments. Aim 3: To determine the threshold and time course of a delayed increase in membrane permeability after axonal stretch injury, examine if this permeability changes is responsive to delayed treatments, and to test if this permeability change leads to a detectable release of 'biomarkers' for detecting axonal injury in vitro. Our overall hypotheses are that (a) sodium channel mechanoactivation occurs during mild stretch injury to unmyelinated axons, (b) sodium channel proteolysis is calpain mediated, (c) the primary pathway(s) for sustained calcium elevation in axons after stretch is a critical target in controlling sodium channel proteolysis after axonal injury. Relevance: One common type of injury in head injured patients is the swelling and disconnection of axons throughout many brain regions. We will study how this injury occurs, evaluate if we can treat this injury, and will assess if there specific biomarkers that can serve as prognostic indicators of axonal degeneration.

DESCRIPTION (provided by applicant): This shared instrumentation proposal for a customized multiphoton microscope is part of a larger institutional effort at Penn in cellular programming. Strategically, this effort lies at the intersection between five of Penn's schools - Medicine, Dental, Veterinary, Arts and Sciences, and Engineering and Applied Science. We request a multiphoton microscope equipped to perform three separate, but related efforts, in cellular programming. The efforts include: At the single cell level, where we use a novel technology developed by one of the project PIs (Jim Eberwine, Pharmacology, School of Medicine) to controllably deliver a define mRNA population to living cells to redirect their cellular phenotype, At the multicellular scale, where we use novel photopolymer formulations to assemble complex, three-dimensional cell culture substrates (Chris Chen, Bioengineering, School of Engineering and Applied Science) with tunable microenvironments for building vascularized tissue and cartilage, and At the tissue scale, where we use widely available optical activation techniques to study the in vivo programming of neural circuits in the cortex and hippocampus to understand changes that occur during disease or injury (David Meaney, Bioengineering, School of Engineering and Applied Science). This new microscope system will replace an existing 12 year old BioRad multiphoton microscope in the engineering complex at the University of Pennsylvania. The current BioRad system does meet the high technical demands of the above applications. Moreover, there is no widely available existing system on the Penn campus to perform this work. Therefore, there is substantial need for this microscope system. The combination of these three 'base technologies'on one microscope platform can significantly advance research topics in broadly diverse areas such as cellular differentiation, regenerative medicine, and the etiology of neurological disease and neurobehavior. The potential of integrating two or three of the base technologies into a single topic area provides nearly limitless possibilities for cutting edge advances in how living systems form and regenerate tissue, as well as developing a platform for assembling novel tissue replacement or RNA-based therapeutic approaches.

? DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) remains a major public health problem and is on pace to become the third leading cause of death and disability in the world population by 2020. Although we know that both neuronal degeneration and cognitive deficits are common features of human TBI, we are only beginning to appreciate an entirely new dimension of the disease: how brain networks change immediately after injury, and how cognitive recovery may depend critically on rebuilding these networks. The broad, long-term goal of our work identifies the fundamental mechanisms on how neural activity and intracellular signaling pathways together contribute to rebuilding a circuit after TBI. To our knowledge, our preliminary data is the first evidence showing how the structure of neural circuits changes after TBI, and the first observation on how combining circuit activation with pharmaceutics can together rebuild a network. We build on our preliminary data and propose the following aims: Specific Aim 1: To examine mechanisms of neuronal disconnection from a network after mechanical injury in vitro and in vivo. Specific Aim 2: To determine mechanisms for the activity-induced re-integration of neurons into an injured microcircuit over time, extending this into measuring circuit remodeling in awake, behaving animals subjected to TBI. Our general hypotheses are: (a) Connectivity directly influences neuronal disconnection from a network following injury, as well as network recovery (b) neuronal re-integration into the network is mediated by calcineurin activity and CREB-phosphorylation, (c) neuronal disconnection is mediated by mitochondrial signaling, and (d) prolonged circuit activation, in combination with pharmaceutics, can optimally control the reconstruction of networks. Impact: Knowing the mechanisms for remodeling neuronal connections in a circuit over time after TBI will give us insight into treatments protecting the network structure. Once the mechanisms for remodeling circuitry in the living brain after TBI are better understood, we envision testing therapies in preclinical TBI models within the next 5-10 years. Broadly, we believe this work will shift the therapeutic focus away from reducing neuronal death and towards approaches to rebuild functional circuits after TBI.

For proper neuron function, the neuron must have the correct number of input dendrites, which look like branches on a tree. Very little is currently known about how the pattern of these branches is determined or how these branches change when a person learns. To make new dendrite branches, a cytoskeleton must be produced, much like the skeleton in fingers. Dr. Firestein discovered a protein called cypin that plays a critical role in making this cytoskeleton, and hence dendrites. It is hypothesized that without cypin, neurons will not form properly, and the brain will not function correctly. Using nerve cells in a dish, undergraduate and graduate students will perform experiments to understand the mechanism by which cypin acts to yield functioning neurons. By investigating how cypin gives nerve cells their shape and how these nerve cells integrate into simple circuits, this research will aid in our understanding of principles by which circuits may be modified during events, such as learning. Dr. Firestein and colleagues will include a diverse group of undergraduate and graduate students and will establish an exchange program with University of Puerto Rico. This proposal also encompasses activities to excite a younger generation of students (K-12) about neuroscience by producing a video series "Teach Me Neuroscience" and training K-12 teachers at the bench. It is Dr. Firestein's hope to establish a program to bring neuroscience to the community in Puerto Rico via seminars, workshops, and exchange programs.

The specific goal of the current work is to evaluate how cypin and its binding partner PSD-95 affect neural circuit dynamics. Experiments aim to determine the mechanism by which cypin decreases synaptic PSD-95 using viral-mediated gene expression in cell culture to alter cypin levels. The role of the proteasome in cypin-mediated changes in PSD-95 will be assessed. It will also be determined whether cypin and PSD-95 levels affect function and activity of neural circuits in vitro. Dr. Firestein has a comprehensive set of tools available to manipulate cypin functionality at the molecular level and will assess effects of altered cypin levels on dendrite number, spine number, and size. Experiments will make use of electrophysiology techniques to determine whether cypin affects neuronal signaling. The proposed work uses interdisciplinary approaches - molecular/cellular, biochemical, and electrophysiological - to understand how morphological changes to neurons result in changes in synaptic function, making a large advance from previous cell culture work and defining a mechanism by which cypin acts to regulate dendritogenesis and determine effects on neural circuitry.

? DESCRIPTION (provided by applicant): Finding precise reasons why some patients take much longer to recover from a mild traumatic brain injury (TBI) and why some brains are more vulnerable to rapid head rotation or impact will significantly improve our ability to identify at-rsk populations for TBI and help patients safely recover from these injuries. Our past work showed us the GluN2B subunit of the NMDA receptor confers a `force sensing' property to the receptor, and we determined this feature is controlled by phosphorylation of a serine residue on the GluN2B receptor subunit. We use these past findings to ask a broad question - does the GluN2B-based NMDAR mechanosensitivity provide a biomechanics-based reason for susceptibility and vulnerability of the brain to TBI? Our goal in this R21 proposal is to develop the transgenic tools to answer this question. To this end, we hypothesize that transgenic animals with reduced NMDAR mechanosensitivity will show a significant reduction in cognitive deficits and neuronal degeneration after both a single and repeated TBI. Our proposal examines this hypothesis in two aims: Aim 1: To test if GluN2B-S1323 site mutation affects neural development, behavior, hippocampal function, and neural architecture. Aim 2: To study if animals with mutations in the GluN2B subunit show improved (NMDA1323A) or worse (NMDA1323E) outcome after experimental TBI. We expect NMDAM1323A and NMDA1323E mice will develop normally and exhibit normal cognitive functions. However, because they have significantly reduced `force sensing' ability, NMDA1323A mice will show significantly less cognitive deficits and faster recovery after mild TBI. Conversely, NMDA1323E mice will show enhanced deficits following TBI. Impact: To our knowledge, this work will be the first to change the biomechanics of brain trauma at the molecular level. We expect two broad scientific themes emerging from this work. First, we would be positioned to examine epigenetic factors that enhance the expression of the GluN2B subunit, lending individuals more susceptible to TBI. Second, we would test the possibility that expression of the force-sensitive GluN2B subunit makes the recovering brain more vulnerable to a second injury.